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OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010

Page 99
Page 100 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POSTERS MONDAY & TUESDAY

POS-MON-01 POS-TUE-02
DIGESTIVE VACUOLE GENESIS AND ENDOCYTIC ROLES OF WNT SIGNALLING IN SPERMATOGENESIS
PROCESSES IN THE EARLY INTRAERYTHROCYTIC
STAGES OF PLASMODIUM FALCIPARUM Abud H.E.1, Kerr G.1, Horvay K.1 and Loveland K.L.1, 2, 3
1
Monash University, Department of Anatomy and Developmental
Abu Bakar N.1, Klonis N.1, 2, Hanssen E.1, 2, Chan C.1 and Tilley L.1, 2 Biology, Clayton Vic 3800. 2Monash University, Department of
1
Department of Biochemistry, La trobe University. 2Centre of Biochemistry, Clayton Vic 3800. 3ARC Centre of Excellence in
Excellence for Coherent X-ray Science, La Trobe University. Biotechnology and Development.

The digestive vacuole of the malaria parasite Plasmodium falciparum Male infertility is a worldwide problem with increasing incidence and
is the site of hemoglobin digestion and heme detoxification and is the may be caused in part by disruptions in the development of male
target of chloroquine and other antimalarials. The mechanisms for germ cells and their supporting somatic cells. We have discovered
genesis of the digestive vacuole and transfer of hemoglobin from the that testicular cell communication via the Wnt signalling pathway is
host cytoplasm are still debated. In the present study, we use live-cell required for sperm development, because genetically altered mice
imaging and photobleaching to monitor the uptake of the pH-sensitive with perturbed Wnt signalling exhibit interrupted spermatogenesis and
fluorescent tracer SNARF-1-dextran from the erythrocyte cytoplasm in appear to have reduced fertility. We are currently using two unique
ring-stage and trophozoite-stage parasites. We compare these results mouse models to understand the precise functional role of Wnt signalling
with electron tomography of serial sections of parasites at different during spermatogenesis. We are studying the effect of blocking Wnt
stages of growth. We show that uptake of erythrocyte cytoplasm is signalling by conditional mutation of beta-catenin, a key mediator
initiated in mid-ring-stage parasites. The host cytoplasm is internalised of canonical Wnt signalling. In contrast, the effects of constitutively
via cytostome-derived invaginations and concentrated into several active Wnt signalling will be studied by conditional mutation of the
acidified peripheral structures. Hemoglobin digestion and hemozoin negative regulator, Adenomatous Polyposis Coli (APC). In addition, the
formation take place in these vesicles. The ring-stage parasites can endogenous Wnt signalling components and downstream target genes
adopt a deeply invaginated cup shape but do not take up hemoglobin via present in the mouse testis are being examined using a combination
macropinocytosis. As the parasite matures, the hemozoin-containing of immunohistochemical and qRT-PCR techniques. Our results to date
compartments coalesce to form a single acidic digestive vacuole that implicate canonical Wnt signalling in post-mitotic germ cell development,
is fed by hemoglobin-containing vesicles. There is also evidence for and ongoing work with human clinical specimens will reveal the potential
hemoglobin degradation in compartments outside the digestive vacuole. contribution of disturbances in this pathway to male infertility.
The work has implications for the stage specificity of quinoline and
endoperoxide antimalarials.

POS-MON-03 POS-TUE-04
L1CAM ACTS AS A SOX10 MODIFIER GENE DURING SEXUALLY-DIMORPHIC EXPRESSION OF MIR202* IS
ENTERIC NERVOUS SYSTEM DEVELOPMENT ASSOCIATED WITH TESTIS DIFFERENTIATION IN THE
MALE CHICKEN EMBRYO
Anderson R.B.1, Wegner M.2 and Wallace A.S.1
1
University of Melbourne, Australia. 2University of Erlangen-Nurnberg, Bannister S.C.1, 3, Smith C.A.2, Buermans H.4, Doran T.J.1, Sinclair A.H.2, 3
Germany. and Tizard M.L.V.1
1
CSIRO Livestock Industries, Australian Animal Health Laboratory,
Geelong, Australia, 3220. 2Early Development & Disease, Murdoch
During development, the enteric nervous system (ENS) is derived from Children’s Research Institute, Royal Children’s Hospital, Parkville,
neural crest cells that emigrate from the hindbrain, enter the foregut and Victoria, Australia, 3052. 3Department of Paediatrics, The University of
migrate caudally to colonise the entire gut. Failure of neural crest cells Melbourne, Parkville, Victoria, Australia, 3052. 4Center for Human and
to fully colonise the gastrointestinal tract results in an ‘aganglionic zone’ Clinical Genetics, Leiden University Medical Centre, Leiden, Netherlands.
that lacks an enteric nervous system over a variable length of the distal
bowel, a condition in humans known as Hirschsprung’s disease. The In the chicken, sex is determined genetically by the inheritance of sex
variability observed in the penetrance and severity of Hirschsprung’s chromosomes at fertilization. In birds the sex chromosomes are Z and
disease strongly suggests a role for modifier genes. Human clinical and W and assort as ZW in females and ZZ in males. Establishment of the
animal model studies have suggested that the X-linked gene, L1CAM, sexual phenotype of the embryonic gonads is subsequently driven by the
may act as a modifier gene for the development of Hirschsprung’s expression of sex-determining genes. In the chick, the gonads begin to
disease. To examine whether L1cam interacts with the Hirschsprung’s differentiate into testes and ovaries at embryonic day 6.5 (E6.5). After this
associated gene, Sox10, we used a two-locus complementation point, testes develop bilaterally in the male, whilst asymmetric development
approach. To assay the effects of an interaction, we examined whether proceeds in the female, with only the left gonad forming a functional ovary.
the migration of enteric neural crest cells was altered in L1cam null We are studying the chicken embryo as a model for vertebrate gonadal
mutant mice when combined with a heterozygous mutation in Sox10. development and aim to identify how microRNAs (miRNAs) may be involved
We show that interactions between L1cam and Sox10 significantly delay in defining, or maintaining the differentiation of embryonic testes and
ovaries. MiRNAs are 21-24nt non-coding RNAs which potentiate sequence-
neural crest migration within the developing gut, and that neural crest specific, post-transcriptional gene repression during development. We
cells undergo excessive cell death prior to gut entry. Using a doxycycline have used microarray and next-generation sequencing technologies to
inducible system, we show that Sox10 can regulate the expression of compare male and female miRNA expression profiles across three stages
L1cam. Thus, L1cam can act as a modifier gene for the Hirschsprung’s of gonadal sex differentiation in the chick. We have since focused our
associated gene, Sox10, and is likely to play a role in the aetiology of studies on chicken MIR202*, which was found to be up-regulated in testes
Hirschsprung’s disease. cords of male gonads, from the onset of sex differentiation. Our recent
work shows MIR202* expression is suppressed during estrogen-induced
feminization of male embryonic gonads and promoted in female gonads
when estrogen synthesis is blocked by an Aromatase inhibitor. These
findings suggest MIR202* expression is associated with male embryonic
testes development. Our continuing studies are focused on elucidating the
cellular and molecular function of MIR202* during sexual differentiation.
We are currently using in ovo retroviral-mediated delivery to optimise
over-expression of MIR202* during early gonadogenesis. This will allow
us to identify and validate MIR202* gene targets and understand the sex-
specific regulation of MIR202* expression and processing.

OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 101


POSTERS MONDAY & TUESDAY

POS-MON-05 POS-TUE-06
TWIST1 IS REQUIRED BOTH IN THE CRANIAL NEURAL FGF9 ANTAGONISES FEMALE AND PROMOTES MALE
CREST AND THE CRANIAL MESODERM FOR PROPER GERM CELL FATE IN MICE
HEAD DEVELOPMENT
Bowles J., Feng C.-W., Davidson T.-L., Spiller C. and Koopman P.
Bildsoe H.1, 2 , Loebel D.A.F.1, 2, Jones V.1, Hor A.1, Steiner K.1, Chen Y.T.3, Institute for Molecular Bioscience, University of Queensland, Australia.
Beringer R.R.3 and Tam P.P.1, 2
1
Embryology Unit, Children’s Medical Research Institute, Locked Bag In the fetal gonad, germ cells commit to a female or male sexual fate
23 Wentworthville, NSW 2145, Australia. 2Sydney Medical School, on the basis of environmental cues, rather than XX or XY chromosome
University of Sydney, NSW, Australia. 3Department of Molecular constitution. Germ cells in a developing ovary enter meiosis, hence
Genetics, University of Texas, M.D.Anderson Cancer Center, 1515 committing to the female fate or oogenesis. In a developing testis, germ
Holcombe Blvd, Houston, TX 77030, USA. cells do not enter meiosis during fetal life but they do stop proliferating
and arrest in G0/G1 (mitotic quiescence), hence committing to the male
The basic helix-loop helix transcription factor Twist1 is a key regulator fate or spermatogenesis. In recent years we showed germ cells in a
of craniofacial development. Twist1-null mouse embryos exhibit failure female mouse embryonic gonad are triggered to enter meiosis by the
of cephalic neural tube closure and abnormal head development. The potent signaling molecule retinoic acid (RA). RA induces germ cells to
mutant embryos die at E11.0 preventing studies beyond midgestation. express a key gene, Stra8, which encodes a protein essential for initiation
We have used Cre-loxP conditional deletions to dissect the function of of meiosis. In the developing testis, germ cells avoid entering meiosis
Twist1 specifically in the cranial neural crest (CNC) and cranial mesoderm because RA is actively degraded by a cytochrome P450 enzyme,
(CM). Deletion of Twist1 in CNC cells resulted in loss or malformations CYP26B1. Hence, CYP26B1 is a ‘meiosis-inhibiting’ substance by virtue
of the CNC-derived skeleton, including the frontal bone, upper jaw and of its ability to degrade the meiosis inducer, RA. Here, we report that a
snout, due to impaired CNC cell survival and differentiation. Parietal second ‘meiosis-inhibiting’ substance, FGF9, acts directly on germ cells
and interparietal bones are also affected although these structures to prevent up-regulation of Stra8 and hence their entry into meiosis.
are primarily of mesodermal origin. Loss of Twist1 in the CM leads to In addition, FGF9 maintains germ cell pluripotency and promotes
incomplete closure of the cephalic neural tube but normal development expression of male fate markers. Since RA is more abundant in the
of 1st branchial arch and frontonasal tissues. The majority of these developing ovary and FGF9 is more abundant in the developing testis,
embryos died at mid-gestation. Embryos developing past midgestation the model we propose allows for ‘back-up’ and hence robustness in the
show loss or reduction of parts of the chondrocranium and poor oogenic/spermatogenic fate decision. Antagonistic interplay between
development of the posterior skull base, while the viscerocranium was FGFs and RA is proving to be a recurring theme in development, in each
not affected. Taken together, the phenotypes of these two tissue-specific instance being associated with key cell lineage decisions. Our findings
conditional deletions recapitulate that of the null mutation. Our results provide a new example of this phenomenon.
further show that tissue-specific loss of Twist1 function in one of the two
cell populations that make up the craniofacial skeleton can impact on
both tissues, suggesting that interaction between primordial tissues is
critical for proper craniofacial development.

POS-MON-07 POS-TUE-08
SIDEROPHORE DISCOVERY BY APPLICATION TWIST FUNCTION IN CRANIOFACIAL MORPHOGENESIS
OF IMMOBILIZED METAL ION AFFINITY
CHROMATOGRAPHY Brinas I.B., Wade C.W. and Farlie P.F.
Murdoch Childrens Research Institute, Royal Children’s Hospital,
Braich N.E. and Codd R. Flemington Rd, Parkville, Victoria, 3052.
School of Medical Science (Pharmacology) and Bosch Institute, The
University of Sydney, NSW 2006, Australia. Twist1 has been demonstrated to play critical roles in the early
development of neural crest and mesodermally derived tissues. Twist2
Siderophores are low-molecular-weight Fe(III) binding secondary has been less well characterised but its relatively late onset of expression
metabolites produced by bacteria and plants. Siderophores are suggests specific roles in the development of a number of sites. We
pharmacologically important and have numerous applications. Currently have used RCAS-mediated overexpression to investigate the function of
they are clinically used for the treatment of iron overload diseases. Twist genes in craniofacial development. Sustained expression of Twist1
However, they are difficult to synthesize and/or purify. Thus, studies or 2 results in failure of fusion between the maxillary, lateral nasal and
were conducted on the use of Ni2+-charged immobilized metal ion frontonasal processes. The Twist1 phenotype tends to be more severe
affinity chromatography (IMAC) for the capture of hydroxamate-based than the Twist 2 phenotype. Analysis of the craniofacial skeleton reveals
siderophores as a fast and effective means of biodiscovery. Results a failure of fusion between the premaxilla and the maxillary/palatine
showed the successful capture of monohydroxamate, dihydroxamate, bones. In addition, the premaxilla, maxilla and palatine bones were
and trihydroxamate based siderophore standards and optimal capture hypoplastic. These facial clefts were either uni- or bilateral in nature and
conditions were also established. IMAC was successfully used for the exhibited substantial variation in the severity in terms of the width of the
purification of desferrioxamine B (DFO B), a trihydromate, from crude cleft and the extent of hypoplasia. Apart from some skeletal hypoplasia,
samples of culture supernatant of the DFOB-producing bacterium the mandible appears to be predominantly unaffected by Twist1 or 2
Streptomyces pilosus. RP-HPLC traces indicate a significant purification overexpression. There are no consistent changes in either Fgf or Shh
of >50 species reduced to 3 species, of which 2 are media derived. or Bmp signalling around the time of facial fusion. The molecular basis
Salinispora triopica is an actinomycete bacteria which is predicted by of this phenotype remains unclear. Apoptosis and proliferation was
bioinformatics to produce multiple siderophores including DFO and quantified to determine the cause of facial clefts in Twist1 overexpressed
yersiniabactin like siderophores. Preliminary results show that S. tropica embryos. There was no change in apoptosis of Twist1 overexpressed
produces DFOB, but also produces several unidentified siderophore- and control embryos. There was, however, an unexpected 3-fold
like molecules. Currently, investigations are underway on identifying the increase in the number of proliferating cells within the lateral nasal and
remaining siderophores. This work opens the door for the use of IMAC frontonasal masses of Twist1 overexpressed embryos. It is likely that
in discovering a range of secondary metabolites with metal ion binding Twist2 overexpression is also upregulating proliferation but we will test
affinity. this. Overall, Twist1 and 2 appear to have important roles in craniofacial
morphogenesis and particularly in the fusion of the facial processes.

Page 102 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010


POSTERS MONDAY & TUESDAY

POS-MON-09 POS-TUE-10
DISCOVERY OF A NOVEL A-TYPE LAMIN PROLIFERATION OF NEURONS AND NON-NEURONAL
INTERACTING TRANSCRIPTION FACTOR, LITF, CELLS IN THE DEVELOPING MOUSE STELLATE
REQUIRED FOR MYOGENESIS GANGLION
Ahmady E.1, Deeke S.A.1, Rabaa S.1, Kouri L.1, Krotneva S.1, Kenney Gonsalvez D.G., Cane K.N. and Anderson C.R.
L.1, Blais A.2, Stewart A.F.R.1 and Burgon P.G.1 University of Melbourne. Parkville VIC, 3010.
1
University of Ottawa Heart Institute, Ottawa ON Canada. 2University
of Ottawa, Ottawa ON Canada. Mouse sympathetic ganglia appear around embryonic day (E)9.5,
with differentiated neurons present at E10.5 and the first glial cells
Lamins are intermediate filament proteins of the inner nuclear at E11.5. From E10.5 onwards, the number of cells in the ganglion
membrane fundamentally important for nuclear architecture, chromatin increases dramatically. We have used immunoreactivity to Sox10, to
organization and transcriptional regulation of gene expression. mark uncommitted neural crest cells and glia, and tyrosine hydroxylase
However, the molecular mechanisms that couple lamins to transcription (TH), to mark sympathetic neurons in E10.5 to E18.5 stellate ganglia
are poorly understood. Here, we report the identification of an A-type (n=4 in each case). We have also estimated cell division rates with
Lamin-interacting Transcription Factor, LITF, a unique single copy bromodeoxyuridine (BrdU) and S-phase length with BrdU and
amniota gene. Chromatin immunoprecipitation and gel mobility shift ethyldeoxyuridine(EdU). At E10.5, 99% of all cells are positive for Sox10
assays demonstrated that LITF binds to DNA within close proximity of expression, with half of this population also expressing TH; only 1% of
genes encoding many transcription factors that control tissue-specific cells express TH alone. By E11.5, 19% of cells express Sox10 alone,
differentiation. Transient transfections of GAL4-LITF fusion constructs 6% express both Sox10 and TH and 76% of cells express TH only.
with a GAL4 responsive reporter showed that LITF contains a strong The proliferation rate of both TH-IR and Sox10-IR cells was highest
transactivation function within its conserved C-terminal domain. LITF around E12.5, when around 50% of both neuronal and non-neuronal
protein is primarily expressed in cardiac and skeletal muscle and cells in the ganglion contained BrdU after a two hour pulse. We have
is expressed as early as E8.5 in the mouse embryo. Using the well- determined that S-phase lengths for neuronal and non-neuronal cells
characterized C2C12 model of in vitro muscle differentiation, we differ during this period (TH+-6hrs, Sox10+-3hrs). Total cell cycle lengths
found that blocking LITF expression in myoblasts also prevented the for the two cell populations do not differ greatly at either of the two time
expression of myogenic transcription factors (Mef2C, MyoD, Myf6 & points considered. Proliferation of non-neuronal cells overtook that of
MyoG) and subsequently inhibited myogenic differentiation. In addition, neurons on E16.5, when neuronal division had dropped to a low rate.
LITF mediated chromatin immunoprecipitation studies revealed that Between E11.5 and E14.5, the growth of the ganglion is largely due to
LITF interacts with many DNA regions in close proximity of transcriptions the division of existing neurons. The increasing disparity between the
factors (GATA4, Pitx2, Mef2c, RXRα and Sox4) that are important for number of neurons and non-neuronal cells during this period does not
normal muscle formation and differentiation. Data will be presented depend on differences in proliferation rate or cell cycle length, but solely
from the phenotypic analysis of a muscle specific LITF null. These on the relative starting numbers of neurons versus non-neuronal cells,
observations coupled with the molecular uniqueness of LITF suggest which is established on E10.5 when most, but not all, of the neural crest
that LITF lies in a prominent position within the regulatory process of precursor cells differentiate into neurons.
muscle development. Our discovery of LITF provides the first direct
molecular link between the Lamin A/C gene and gene expression.

POS-MON-11 POS-TUE-12
POSITIONAL CLONING OF LYMPHATIC MUTANTS IN CHARACTERISATION OF THE DEVELOPMENTAL
ZEBRAFISH ROLES OF THE DROSOPHILA MACPF PROTEIN
TORSO-LIKE
Coxam B.1, Neyt C.1, Shulte-Merker S.2 and Hogan B.1
1
Institute for Molecular Bioscience, The University of Queensland, 306 Crossman T.1, 2 , Johnson T.K.1, 2, Herr A.1, 2, Whisstock J.C.2 and Warr
Carmody Road, St Lucia, 4072, Brisbane, QLD, Australia. 2Hubrecht C.G.1
Institute Uppsalalaan 83584 CT UTRECHT,The Netherlands. 1
School of Biological Sciences, Monash University, Clayton, Victoria,
Australia 3800. 2Department of Biochemistry and Molecular Biology,
Lymphatic vessels play an essential role in fluid homeostasis, immune Monash University, Clayton, Victoria, Australia 3800.
responses, fat absorption, and in pathological processes such as
lymphedema and cancer metastasis. Despite recent breakthroughs Torso-like (tsl) is the sole Drosophila member of the Membrane Attack
in unravelling the complexity of lymphatic vessel development (called Complex and Perforin (MACPF) protein superfamily. While members
lymphangiogenesis), a lot is left to discover about the early steps of this of the MACPF family are typically involved in immunity and defence,
process and its underlying cellular dynamics and molecular regulation. tsl has long been established as a maternally expressed gene and key
Using a genetic screen in zebrafish, we have identified two mutants component of the pathway responsible for patterning the anterior and
presenting novel lymphatic phenotypes. One mutant presented a block posterior poles of the developing oocyte. However, our recent research
in lymphangiogenesis associated with larval stage cardiac dysfunction has indicated additional roles for tsl in development. Expression studies
and the other exhibited pleiotropic developmental defects encompassing using in situ hybridization experiments and a reporter gene line have
a block in lymphangiogenesis, blood vasculature hyper-branching shown that tsl is expressed in a number of specific tissue and cell types.
and a dysmorphic head. Initial molecular genetic data and a detailed In the developing embryo we see tsl expression in the midline glia of the
phenotypic analysis at the cellular level using vascular transgenic central nervous system, a specialized set of glia that are analogous to
fish lines will be presented. Keywords: Lymphatic system, Zebrafish, the vertebrate floor plate. During larval stages we see tsl expression in
Positional Cloning. the prothoracic gland, a part of the ring gland, which is the hormonal
control centre of the developing fly. In adults we see expression of tsl in
a subset of cells in the retina. Functional studies on tsl mutant strains
have shown serious central nervous system defects, indicating a role
for tsl in midline glia survival or function. To study the role of tsl in the
prothoracic gland we are performing tissue-specific RNA interference
experiments. The role of tsl in the adult retina is being characterised
using available mutant strains and the many markers available for
different cell types in the retina. Overall these approaches aim to enable
the complete characterisation of the roles of tsl from embryogenesis to
adulthood, and to provide insight into the roles of MACPF proteins in
development.

OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 103


POSTERS MONDAY & TUESDAY

POS-MON-13 POS-TUE-14
INTEGRIN LINKED KINASE (ILK) IS REQUIRED NARINGIN PROMOTES ANTICARCINOGENIC EFFECTS
FOR LENS EPITHELIAL CELL SURVIVAL AND ON RATS BEARING WALKER 256 TUMOR
PROLIFERATION
Camargo C.A.1, Wutzki N.C.1, Bueno L.G.M.1, Gomes-Marcondes
Teo J.1, McQueen-Miscamble L.1, Turner K.1, Martinez G.1, Reneker L.2, M.C.C.2, Da Silva M.E.F.3 and Aoyama H.1
Dedhar S.3, Robinson M.L.4 and De Iongh R.U.1 1
Departamento de Bioquímica, Instituto de Biologia, UNICAMP –
1
Anatomy & Cell Biology, University of Melbourne, Parkville, Victoria, Campinas, São Paulo, Brazil. 2Departamento de Anatomia, Biologia
Australia. 2Ophthalmology, University of Missouri. 3British Columbia Celular e Fisiologia, Instituto de Biologia, UNICAMP – Campinas, São
Cancer Research Center, Vancouver, Canada. 4Zoology, Miami Paulo, Brazil. 3Instituto de Ciências da Saúde, UNIP - Campinas, São
University, Oxford, Ohio. Paulo, Brazil.

The role of growth factors in lens development has been investigated Naringin (NAR), a plant polyphenolic compound, is a naturally occurring
extensively; the role of ECM signaling is less well understood. The laminin- citrus flavonone (oranges and grapefruits). Flavonoids have been
binding integrins (α3β1, α6β1) are cooperatively required for epithelial cell reported to exhibit numerous biological and pharmacological effects, such
survival during embryonic development. In this study we investigated as antioxidant, anticarcinogenic, antiinflammatory and cardioprotective.
the role of integrin linked kinase (ILK), a downstream mediator of ECM- Cachexia is a poorly understood syndrome characterized by generalized
integrin signaling, in lens development. We generated mice that were host wasting, anorexia and a variety of metabolic alterations that result
conditionally null for Ilk in the lens or expressed a hyperactive ILK in the in death. One characteristic of Walker 256 tumor (W256) is to provoke
lens. Consistent with its epithelial expression, Ilk deletion only resulted in cachexia in infected animals. More than 80% of the patients with
a phenotype if deleted in lens epithelium, not when deleted in fibres only.
The phenotype was characterised by thinning of the epithelium by E17.5, malignant disease present features of malnutrition and cachexia as the
loss of central epithelial cells by P2 and extensive fibre cell degeneration cause of death. This study was designed to describe an in vivo naringin
and apoptosis by P10. At E17.5 there was significant inhibition (~50%) therapeutic treatment of rats bearing W256. Rats bearing W256 were
of epithelial cell cycle progression (reduced BrdU, cyclin D1, cyclin D2 treated with different daily doses (10, 25 and 35 mg/kg) of naringin
and phospho-histone3 staining), but no significant changes in epithelial i.p. for 50 days or until the obit. Survival, dose-response (ED50) and
markers (E-cadherin, Pax6). Loss of Ilk also affected deposition of ECM tumor regression curves were obtained and 25 mg/kg was taken as the
(laminin and collagen IV), with retention of collagen in exocytic pathway. effective dose. Our studies showed that daily administrations of naringin,
Erk and Akt phosphorylation were markedly decreased in Ilk null lenses. in low doses (10-25 mg/kg), had inhibited tumors growth (about 50%)
Postnatally, there was reduced and delayed expression of fibre cell in comparison with the control animals. Moreover, two animals of this
markers, β-crystallin, c-Maf, p57kip2 and p27kip1, but not Prox1, and abnormal group had presented complete tumor regression and when inoculated
accumulation of fibronectin and α-smooth muscle actin. Ectopic expression again with the carcinosarcoma, no tumor development was observed.
of hyperactive kinase ILK (S343D) did not affect lens development However, at a higher concentration (35 mg/kg) this flavonoid provoked
with only subtle changes in epithelial cell morphology detected in vitro corporal weakening and cachexia process in the animals. Our studies
(increased formation of lamellipodia, enriched ILK at focal contacts). The suggest that naringin can be used as an effective drug to treat cancer
hyperactive ILK transgene completely rescued lens morphology in Ilk null and to prevent against the effects of cachexia. Financial Support:
mice cell, but proliferation was incompletely rescued. These data indicate CAPES, CNPq and FAPESP.
IK is required for epithelial proliferation, survival and subsequent fibre cell
differentiation. ILK is required for epithelial proliferation and survival, but is
insufficient to initiate cell proliferation.

POS-MON-15 POS-TUE-16
REGULATION OF INTERNEURON DEVELOPMENT BY REPAIRING HYPEROXIA-MEDIATED RESPIRATORY
SUPPRESSOR OF CYTOKINE SIGNALLING-2 (SOCS2) DEFICIT WITH ENDOTHELIAL PROGENITOR CELLS
Faux C.H. and Turnley A.M. Firsova A.B., Cole T.J. and Mollard R.A.
Centre for Neuroscience, The University of Melbourne. Monash University, Victoria, Australia.

Gamma-aminobutyric acid (GABA)ergic interneurons play a vital role in High tension oxygen treatment is performed to assist breathing of
modulating the activity of the cerebral cortex. Comprising approximately very prematurely born babies. However, such treatment can result in
20% of the total cortical neuron population, they are an extremely disruptions to lung vascularization and alveolarization and eventually lead
diverse population of cells, differing in their morphology, physiology and to chronic lung disease. Steps are taken to reinforce cell differentiation
molecular characteristics. Recent studies have shown that a disruption and repair lung tissue injured following oxygen treatment, but these
to the function of GABAergic interneurons can directly contribute to steps remain sub-optimal. Previous results suggest that administration of
neurological and mental health issues such as schizophrenia, epilepsy endothelial progenitor cells (EPCs) from bone marrow to the diabetic foot
and depression. Often such disruptions arise during development, with can stimulate neovascularisation and thus lead to beneficial functional
alterations to interneuron number, distribution or connectivity leading to outcomes. We have hypothesized therefore that EPCs may engraft the
functional neural impairment. Currently, however, little is known about site of hyperoxia-induced lung damage to similarly offer benefit. In this
the signalling mechanisms that regulate interneuron specification, study, a model of hyperoxia-mediated deficit of the neonatal mouse lung
migration or maturation. We have previously shown that the regulatory has been established to study such EPC engraftment and investigate
protein suppressor of cytokine signalling-2 (SOCS2) is a key player associated functional outcomes. Newborn mice were treated with 90%
in interneuron development. SOCS2-overexpressing mice have large oxygen for four days at birth and parameters of lung development were
increases in numbers of calbindin and calretinin-expressing interneurons monitored for an eight week period. Relative to untreated controls, we
in the adult cortex. In contrast, SOCS2 knockout mice show a reduction observed (i) temporary changes in lung vascularization (blood vessel
in the number of parvalbumin positive cells. We have found that during number and Pecam1 protein levels decreased) and (ii) persistent
development SOCS2 is highly expressed in the ganglionic eminence changes in lung septation (alveolar number and tissue area decreased
(GE) of the ventral telencephalon, which is the primary source of cortical and alveoli diameter increased). Treated animals also displayed an
interneurons. In addition, SOCS2 is expressed in the developing cortical accelerated increase in the number of secondary septa between one day
plate and in the subventricular zone, one of the main regions through and eight weeks after hyperoxia exposure, such that an early secondary
which interneurons tangentially migrate to enter the cortex. These data septal number deficit was later returned to control levels. Data from
suggest that SOCS2 is involved in the early specification and/or in the bone marrow sorting and subsequent culture have identified Kdr and
migration of particular interneuron subtypes. Further elucidation of the Tek as key cell surface markers for vessel-like structure formation in
precise role that SOCS2 plays in interneuron development will greatly vitro. This model recapitulates many aspects of mechanical ventilation-
enhance our understanding of the complex mechanisms underlying associated alterations to the very preterm birth human lung and permits
various neurological disorders. study of the effects of attenuations to neovascularisation upon high
oxygen damaged alveoli.

Page 104 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010


POSTERS MONDAY & TUESDAY

POS-MON-17 POS-TUE-18
GENETIC INTERACTION BETWEEN TRANSCRIPTION DOES GENETIC VARIATION IN METABOLITE
FACTOR (Lhx1) AND WNT INHIBITOR (Dkk1) IN HEAD PROFILES CORRELATE WITH SUCROSE CONTENT?
FORMATION IN MOUSE EMBRYOS
Glassop D.1, 2 , Zwart A.3 and Bonnett G.D.1, 2
Fossat N., Khoo P.-L., Jones V., Lewis S.L. and Tam P.P.L.
1
CSIRO Plant Industry, St Lucia, Queensland, 4067. 2CRC Sugar
Embryology Unit, Children’s Medical Research Institute, University of Industry Innovation through Biotechnology, St Lucia, Queensland,
Sydney, Westmead, NSW 2145, Australia 4067. 3CSIRO Mathematics Informatics and Statistics, Acton,
Australian Captial Territory, 2601.
In mouse embryos, Lhx1 gene encoding the LIM homeobox 1 transcription
factor is expressed in the anterior visceral endoderm (AVE) and the Sugarcane provides most of the world’s supply of sugar. Sugarcane
anterior axial mesendoderm (AME). The expression pattern of Lhx1 is able to accumulate sucrose in mature stem tissue, reaching
partly overlaps with that of Dkk1 (Dickkopf-1) that encodes an inhibitor of concentrations of ~50% of the dry weight. In recent years the yield of
the WNT signalling pathway. Loss of Lhx1 function in the whole embryo sucrose from sugarcane has only increased due to increases in the yield
leads to truncation of the head at the mid- to hindbrain level. However, of biomass rather than increased sucrose concentration of the biomass.
chimeric embryos containing wild type visceral endoderm (including This has lead the search for new approaches to break this upper limit
AVE) and Lhx1-deficient embryonic tissues also display head defects, for the concentration of sucrose stored. The evolution of the ‘omics’
suggesting that Lhx1 function is required more than in the AVE, which sciences provides an opportunity to extensively examine sugarcane
is likely to involve the AME. To better characterise the non-AVE related via genomics, transcriptomics, proteomics and metabolomics in
function of Lhx1, the transcriptome of Lhx1-/flox;Meox2+/Cre (Lhx1 CKO) the search for alternative strategies for increasing sucrose content.
embryos in which Lhx1 is inactivated in the embryo proper but not in the Previous research revealed metabolic profiles specific to stem tissues
visceral endoderm was analyzed. In the Lhx1-deficient tissues, the gene of different developmental age. Those results encouraged research
expression profile was reminiscent of an increase of the WNT signalling to determine the variation in metabolite profiles between sugarcane
activity. Testing the expression of the BATGal transgene where LacZ progeny segregating for sucrose content. Using gas chromatography
expression reports WNT signalling, ectopic activation of WNT activity mass spectrometry we have detected and quantified 105 metabolites
was found in the anterior region of the Lhx1 CKO mutant embryos. A in sugarcane stem tissue from internodes four (immature) and nine
functional connection between the transcriptional function of Lhx1 and (mature) from five high and five low sucrose genotypes. Of the 105
WNT signalling was further demonstrated by the manifestation of the metabolites detected and measured, 46 could be assigned an identity on
head truncation phenotype in compound heterozygous Dkk1+/-;Lhx1+/CKO the basis of mass spectra and retention time; 59 remained unidentified.
mutant embryos which was not found in either Dkk1+/- or Lhx1+/CKO simple Some of the identified metabolites have not been previously identified in
heterozygous mutants. The synergistic interaction between Lhx1 and sugarcane metabolite profiles. This poster will report on the relationship
Dkk1, principally in the AME, is therefore essential for the formation of between metabolites and sucrose content and identify pathways which
the embryonic head through the negative modulation of WNT signalling could potentially be manipulated for increased sucrose production.
activity.

POS-MON-19 POS-TUE-20
DNA DAMAGE-INDEPENDENT FUNCTIONS OF ASCIZ FUNCTIONAL IDENTITY OF THE GAMMA
AS AN ESSENTIAL REGULATOR OF PULMONARY TROPOMYOSIN (γ-TM) GENE: SPECIFIC ROLES IN
ORGANOGENESIS EMBRYONIC DEVELOPMENT, REPRODUCTION AND
CELL VIABILITY
Jurado S.1, Smyth I.2, Van Denderen B.1, Tenis N.1, Hewitt K.1, Cole
T.J.2 and Heierhorst J.1, 3 Hook J.1, Schevzov G.1, Hardeman E.C.2 and Gunning P.W.1
1
St. Vincent’s Institute of Medical Research. 2Monash University. 3Dept 1
Oncology Research Unit, School of Medical Sciences, University of
of Medicine, The University of Melbourne. New South Wales. 2Neuromuscular & Regenerative Medicine Unit,
School of Medical Sciences, University of New South Wales.
Zn2+-finger proteins comprise one of the largest protein superfamilies
with diverse biological functions. The Zn2+-finger protein ASCIZ (ATMIN/ Tropomyosins (Tm) are highly conserved components of actin
ZNF822) was originally discovered as a Chk2-interacting ATM substrate filaments which differentially regulate filament stability and function.
with functions in the DNA base damage response and also proposed to The mammalian Tm family consists of four genes; α-Tm, β-Tm, γ-Tm
be an essential cofactor of the ATM kinase. Here we show that absence and δ-Tm. Multiple Tm isoforms (>40) are generated by alternative
of ASCIZ leads to p53-independent late embryonic lethality in mice. splicing, and expression of these isoforms is highly regulated during
ASCIZ-deficient primary fibroblasts exhibit increased sensitivity to DNA development. We tested the specificity of function of products from the
base damaging agents MMS and H2O2, but ASCIZ deletion or knock- γ-Tm gene in mice using a series of gene knockouts. Mice homozygous
down does not affect ATM levels and activation in mouse, chicken or for the knockout of all cytoskeletal products from the γ-Tm gene
human cells. Unexpectedly, ASCIZ-deficient embryos also exhibit (Tm5NM1-11) were not viable and could not be detected as early as
severe respiratory tract defects, where lung buds never emerge from the blastocysts. Elimination of just two cytoskeletal products from the γ-Tm
respiratory endoderm and where ascending separation of the tracheal gene (NM1,2) resulted in a 50% reduction in embryo viability. We re-
bud from the ventral foregut stalls early. Genetically, the complete targeted the γ-Tm gene in embryonic stem (ES) cells hemizygous for the
pulmonary agenesis and severe tracheal atresia place ASCIZ between knockout of all or subsets of isoforms from this gene. It was not possible
Wnt/ß-catenin and FGF10 signaling pathways. The data indicate that, in to create knockout ES cells for the targets which eliminated or reduced
addition to its role in the DNA damage response, ASCIZ has separate embryo viability in mice. In contrast, homozygous ES cells were created
developmental functions as an essential regulator of respiratory for a different set of isoforms (NM3,5,6,8,9,11) which were not required
organogenesis. for embryogenesis. We also observed that males hemizygous for the
knockout of all cytoskeletal products from the γ-Tm gene preferentially
transmitted the minus allele with 80-100% transmission. Since all four
Tm genes are expressed in early embryos, ES cells and sperm, we
conclude that isoforms of the γ-Tm gene perform specific functions in
embryogenesis, ES cell viability and sperm function that cannot be
compensated by the other Tm genes.

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POS-MON-21 POS-TUE-22
AMOUNT OF DNA METHYLTRANSFERASE 1 REGULATION OF WNT SIGNALLING IS CRITICAL IN
TRANSCRIPTS IN VARIOUS DEVELOPMENTAL EARLY EYE DEVELOPMENT
STAGES OF MOUSE TISSUES AND THEIR
EXPRESSION SITES Chen Y.1, Storen R.1, 2, 3, Greenlees R.1, 3, Grigg J.2, 3, Tam P.1, 3 and
Jamieson R.1, 2, 3
Isokane T.1, Ghanem.E M.2, Nishibori M.1, Hiraiwa N.3 and Yasue H.4
1
Eye Genetics Group, Embryology, Children’s Medical Research
1
Grad. Sch. of Bio Sci., Hiroshima Univ., Hiroshima, Japan. 2Suez Institute. 2The Children’s Hospital at Westmead & Save Sight Institute.
Canal Univ, Egypt. 3RIKEN, Japan. 4Nat. Inst. Agrobiol. Sci, Japan.
3
Sydney Medical School, University of Sydney.

DNA cytosine methylation in mammals influences many cellular events, Aberrations in optic vesicle and cup formation lead to disorders affecting
including gene transcription, genomic imprinting, X chromosome vision including microphthalmia (small eyes) and abnormal iris and retinal
inactivation, carcinogenesis and genome stability. A member of DNA development. Distinct domains are specified in the distal part of the optic
methyltransferase (Dnmts) has been identified that catalyzes the vesicle for retinal progenitors and the dorsal part for retinal pigment
reaction of cytosine methylation in DNA. When the maintenance-type epithelium (RPE). In optic vesicles of E9.5 mouse embryos, WNT activity
DNA methyltransferase, designated as Dnmt1 is absent in mouse, is detected in prospective RPE via assaying the expression of TopGal
homozygous mutant embryos cannot survive after mid-gestation as their and BatGal reporters. In contrast, WNT reporter activity is absent in
genomic imprinting being canceled. Epigenetic modifications of chromatin prospective retina, where Dkk1, an inhibitor of canonical/beta-catenin
consist of DNA methylation and post- translational modifications to WNT signalling, is expressed. Hence, regionalization of WNT signalling
histones like acetylation, phosphorylation, glycosylation, ribosylation. activity may be critical for tissue specification in early eye development.
These modifications are key regulators of gene expression during While loss of Dkk1 function and increased canonical WNT signalling
growth and differentiation in all tissues including brain. In the present leads to head truncation precluding investigation of WNT signalling in eye
study, to elucidate the functions of Dnmt1, the amounts Dnmt1 sense/ development, reduction of Wnt3 in compound Dkk1-/-;Wnt3+/- mutant
antisense transcripts in the mouse’s tissue of different developmental mice rescues the head phenotype and reinstates eye development.
stages were measured by real-time RT-PCR. The localization of Dnmt1 Optic vesicle examination of E9.5 compound mutant embryos (Dkk1-
sense transcripts as well as the existence of antisense transcripts was /-;Wnt3+/-) harbouring a WNT reporter revealed ectopic response of
also investigated. In results, mouse brain cells showed a remarkable cells in prospective retina to WNT signalling. Concurrently, there was
Dnmt1 gene expression values during different stages of embryonic ectopic expression of RPE marker (Mitf) and reduced expression of
development. Antisense transcript was largely unaltered. The localization neuroretinal marker (Chx10) in WNT-responding cells. Subsequently,
of Dnmt1 transcripts in adult medulla oblongata, sense transcript was iris and retinal development was disrupted and small eyes formed.
observed remarkably in neuron, however antisense transcript observed These results indicate that active canonical WNT pathway is required
faintly around glia cell. In Cerebral cortex, the expression of these in specification of RPE fate of optic vesicle cells, and suppression of
transcripts was observed. These findings indicated that Dnmt1 has a this signalling pathway in prospective retina is essential for retinal cell
great role in brain development of adulthood. However, its role during fate. Our results demonstrate the role of a key inhibitor, Dkk1, in precise
the embryonic stages still needs more investigation. spatial and temporal regulation of canonical WNT signalling in early eye
development.

POS-MON-23 POS-TUE-24
THE ROLE OF THE DROSOPHILA MEMBRANE SOX18-REGULATED GENES IMPLICATED IN
ATTACK PROTEIN TORSO-LIKE IN EARLY EMBRYONIC LYMPHATIC DEVELOPMENT
PATTERNING
Kartopawiro J., Neyt C., Francois M. and Hogan B.
Johnson T.K.1, 2, Bennett M.2, Herr A.1,2, Warr C.G.2 and Whisstock J.C.1 Institute for Molecular Bioscience, University of Queensland, Brisbane
1
Department of Biochemistry and Molecular Biology, Monash 4072, Queensland, Australia.
University, Clayton VIC 3800, Australia. 2School of Biological
Sciences, Monash University, Clayton VIC 3800, Australia. Recent discoveries have demonstrated the pivotal role of lymphatic
vessels in numerous pathological states, including inflammatory
Membrane Attack Complex/Perforin-like (MACPF) proteins have diseases, tumor metastasis and lymphedema. Identification of new
important roles in vertebrate immunity and often function by forming molecular markers implicated in lymphatic vascular development
oligomeric pores in cell membranes causing cell lysis. However, several (lymphangiogenesis) is therefore essential in order to increase our
MACPF proteins play crucial yet poorly characterised roles in embryonic understanding of the mechanisms that underlie these disease conditions.
patterning and neural development. In Drosophila, correct formation of Recently, the transcription factor Sox18 has been shown to initiate
the terminal anterior and posterior structures of the embryo is governed lymphatic endothelial cell specification during mouse embryogenesis.
by Torso-like (Tsl), the only known Drosophila MACPF. Tsl is expressed The loss of Sox18 function leads to a complete loss of lymphangiogenesis
maternally in specific ovarian follicle cells at the poles of the developing and embryos die in utero completely devoid of lymphatic vasculature.
oocyte, where it is secreted and deposited in the perivitelline space However, the cascade of Sox18-dependent genes that modulates early
between the oocyte and future eggshell. After fertilisation Tsl mediates lymphatic vascular development is yet to be identified. In order to uncover
localised activation of Trunk (Trk), a noggin-like growth factor, and molecular targets of SOX18, a micro-array analysis has been performed
putative ligand of the Torso receptor tyrosine kinase. The mechanism from purified lymphatic endothelial cells, comparing wild type and Sox18
of Trk activation, and the role of Tsl, remains elusive, however one mutant mice. This study has revealed a subset of Sox18-regulated genes
hypothesis is that Trk is proteolytically cleaved by an unidentified with no known function in lymphangiogenesis. Using a zebrafish model
protease. We have used in vivo functional studies such as RNAi and system, our goal is to investigate on a large scale the in vivo expression
overexpression approaches to screen Drosophila serine proteases patterns and function of conserved lymphatic modulators regulated by
and serine protease inhibitors for roles in terminal patterning. In so Sox18. In order to perform this analysis we utlize in situ hybridization
doing we have found that over-expression of Serpin 4 from follicle cells analysis, heat-shock-inducible transgenic zebrafish and transient gene
causes eggshell and possibly terminal patterning defects. We are also knock-down (loss-of-function). We expect to identify novel genes with
expressing predicted cleavage site mutants of Trk in vivo to test whether critical functions in lymphatic vascular development. These discoveries
Trk is proteolytically cleaved and whether this is Tsl-dependent. Finally, may subsequently form the basis for novel therapeutic avenues for
using a known and related MACPF structure, we have designed and lymphatic disorders.
are testing mutations in Tsl that target the putative membrane inserting
region to determine if membrane insertion is crucial for Tsl-mediated Trk
activation and embryonic patterning.

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POS-MON-25 POS-TUE-26
CHIROPTERA TRANSTHYRETIN PLATELET DERIVED GROWTH FACTOR RECEPTOR
ALPHA (PDGFRα) AND CORONARY VASCULATURE
Khwanmunee J. and Prapunpoj P. FORMATION
Department of Biochemistry, Faculty of Science, Prince of Songkla
University,Hat Yai, Songkhla 90112, Thailand. Biben C.1, Ooi F.1, Hartley L.1 and Prall O.W.J.1, 2
1
Walter and Eliza Hall Institute, 1G Royal Parade, Parkville 3052 VIC,
Thyroid hormone (TH) is known playing an important role in development, Australia. 2Royal Melbourne Hospital, Grattan Street, Parkville 3052
reproduction and basal metabolism of vertebrates. In the plasma of VIC, Australia.
larger mammals, thyroxine-binding globulin, transthyretin (TTR) and
albumin take responses to transport/distribute the hormones throughout Coronary artery disease is one of the leading causes of death in the
body of the animals. Among of these plasma proteins, TTR is becoming western world. Formation of coronary arteries and veins is still poorly
the most of interest because of its multifunction and association to a understood. Classical embryology experiments have suggested that
genetic metabolic disorder called amyloidosis. In Chiroptera, TH is they derive from an extra cardiac tissue, the proepicardium (PE), which
crucial not only on basal metabolism but also the reproductive cycle. is located posterior to the heart. PE cells cover the myocardium, forming
However, the TH transporter/distributor and gene expression have not the epicardium, from which cells invade the subepicardial space and
been studied in this eutherian. In this study, the nucleotide sequence of then the myocardium. Whether they contribute most or only some of the
Chiroptera liver TTR cDNA was first reported. The deduced amino acid lineages of the coronary vasculature is unclear, as well as whether they
sequence showed highly conservation in comparison to those of TTRs give rise to additional cardiac lineages, including muscle. We have found
from human and other eutherians, except two amino acid residues in that the gene encoding the receptor tyrosine kinase Platelet Derived
the N-terminal region was absent. Parsimony analysis revealed the Growth Factor Receptor Alpha (PDGFRα) is expressed throughout the
amino acid sequence of the flying mammal TTR is most similar to that of mouse PE. Pdgfrα is required at multiple steps of embryonic development
pig TTR. Sections of genomic DNA in the regions coding for the splice but its role in the heart is uncharacterized. Pdgfrα remains expressed
sites between exons 1 and 2 were synthesized and sequenced, and in epicardial and subepicardial cells but becomes downregulated as
the location and mRNA splicing in Chiroptera TTR gene was identified. those cells migrate out of the subepicardium. To understand the role
Moreover, the recombinant Chiroptera TTR was produced by using the of this gene in formation and maintenance of the coronary vasculature,
heterologous expression system of Pichia pastoris. The recombinant we have engineered mouse models with either low Pdgfrα expression
protein had general physicochemical properties similar to those of or specifically lacking this gene in various tissues, such as the PE,
TTRs from human and other eutherians. Its functions including binding endothelium or cardiac muscle. Our preliminary observations suggest
to retinol binding protein (RBP) and proteolytic cleavage were also that Pdgfrα is specifically required in subepicardial cells and involved in
demonstrated. coronary smooth muscle development.

POS-MON-27 POS-TUE-28
SOX3 OVEREXPRESSION LEADS TO DEFECTIVE THE ROLE OF FYN KINASE IN THE DEVELOPMENT OF
SUBCOMMISSURAL ORGAN DEVELOPMENT WITH PRE-IMPLANTATION EMBRYOS
CONGENITAL HYDROCEPHALUS
Levi M.1, Maro B.1, 2 and Shalgi R.1
Lee K.P.Y., Cheah P.S., Piltz S.G., Rogers N.A. and Thomas P.Q.
1
Department of Cell and Developmental Biology, Sackler Faculty of
The University of Adelaide, Adelaide, SA, Australia. Medicine, Tel Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel.
2
CNRS, Paris, France.
Abnormalities in central nervous system (CNS) development that impede
the rostral to caudal flow of cerebrospinal fluid (CSF) cause congenital Fertilization in mammals triggers the arrested oocytes to exit from
hydrocephalus (CH) in mammals. Blockage of the sylvian aqueduct meiotic metaphase. The extrusion of the second polar body and the
(SA), a narrow constriction connecting the third and fourth ventricles, formation of the pronuclei mark the completion of meiosis and the
is a common cause of CH. Genetic studies in mice have indicated that beginning of pre-implantation embryo development. Several lines of
the subcommissural organ (SCO), a brain gland located at the dorsal evidence imply the involvement of Fyn, a Src family kinase, in somatic
midline immediately anterior of SA, is critical for clear passage of the cell cycle control. In the current study we demonstrated, using live cell
SA. However, the development of the SCO is poorly understood. To confocal imaging and microinjection of Fyn cRNA, the co-localization
investigate the function of the CNS transcription factor Sry-related HMG of Fyn with tubulin at the spindle poles of mouse oocytes. During the
box transcription factor 3 (Sox3), we have generated two independent exit from meiotic metaphase the amount of phosphorylated Fyn was
BAC transgenic mouse lines to overexpress Sox3 in developing CNS reduced, Fyn disappeared from the spindle poles and concentrated
using native control elements. These transgenic lines develop overt around the spindle midzone and co-localized with filamentous actin
CH (dome-shaped cranium) with 20% and 99% penetrance in single at the cleavage furrow and contractile ring area during meiosis and
and double transgenic, respectively. We found endogenous Sox3 mitosis. Inhibition of Fyn by exposure to SFKs inhibitor, SU6656, or
expression in the SCO from inception (approximately 11.5 dpc) through by microinjection of DN-Fyn cRNA inhibited the exit from metaphase,
to adulthood and its pattern is recapitulated in Sox3 transgenic mice nuclear envelope breakdown and preimplantation cell mitosis. Moreover,
spatially and temporally. Double transgenic embryos invariably exhibit although microinjection of cortical DN-Fyn did not affect the initiation of
profound SCO dysmorphology at 14.5 dpc and agenesis at 18.5 dpc. the ingression of the cleavage furrow, it prolonged the average duration
BrdU analysis indicated increased SCO primordium proliferation at 12.5 of ingression, decreased the rates of polar body extrusion and the first
dpc in double transgenic embryos, suggesting Sox3 overexpression cleavage and enlarged the average volume of the polar body and the
may inhibit progenitor differentiation. Comparison of expression profiles length of the meiotic spindle. We propose that Fyn regulate several
of wild type and double transgenic SCO at 12.5 dpc through microarray key pathways leading to the exit from metaphase, nuclear envelope
analysis showed increased dosage of Sox3 alters expression of genes breakdown, and in the ingression of the cleavage furrow during meiosis
implicated in SCO and/or dorsal midline development, including and mitosis.
members of the Wnt signalling pathway. We are extending these studies
using qRT-PCR and in situ hybridisation analyses. Ultimately, our work
will provide a better understanding of the genetic and molecular basis
for CH pathogenesis.

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POS-MON-29 POS-TUE-30
IDENTIFYING NOVEL GENES IN SEX DEVELOPMENT THE CREB1 TRANSCRIPTION FACTOR MODULATES
SCD1 EXPRESSION DURING MOUSE LUNG
Alankarage D.1, 2 , Ludbrook L.1, Svingen T.3, Bagheri-Fam S.1, DEVELOPMENT
Koopman P.3 and Harley V.1
1
Prince Henry’s Institute, Clayton, VIC, Australia. 2Biochemistry Antony N., Bird A.D., Tan K.H. and Cole T.J.
and Molecular Biology, Monash University, Clayton, VIC, Australia. Department of Biochemistry & Molecular Biology, Monash University,
3
Institute for Molecular Bioscience, University of Queensland, Clayton, Victoria, 3800, Australia.
Brisbane, QLD, Australia.
Transcriptional factors play a crucial role during lung development
Disorders of sexual development (DSDs) are surprisingly common (e.g. in regulating gene expression programs which influence branching
XX males, XY females), and often result in infertility, genital abnormalities, morphogenesis and cellular proliferation and differentiation. Cyclic AMP
gender mis-assignment and long-term psychological trauma. The main Response Element-Binding Protein1 (Creb1) is a transcription factor that
pathway of sex development in males centres on the regulation of SOX9 mediates cyclic adenosine 3’,5’-monophosphate (cAMP) signaling in
in the developing XY gonad, which is regulated by SF1, SRY and SOX9, target tissues. Creb1-null mice die at birth due to respiratory failure and
as well as PGD and FGF9 signalling pathways. However, most DSD are phenotypically smaller than their wild type litter mates. The aberrant
conditions remain unexplained genetically suggesting the presence of lung morphology in Creb1-null mice is established by E16.5 and is
additional genes involved in gonadal development that have not yet been clearly detected at E17.5 as unexpanded distal and proximal airways. We
identified. A microarray expression study where SF1, SOX9, SRY and investigated whole genome expression profiles on embryonic day E17.5
DAX1 were over expressed separately in NT2 cells, a human pluripotent in Creb1-null fetal mice lungs to identify novel gene targets under the
testicular EC line, was recently performed in the lab. Microarray analysis control of Creb1 and to elucidate the molecular mechanisms underlying
of FACS-sorted, transiently transfected SOX9 cells identified candidate the role of cAMP signaling during airway development. Microarray
genes responsive to SOX9 overexpression. A bioinformatic filtering analysis has identified several gene targets that are down-regulated
process reduced the list from 2626 genes to 10. Promising candidate compared to wild type mice lungs; these included, genes involved in
genes such as CLCN7, COL18A1 and ETV5 are being evaluated further lipid metabolism such as, enzyme fatty acid synthase, paraoxonase-1
in terms of their gene expression in XY Sox9 knockout embryonic (Pon-1) and a lipogenic enzyme, stearoyl-CoA desaturase 1 (Scd-1). In
mouse gonads, and by CHIP-seq and promoter analysis. This study Creb1-null mice lungs the gene expression was significantly reduced,
can potentially lead to the discovery of novel causative DSD genes and Pon-1 (1.6 fold, p=0.0005, n=4) and Scd-1 (8.6 fold, p=0.0001, n=4); this
regulatory mechanisms during sex development. was also confirmed by quantitative RT-PCR, Pon1 (p=0.0005, n=4) and
Scd-1 (p=0.0317, n=4). The gene expression profiles of Pon-1 and Scd-
1 are developmentally regulated and the expression were increased
dramatically from E16.5 to E18.5, following which Pon-1 levels remained
elevated, while Scd-1 levels decreased after birth. The expression of
Scd-1 protein was restricted to type II pulmonary epithelial cells and the
levels were significantly lower in Creb1-null when compare to wild type
mice lungs. Expression levels of Pon-1 and Scd-1 are also affected in
other transcription factor knockout mice models suggesting overlapping
roles in transcriptional regulatory pathways during lung development.

POS-MON-31 POS-TUE-32
RAPAMYCIN AND ISCHEMIA REPERFUSION INDUCE EXOSOMAL MICRORNA PROFILING IN
HEME OXYGENASE-1 AND PEROXIREDOXIN-1 IN NEURODEGENERATIVE DISEASE
LIVER Bellingham S.A.1, 2, 3, Kuehlich J.1, 2, Coleman B.M.1, 2, Sharples R.1, 2
and Hill A.F.1, 2, 3
Kist A.1, Wakkie J.1, Nikolic A.1, Zeile S.1, Versteeg R.1, Ten Berge J.1, 1
Department of Biochemistry and Molecular Biology, The University
Wilson C.H.1, Nieuwenhuijs V.B.2, Padbury R.T.A.3 and Barritt G.J.1 of Melbourne, Victoria 3010, Australia. 2Bio21 Molecular Science and
1
Department of Medical Biochemistry, School of Medicine, Flinders Biotechnology Institute, The University of Melbourne, Victoria 3010,
University, Adelaide, South Australia. 2Department of Surgery, Australia. 3Mental Health Research Institute of Victoria, Melbourne,
University Medical Centre, Groningen, The Netherlands. 3The HPB Victoria, Australia.
and Liver Transplant Unit, Flinders Medical Centre and School of
Medicine, Flinders University, Adelaide, South Australia. Exosomes are small membrane vesicles of endosomal origin that are
released from a variety of cell types into the microenvironment. The
Liver surgery is associated with ischemia and reperfusion (IR) injury secretion of exosomes was initially thought to be a mechanism for
resulting in loss of liver function and hepatocyte death. The onset of removing unnecessary proteins. However, our research implicates
hepatocyte damage is chiefly due to the deleterious actions of reactive exosomes in the potential pathogenesis of neurodegenerative disorders
oxygen species (ROS). Experimental induction of two anti-oxidant such as prion and Alzheimer’s diseases. We have demonstrated that
enzymes, heme oxygenase-1 (HO-1) and peroxiredoxin-1 (Prx-1), neuronal cells infected with prions release normal and infectious forms
reduces ROS-induced damage to the liver. Rapamycin, employed of the prion protein in associated in exosomes. We have also shown
clinically in liver transplantation as an immunosuppressant has also that exosomes contain the Alzheimer’s disease precursor protein (APP),
been shown to induce the synthesis of HO-1 in non-liver cell types. The the amyloid-β peptide (Aβ), and secretase components required for Aβ
aim of this study was to test whether rapamycin will induce HO-1 and generation from APP. These studies identified previously unknown
Prx-1 in hepatocytes. Incubation of rat hepatocytes with rapamycin or pathways for the conversion and propagation of prion infection and
cobalt protoporphyrin (CoPP) increased HO-1 (assessed by quantitative the processing of APP that may contribute to AD pathogenesis. More
PCR). Maximal effects were observed at 36 h, and half-maximal at recently, exosomes have been shown to contain both mRNA and miRNA,
10-100 ng/ml. Rapamycin and CoPP increased HO-1 expression in termed exosomal RNA (esRNA) that can be transferred between cells
livers removed immediately after laparotomy and in livers subject to in a novel mechanism of cell-cell communication of genetic signals. The
laparotomy and a sham operation. Livers subject to a sham operation transferred esRNA is also functional, as the mRNA can be translated
exhibited higher relative expression of HO-1 mRNA than livers rapidly into new proteins in recipient cells, while the miRNA, a class of small
removed. 2D-DIGE revealed two abundant cytosolic proteins which RNA ~ 22nt long, play important roles in gene regulatory networks by
showed a 3-fold increase in IR (P≤0.05). These were identified as binding to and repressing the activity of specific target mRNAs. We
reduced and hyperoxidised Prx-1. A 1.5-fold increase in expression of therefore hypothesised that esRNA can contribute to the pathogenesis
Prx-1 mRNA was observed in IR. It is concluded that rapamycin and IR of prion and Alzheimer’s diseases. Utilising our cell culture models, we
induce expression of HO-1 and Prx-1 in hepatocytes. Clinical application aim to identify disease specific signatures from isolated exosomes by
of rapamycin pre-treatment may offer protection against IR damage. miRNA profiling with “next-generation deep sequencing”, miRNA Array’s
and quantitative PCR. This research has significant diagnostic potential
for prion, Alzheimer’s and other human diseases since circulating
exosomes can be isolated from a variety of biological fluids including
blood, urine, saliva and CSF.

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POS-MON-33 POS-TUE-34
CYCLIN-DEPENDENT KINASE-LIKE 3 REGULATES TAXOL AND BORTEZOMIB INDUCE APOPTOSIS
ACTIN REMODELLING AND CELL MIGRATION SYNERGISTICALLY IN CHRONIC MYELOGENOUS
LEUKEMIA CELLS
Brown M.K.1, Forrest A.2, Kerr M.3, Wani S.1, Cloonan N.1, Gabrielli B.4
and Grimmond S.1 Bucur O.1, 2 , Stancu A.L.1, Petrescu S.M.2 and Khosravi-Far R.1
1
Queensland Centre for Medical Genomics, Institute for Molecular 1
Beth Israel Deaconess Medical Center, Harvard Medical School,
Bioscience, University of Queensland, Australia. 2Omics Science Boston, MA, USA. 2Institute of Biochemistry of the Romanian
Centre, RIKEN, Yokohama Institute, Japan. 3Institute for Molecular Academy, Bucharest, Romania.
Bioscience, University of Queensland, Australia. 4Cell Cycle
Laboratory, University of Queensland Diamantina Institute, Princess Bcr-Abl fusion protein plays a critical role in the pathogenesis and
Alexandra Hospital, Australia. progression of Chronic Myeloid Leukemia (CML) and in some Acute
Lymphocytic Leukemia cases. We and others have previously
Organisation of the actin cytoskeleton is central to cell migration, established Bortezomib (FDA approved for the treatment of Multiple
and is largely regulated by members of the mammalian Rho GTPase Myeloma) as a potential important treatment in Bcr-Abl positive
family. Members of this family have recently been implicated in dendrite leukemias. Taxol (Paclitaxel), a mitotic inhibitor drug (FDA approved for
formation and branching, with alterations in Rho GTPase-signalling the treatment of a number of cancers) is also now in clinical trials for
reportedly contributing to mental retardation (MR) disorders. Cyclin- the treatment of CML. However, to our knowledge, there are no studies
dependent kinase-like 3 (Cdkl3) is a poorly characterised member of regarding the combined treatment of Bortezomib and Paclitaxel in Bcr-
the serine/threonine family, with homology to the cell cycle regulator, Abl positive CML. RESULTS: Combined treatment of Bortezomib and
Cdk1. Recently, a role for Cdkl3 in neuronal morphogenesis has been Paclitaxel synergistically induces cell death in human Bcr-Abl positive
demonstrated, whereby Cdkl3 knock-down has been shown to decrease K562 cell line, by activating the initiator caspase 9 and effector caspase
dendrite formation and branching. We report the characterisation of 3, which leads to the cleavage of a number of substrates, such as Poly
two alternative transcripts derived from the human Cdkl3 locus. While ADP Ribose Polymerase (PARP), and results in apoptosis. Additionally,
the canonical Cdkl3 product contains two putative ERK binding motifs the exposure of the Bcr-Abl positive leukemia cells to the Bortezomib/
and a Nuclear Localisation Sequence (NLS), the second isoform has a Paclitaxel regimen results in an increase in the activation of the
single putative ERK binding motif and no NLS, suggesting an alternative stress-related MAP kinases (such as p38 MAPK and JNK) versus the
function for this variant. We report a significant decrease in cell migration cytoprotective kinases (ERK1, ERK2), suggesting a possible implication
with Cdkl3 knock-down, as well as hyperphosphorylation of ERK in of JNK and p38 MAPK in mediating the effect of the combined treatment.
HeLa cells. Additionally, Cdkl3 knock-down leads to actin remodelling Moreover, we also show that the combined treatment is effective in
and altered cell morphology. We propose a role for Cdkl3 in the MAPK inducing apoptosis in the murine Baf3 Bcr-Abl and Imatinib-resistant
signalling pathway in a manner that regulates actin cytoskeleton Baf3 Bcr-Abl T315I cell lines. CONCLUSION: Taken together, these
organisation, which affects the ability of cells to migrate correctly. We findings underline that the combined treatment with Bortezomib and
suggest that alternative splice variants from the Cdkl3 locus encode Paclitaxel represents a potentially promising strategy for the treatment
proteins that differ in their biological activities, and that these alternative of Chronic Myelogenous Leukemia in general, and of Imatinib-resistant
components should be considered when investigating Cdkl3 functions. CML in particular.

POS-MON-35 POS-TUE-36
INVESTIGATING THE RNA BINDING PROPERTIES OF IDENTIFICATION OF NFIL3 AS A DIRECT
KRÜPPEL-LIKE FACTORS GLUCOCORTICOID-REGULATED GENE TARGET IN
T-LYMPHOCYTES
Burdach J.G.1, 2 , Mackay J.1 and Crossley M.2
1
School of Molecular Bioscience, University of Sydney, Australia. Carey K.T.1, Tan K.1, Ng J.1, Liddicoat D.R.1, 2, Godfrey D.I.2 and Cole T.J.1
2
School of Biotechnology and Biomolecular Sciences, University of 1
Department of Biochemistry & Molecular Biology, Monash University,
NSW, Australia. Clayton, Victoria, 3800. 2Department of Immunology and Microbiology,
University of Melbourne, Parkville, Victoria, 3010.
The Krüppel-like factors (KLFs) are a family of mammalian transcription
factors involved in the regulation of a diverse range of biological Glucocorticoids (GCs) are homeostatic steroid hormones with
processes including proliferation, apoptosis, differentiation and essential roles in the regulation of development, integrated metabolism,
development (Pearson et al., 2008). These proteins bind to a common immune and neurological responses. In the immune system the strong
CACCC box element within GC-rich regions of DNA to activate or repress lymphocytolytic actions of GCs are central in treatment of lymphocytic
transcription (Suske et al., 2005). DNA interaction is mediated by three leukemias and lymphomas such childhood acute lymphoblastic leukemia
C-terminal Cys2His2 zinc finger domains which are highly conserved (ALL). GCs act via the glucocorticoid receptor (GR) which is expressed
across the family. The N-termini are highly variable and can contain from multiple untranslated exon 1s to yield at least 11 alternatively spliced
either transcriptional activation or repression domains. Cys2His2 zinc transcripts in humans and at least five in mice (1A-1H). GR transcripts
finger domains are known to have a variety of functions including DNA, initiating from the GR1A promoter have previously only been localised to
RNA and protein binding (Iuchi, 2001). As transcription factors, the DNA T-lymphocytes and brain cortex. In T-lymphocytes the GR1A promoter
binding characteristics of the KLFs are well established. Preliminary in is implicated in increasing sensitivity to Glucocorticoid Induced Cell
vitro data from our laboratory suggests that these proteins may also Death (GICD). CD4+CD8+ Double Positive (DP) cells as well as NK cells
be capable of binding RNA, though the specificity and affinity of these in particular have been shown to be hypersensitive to GICD. To explore
interactions is still unclear. We are using RNA immunoprecipitation the molecular pathway driving GCID in T-cells we have performed whole
coupled with next generation sequencing technology (RIP-seq) genome microarray analysis in mouse GR null T-cells. Interesting direct
to determine whether these proteins are capable of specific RNA GR targets included P21 and Bim, in addition to many not previously
interactions in a cellular context. This powerful technique may shed well characterised, such as Nfil3. Regulation of these targets by GCs
light on the biological role of such an interaction, leading to a better has been validated using qRT-PCR in WT T-cells. Nfil3 in particular has
understanding of the role of KLFs in gene regulation. Iuchi, S. (2001). been studied further. Previous studies suggest that the development and
Three classes of C2H2 zinc finger proteins. Cell Mol Life Sci 58, 625- functional maturation of NK cells requires Nfil3 expression, in addition
635. Pearson, R., Fleetwood, J., Eaton, S., Crossley, M., and Bao, it has been demonstrated that GC-mediated upregulation of Nfil3 is
S. (2008). Kruppel-like transcription factors: a functional family. Int J dependent on intracellular calcium levels, and correlates with GCID
Biochem Cell Biol 40, 1996-2001. Suske, G., Bruford, E., and Philipsen, of GC-sensitive leukemic cells. In silico promoter analysis revealed a
S. (2005). Mammalian SP/KLF transcription factors: bring in the family. putative Glucocorticoid Response Element in the Nfil3 promoter region
Genomics 85, 551-556. which was confirmed by ChIP. Immunohistochemical staining of Nfil3 in
whole thymus has localised Nfil3 protein primarily to the medullary region,
which contains developing NK cells. It will be of interest to investigate
the involvement of GICD in negative selection of sensitive DP cells and
correlate this to expression of Nfil3.

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POS-MON-37 POS-TUE-38
A NOVEL FUNCTION OF TRISTETRAPROLIN : INTESTINAL STEM CELL GENE LGR5 IS REGULATED
INHIBITING THE ACTIVITY OF POLY(A) POLYMERASE BY SYNERGISTIC C-MYB AND β-CATENIN
(PAP) BY DIRECTLY INTERACTING WITH PABPN1 AND COOPERATION
PAP
Cheasley D.A.1, 2 , Malaterre J.1, Vincan E.3, Lightowler S.1, Pereira L.1
Wang S.-C.1, Su Y.-L.2, Chiang P.-Y.2, Lin N.-Y.1, Shen Y.-F.2 and and Ramsay R.G.1
Chang C.-J.1, 2
1
Differentiation and Transcription Laboratory, Peter MacCallum
1
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. Cancer Institute, East Melbourne and the Pathology Department,
2
Graduate Institute of Biochemical Sciences, National Taiwan The University of Melbourne. 2The Department of Genetics, La Trobe
University, Taipei, Taiwan. University, Bundoora. 3The Department of Anatomy and Cell Biology,
The University of Melbourne, Parkville.
Tristetraprolin (TTP) is one of AU-rich element (ARE)-binding proteins,
which facilitates mRNA destabilization. To realize the detailed The regulation of tissue development and the establishment of
mechanism of TTP upon mRNA degradation control, it is necessary homeostasis is achieved through the orchestration of transcription
to find out its direct interaction proteins. In this report, we performed factor action and co-ordination of stem and progenitor cell populations.
phage-display biopanning to search the TTP-interacting proteins. One The transcription factor c-Myb has emerged as a key regulator of stem/
of them, Pabpn1 (poly (A) binding protein nuclear 1), was identified and progenitor cells within the gastrointestinal tract crypt. These structures
further characterized. Moreover, their interacting domains were verified line the epithelium of the colon and small intestine. This conclusion
and determined with their deletion constructs by pull-down assays. is based upon our exploitation of c-myb knock-out and hypomorphic
Pabpn1 plays a major role in the polyadenylation of mRNAs, which binds mutant mouse models. Recent studies have found that the Wnt target
to poly(A) RNA and increases the affinity of the poly(A) polymerase gene, Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor
(PAP) to promote the formation of the polyadenylate tail of the 3’ end 5), along with being an important intestinal and colon crypt stem cell
of mRNA. Interestingly, we found that TTP could bind to both Pabpn1 marker, is also expressed in colon cancer cells. Based on co-incidental
and PAP and inhibit polyadenylation of ARE-containing RNA in vitro. expression of lgr5 and c-myb in the GI, we investigated whether lgr5 is
The poly(A) tail is important for the mRNA export, mRNA stabilization a c-Myb target gene. Using both in vitro and in vivo studies, we show
and translation efficiency. Consequently, except the mRNA-degrading that the proto-oncogene c-Myb in combination with β-catenin, is bound
function, inhibition of polyadenylation activity of PAP is a novel function to (by chromatin immunoprecipitation studies), and is a more potent
of TTP to regulate ARE-containing mRNA expression. regulator of, the lgr5 promoter (by reporter experiments) in the presence
of activated β-catenin. These observations parallel our previous studies
of the regulation of the c-myc gene by both of these transcription
factors (Ciznadija et al 2009) suggesting this is a more general mode
of transcriptional co-operation. How c-Myb and β-catenin co-operate
at the genetic and physical level will be presented. Collectively our data
indicate that the Wnt pathway through β-catenin converge with c-Myb in
regulating lgr5 expression in the GI stem/progenitor cells.

POS-MON-39 POS-TUE-40
ANALYSIS OF THE RELATIONSHIP BETWEEN REGULATION OF SPLICEOSOMAL GENES IN
BIOENERGETIC SIGNATURES AND DIFFERENTIATION SACCHAROMYCES CEREVISIAE
POTENTIALS OF HUMAN MESENCHYMAL STEM
CELLS FROM DIFFERENT ORIGINS Chen S.-C.E., Kornfeld G.D. and Dawes I.W.
School of Biotechnology and Biomolecular Sciences, University of
Chen C.T.1, Lan Y.W. 1, Hsu S.H.1, Hwang S.M.2 and Wei Y.H.1, 3 New South Wales, NSW Australia.
1
Department of Biochemistry and Molecular Biology, National Yang Ming
University, Taipei, Taiwan. 2Bioresource Collection and Research Center, Sm-like (Lsm) proteins function in a variety of RNA-processing
Food Industry Research and Development Institute, Hsinchu, Taiwan. events, including splicing, post-transcriptional modification and RNA
3
Department of Medicine, Mackay Medical College, Taipei, Taiwan. degradation. In yeast, the proteins Lsm2-Lsm8 form a heteroheptameric
ring complex and interact with the spliceosomal U6 snRNA to facilitate U4/
Our previous studies indicate that the metabolic shift from anaerobic U6 snRNP formation during the mRNA splicing process; whilst another
glycolysis to mitochondrial respiration plays critical roles during complex comprised of Lsm1-Lsm7 is involved in mRNA degradation
osteogenic differentiation of human mesenchymal stem cells (hMSCs). via decapping in the cytoplasm. An increasing number of studies have
Hypoxic signals suppressed the activation of mitochondria and attenuated suggested that there are important functions of introns acting in parallel
differentiation of hMSCs. Recent studies also showed the necessity of with the protein-based regulatory systems, possibly related to the
mitochondrial function for stem cells to differentiate into cardiomyocytes complexity of organism. Studies from our laboratory showed that the
and other types of somatic cells. Therefore, we have hypothesized that intron of the LSM7 gene along with the coding sequence is required for
the metabolic signatures, namely, the relative contribution of aerobic and the regulation of LSM1-LSM8 expression on different carbon sources.
anaerobic metabolism to energy production, of stem cells may affect The aim of our study is to identify the mechanism whereby the LSM7
their differentiation potentials. We isolated hMSCs from four different intron regulates the expression of LSM genes in Saccharomyces
origins including cord blood, bone marrow, amniotic membrane and cerevisiae. The approach involved targeted mutagenesis of the LSM7
amniotic fluids. Their metabolic features were analyzed by an automatic intron sequence, in particularly, the splicing elements in the intron, and
bioenergetic analyzer, Seahorse XF24 Extracellular Flux Analyzer. We qRT-PCR analysis for the expression profiles of the LSM genes in these
found that hMSCs form different origins displayed distinct metabolic mutants. In addition, microarray analysis has also conducted to examine
signatures as revealed by their oxygen consumption rate (OCR) and the genome-wide expression profiles in response to deletion of LSM7
extracellular acidification rate (ECAR) despite their expression of similar intron.
panels of surface markers. These hMSCs also showed distinct preference
in differentiating into multiple lineages of progenies such as osteoblasts
and adipocytes. Manipulation of their aerobic and anaerobic metabolism
by inhibitors against either mitochondrial or glycolytic activities affected
their differentiation abilities. Taken together, the distinct metabolic
signatures of hMSCs from different origins may serve as an additional
biomarker for the evaluation of stem cell properties in terms of their
differentiation potentials. Characterization of metabolic signatures could
be of great value in the selection of stem cells with superior quality to
facilitate future clinical application of stem cells in tissue regeneration.

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POS-MON-41 POS-TUE-42
DISTINCT ROLES OF TTP, HNRNP K AND HUR IN COX- PHOSPHORYLATION ADJACENT TO THE BZIP
2 MRNA STABILITY INDUCED BY LPS DOMAIN OF FOS RELATED ANTIGEN-1 REGULATES
DNA BINDING
Chiang P.Y.1, Shen Y.F.1 and Chang C.J.1, 2
1
Graduate Institute of Biochemical Sciences, National Taiwan Diesch J.1, Low D.1, Morrice N.2, Tulchinsky E.3, Hannan R.D.4 and
University, Taipei, Taiwan. 2Institute of Biological Chemistry, Acdemic Dhillon A.S.1
Sinica, Taipei, Taiwan. 1
Department of Biochemistry and Molecular Biology, University
of Melbourne, Vic, Australia. 2MRC Protein Phosphorylation Unit,
Cyclooxygenases (COXs) are key regulatory enzymes catalyzing University of Dundee, UK. 3Department of Cancer Studies and
the rate-limiting step of prostaglandin formation in various biological Molecular Medicine, University of Leicester, UK. 4MacCallum Cancer
processes, such as inflammation, and overexpression of the inducible Centre, Melbourne, Vic, Australia.
isoform COX-2 is associated with carcinogenesis. In macrophage Raw
264.7 cells, lipopolysaccharide (LPS) induces COX-2 expression at both The Activator Protein-1 (AP-1) transcription factor is a sequence-
transcription and posttranscriptional level. To understand the mechanism specific DNA binding protein complex that orchestrates gene
of posttranscriptional regulation, the RNA binding proteins (RNA-BPs) expression programs directing cell fate decisions (e.g. proliferation,
bound to the 3’ untranslated region (3’UTR) of COX-2 mRNA were survival, differentiation, migration). It has a dimeric core, consisting
studied firstly. By using RNA pull-down and RNA immunoprecipitation mainly of members of the Fos, Jun and ATF protein families, and
assay, tristetraprolin (TTP), heterogeneous nuclear ribonuclear protein operates downstream of many cancer-associated signal transduction
K (hnRNP K) and HuR were proven to bind to the distinct regions of pathways. The Fos related antigen-1 (Fra-1) is a Fos family protein
COX-2 mRNA 3’UTR respectively. Further reporter assay indicated that frequently over-expressed in cancers of epithelial origin, and is linked
LPS-induced TTP destabilizes COX-2 mRNA by binding to the AU-rich to increased tumour cell migration and invasion. There is thus much
element (AREs) located within the first sixty nucleotides of 3’UTR in interest in understanding how the functions of Fra-1 are regulated.
COX-2 mRNA. On the other hand, HuR binds to both ARE and distal Using mass spectrometry, we have identified a novel phosphorylation
region of COX-2 mRNA 3’UTR and increases mRNA stability. Moreover site in Fra-1 (Ser101) that lies adjacent to the region of the protein
the RNA stability and translation modulated by hnRNP K are mediated involved in DNA binding. We substituted Ser101 with alanine (S101A)
by binding to the distal region of COX-2 mRNA 3’UTR. Although these or aspartic acid (S101D) residues, and examined the effects of these
RNA-BPs function in distinct ways, the combination effect mediated by mutations on the subcellular localisation of Fra-1, its capacity to form
TTP, hnRNP K and HuR causes the tight control of COX-2 expression dimers with c-Jun in cells, and its ability to bind to oligonucleotides that
during inflammation reaction. contain an AP-1 consensus sequence (TRE). While the localization and
dimerisation of Fra-1 with c-Jun were not affected by the substitutions,
we noted a significant increase in the binding of Fra-1S101A, and decrease
in the binding of Fra-1S101D, to the TRE. As these Fra-1 proteins were
part of dimer containing endogenous c-Jun, our results suggest that
phosphorylation of Ser101 can disrupt the recognition or binding of Fra-
1/c-Jun complexes to consensus sites in AP-1 target genes.

POS-MON-43 POS-TUE-44
BIOPHYSICAL AND STRUCTURAL EXAMINATION OF PROTEOLYSIS OF AN ANTI-SIGMA FACTOR
THE LER TRANSCRIPTION FACTOR CONTROLS SIDEROPHORE SYNTHESIS AND UPTAKE
IN PSEUDOMONAS AERUGINOSA
Stone R.D.1, 2, Dogovski C.1, 2 , Bailey M.F.1, 2, Ji Y.3, Robins-Browne
R.M.3 and Perugini M.A.1, 2 Draper R.C. and Lamont I.L.
1
Department of Biochemistry and Molecular Biology, The University of University of Otago, Dunedin, New Zealand.
Melbourne, Parkville VIC 3010, Australia. 2Bio21 Molecular Science
and Biotechnology Institute, The University of Melbourne, Parkville, Pseudomonas aeruginosa is an important human pathogen that
VIC 3010, Australia. 3Department of Microbiology and Immunology, requires iron for growth and pathogenesis. To acquire iron from the
The University of Melbourne, Parkville, VIC 3010, Australia. environment or host tissues, this bacterium secretes the iron-binding
molecule pyoverdine. Pyoverdine is also a signalling molecule, inducing
Pathogenic strains of Escherichia coli cause acute and persistent the expression of genes for pyoverdine synthesis and uptake. In
diarrhoea in humans. Several pathotypes express specific virulence the absence of pyoverdine, sigma factor proteins required for gene
factors that mediate adhesion to the intestinal epithelium and expression are inhibited by an anti-sigma factor, FpvR, which spans
production of distinctive pathological changes in the intestine mucosa the inner membrane. When pyoverdine binds to a cell surface receptor,
(A/E phenotype). Virulence genes in pathogenic E. coli are typically this inhibition is relieved, allowing sigma activity and the expression of
organised in operons that are tightly controlled by a network of regulatory target genes. This project investigated the role of FpvR in this signalling
proteins. The genes involved in the A/E phenotype are encoded by a 36 pathway, and sought to determine the molecular mechanism underlying
kb chromosomal pathogenicity island, named the locus of enterocyte this system. We have shown that the FpvR protein is only detectable in
effacement (LEE). LEE carries more than 40 genes which form five strains where sigma factor activity is inhibited. When pyoverdine was
transcriptional units, termed LEE1 to LEE5. A number of global and added we observed a rapid disappearance of FpvR, suggesting that
specific regulatory proteins are known to play an essential role in proteolysis regulates FpvR activity. Candidate proteases were screened
gene expression and pathogenesis. The transcription factor Ler (LEE for involvement in the degradation of FpvR. The intra-membrane
encoded regulator) a homologue of H-NS, is essential for anti-silencing protease RseP was implicated in this process by the accumulation of
of LEE operons LEE2-LEE5. Ler is considered to be a key regulatory intermediate FpvR fragments in strains lacking RseP. These strains also
element and novel antibiotic target. Although much work has been done exhibited reduced sigma activity as measured by qRT-PCR. Together
to establish the role of Ler in E. coli pathogenicity, little is known about these data suggest a model whereby the binding of pyoverdine at the
the solution properties of the protein and how it prevents silencing of the cell surface causes the degradation of FpvR, resulting in changes in
LEE pathogenicity island. The purpose of this study is to characterize the gene expression.
solution properties and structure of Ler via analytical ultracentrifugation,
circular-dichroism spectroscopy and X-ray crystallography.

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POS-MON-45 POS-TUE-46
MECHANISM OF SYNERGISTIC INDUCTION OF GLUT1 FUNCTIONAL ANALYSIS OF THE
TRANSCRIPTION BY ENDOTHELIN-1 AND CYCLIC AMP PROTEIN L-ISOASPARTYL/D-ASPARTYL
IN 3T3-L1 ADIPOCYTES O-METHYLTRANSFERASE (PIMT) GENE PROMOTER
Fong J.C. and Kao Y.S. Furuchi T.1, Harada S.1, Shimizu Y.1, Kosugi S.2, Katane M.1, Sekine M.1
Institute of Biochemistry and Molecular Biology, School of Life and Homma H.1
Sciences, National Yang-Ming University, Taipei, Taiwan, ROC. 1
Sch. of Pharm. Sci., Kitasato Univ., Tokyo, Japan. 2Sch. of Med.,
Kanazawa Univ., Kanazawa, Japan.
We have shown previously that chronic exposure to both endothelin-1
(ET-1) and cAMP results in a synergistic increase in Glut1 transcription L-Aspartyl (L-Asp) and L-asparaginyl residues in proteins spontaneously
in 3T3-L1 adipocytes. In the present study, we have further examined isomerize or racemize to D, L-isoaspartyl (D, L-isoAsp) or D-aspartyl
the molecular mechanism involved. Experimental results indicate that (D-Asp) residues under physiological conditions. These atypical Asp
ET-1 through ETAR/Gi/PKCε interacts with cAMP to enhance Glut1 residues can interfere with the protein function and lead to malfunction
transcription. Although p42/p44 MAPK is required for the stimulatory of cells. Protein L-isoaspartyl (D-aspartyl) methyltransferase (PIMT) is a
effect of ET-1 alone, it is not involved in the synergistic effect of ET-1 cytosolic protein repair enzyme that initiates the conversion of L-isoAsp
and cAMP on Glut1 transcription. Further investigation demonstrated and D-Asp to normal L-Asp residues. Mice lacking this enzyme suffer
that a ternary complex containing transcription factors Sp1, pCREB from epileptic seizures and die at early age; thus, formation of the
and AP-1 was mediating the synergistic effect of ET-1 and cAMP on abnormal residues and their subsequent correction by PIMT is widely
Glut1 transcription. While an increase in nuclear AP-1 can be achieved believed to constitute an important pathway of protein damage and
by either ET-1 or cAMP, the accumulation of nuclear pCREB is only repair. However, regulatory mechanisms of PIMT gene expression have
sustainable by stimulation with cAMP. An increase in nuclear Sp1, on remained unclear. To analyze the regulatory mechanisms controlling the
the other hand, is dependent on the activation of both PKCε and CREB. expression of human PIMT, we characterized the 5’-flanking region of
Thus it seems that chronic exposure to both ET-1 and cAMP provides the gene. About a 1 kbp fragment of the putative promoter region was
a circumstance favoring increases in nuclear Sp1, pCREB and AP-1, PCR-amplified and ligated into a luciferase-expression vector, pGL3-
and their interaction to form a ternary complex which in turn can greatly basic. Transfection in HEK293 cells indicate that the 5’-flanking region
facilitate the transcription of glut1 gene. contains regulatory elements for constitutive expression of PIMT. The
minimal region required for the basal activity of the PIMT promoter
were determined by generating a series of deletion and point mutation
constructs, and were found to be encoded by a sequence around –190
relative to the translation initiation codon. Electrophoretic mobility shift
assay suggested the presence of factors that bind to the minimal region
in a sequence-specific manner in the nuclear extract of HEK293 cells.
Further study is currently in progress to identify the binding factors using
liquid chromatography/tandem mass spectrometry (LC-MS/MS).

POS-MON-47 POS-TUE-48
ARABIDOPSIS GLYCINE-RICH RNA BINDING FUNCTIONAL REGULATION OF THE FRA-1/AP-1
PROTEIN 8 (ATGRP8) EXPRESSION IN RESPONSE TO TRANSCRIPTION FACTOR VIA INTERACTIONS WITH
PHOSPHATE STARVATION PROTEIN PHOSPHATASE 2A
Gaza H.L.1, Jost R.1, Ludwig M.2 and Finnegan P.M.1 Gilan O.1, Jastrezebski K.2, Diesch J.1, Verrills N.3, Hannan R.D.2 and
1
School of Plant Biology, UWA. 2School of Biomedical, Biomolecular Dhillon A.S.1
and Chemical Sciences,UWA. 1
Bio21 Institue Department of Biochemistry and Molecular BIology.
2
Research, Peter MacCallum Cancer Centre. 3Faculty of Health,
Plant growth and development is restricted by phosphate (Pi) deficiency. University of Newcastle.
This leads to an adaptive response by altering gene expression and
metabolism as a result of cell signaling (Schmidt et al, 2010). The The Activator Protein-1 (AP-1) transcription factor complex regulates
expression of the Arabidopsis glycine-rich RNA-binding protein 8 gene expression downstream of signal transduction pathways activated
(AtGRP8) gene is regulated by a number of external stimuli including by a variety of growth factors, cytokines, hormones and cellular stresses.
cold, circadian rhythm, ABA, drought and salinity (Carpenter et al., 1994; The products of AP-1-regulated genes are required for the execution
Kwak et al, 2005). The altered gene expression of AtGRP8 under different of fundamental cellular processes, including proliferation, survival and
stress conditions has led to the hypothesis that it may be involved in the differentiation. AP-1 complexes consist of a dimeric core, formed mainly
response of plants to phosphate starvation. Using quantitative reverse by members of the Fos, Jun and ATF protein families. Fos related
transcription polymerase chain reaction (qRT-PCR) the expression of antigen-1 (Fra-1) is a Fos family protein that is frequently over-expressed
AtGRP8 gene during phosphate deficiency was determined. Roots in cancers, and is strongly linked to cancer progression. The major post-
from A. thaliana plants grown under phosphate-deficient conditions for translational modification regulating Fra-1 is phosphorylation. Using a
seven days showed a decrease in expression compared to the plants proteomics-based approach, we have found that Fra-1 associates with
grown continuously in phosphate-sufficient media. The transcript level components of the hetero-trimeric protein phosphatase 2A (PP2A)
of GRP8 in the shoots however, remained unchanged. GRP8 may complex. This complex consists of a holoenzyme formed by the binding
function similarly to WRKY6 and WRKY75 transcription factors as a of a 36 kDa catalytic subunit (PP2Acat) to a 65 kDa scaffold subunit,
negative regulator of genes whose expression is up-regulated during whose activity and targeting to substrates is specified by one of at least
phosphate starvation. Experiments using knock-out lines to determine 25 regulatory subunits. We specifically identified the PR65a scaffold and
the role of GRP8 in the regulation of the phosphate starvation response PR55alpha regulatory subunits in Fra-1 complexes and have confirmed
are underway. that both proteins, as well as the catalytic subunit, interact with Fra-1 in
HEK293 cells and in vitro. We show that the PR55alpha regulates the
recruitment of the PP2A catalytic subunit to Fra-1, and that inhibition of
PP2A catalytic activity, or silencing of B55alpha expression perturbs
proteasome-mediated degradation of Fra-1. Our results provide new
insights into how the functions of Fra-1 are regulated in cells, and reveal
a novel mechanism regulating Fra-1 turnover.

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POS-MON-49 POS-TUE-50
HEPARANASE AS A SIGNALING MOLECULE IN A NOVEL ROLE OF DIPEPTIDYL PEPTIDASE 9 IN PI3K/
CANCER AND INFLAMMATION AKT PATHWAY REGULATION
Goodall K.J., Poon I.K.H. and Hulett M.D. Yao T.W.1, 2, Xia P.1, 2, Kim W.S.3, Yu D.M.T.1, 2, Choi K.Y.3 and Gorrell M.D.1, 2
Department of Biochemistry, LaTrobe University, Bundoora, VIC, 1
Centenary Institute. 2Sydney Medical School, University of Sydney.
Australia 3
Yonsei University, Seoul, Korea.

Heparanase is a β-D-endoglucuronidase which degrades heparan Dipeptidyl peptidase (DP) IV, DP8, DP9 and FAP (fibroblast activation
sulfate, a key component of the extracellular matrix and basement protein) are DPIV gene family members of particular interest due to
membrane. The degradation of the ECM is essential for both their peptidase and extra-enzymatic activities that have been implicated
physiological and pathological processes, including inflammation, in various diseases. We report here a novel role of DP9 in cell signal
wound healing, tumour angiogenesis and metastasis. Heparanase is transduction. We found that DP8 and DP9, but not DPIV or FAP, interact
also known to have non-enzymatic functions by regulating cell adhesion, with H-Ras, a key signalling molecule that mediates multiple signalling
cell signalling and differentiation. Although heparanase has been pathways, especially of growth factors (GFX), in both liver tissue and
proposed to facilitate leukocyte migration through degradation of the human cervical carcinoma cell line, HeLa. The ERK1/2 and Akt pathways
ECM and basement membrane, its role in generating an inflammatory are two major downstream effectors of Ras. Interestingly, Akt activation
response by mediating the release of chemokines, cytokines and growth (determined by Akt phosphorylation status) was significantly inhibited
factors have not been well established. In this study, peripheral blood by DP9 overexpression in human hepatoma cells (HepG2), whereas
monocyte cells were stimulated with Heparanase, and cytokine release ERK1/2 activity was unaffected, revealing a pathway-specific effect.
was examined. Heparanase treatment of cells resulted in the release DP9 induced a significant reduction in GFX - induced Akt activation in
of a range of pro-inflammatory cytokines including IL-8, IL-10, TNF, HepG2 cells. This is consistent with the observation that DP9 markedly
IL-6 and IL-1β. Similar results were obtained following the treatment attenuated GFX-dependent mitogenic effects, including apoptosis and
of MyD88-/- mouse spleen cells with Heparanase, suggesting that proliferation. These findings, along with our previous observation that
the cytokine release was not simply due to endotoxin contamination. DP9 overexpression is pro-apoptotic, suggest an important signalling
These data suggests that Heparanase can promote inflammation via role of DP9 in the regulation of a GFX - dependent survival and/or
the release of proinflammatory cytokines. Whether cytokine release is proliferation pathway.
due to the enzymatic or signalling role of Heparanase is currently being
investigated.

POS-MON-51 POS-TUE-52
EVIDENCE THAT 4E-BP, KNOWN AS A COMPLETELY ARABIDOPSIS POLLEN TRANSCRIPTION FACTORS
DISORDERED PROTEIN, PARTIALLY FOLDS UPON
COMPLEX FORMATION : NEW PERSPECTIVES FOR Gibalova A.1, 2, Renak D.1, 2, 3, Duplakova N.1, 2, Solcova K.1, 2 and Honys D.1, 2
THE REGULATION OF TRANSLATION
1
Laboratory of Pollen Biology, Institute of Experimental Botany ASCR,
Rozvojová 263, 165 02 Praha 6, Czech Republic. 2Department of
Gosselin P.1, Oulhen N.1,3, Czjzek M.2, Cormier P.1and Cosson B.1 Plant Experimental Biology, Faculty of Science, Charles University in
1
UPMC Univ Paris 06, CNRS, UMR 7150, Traduction Cycle Cellulaire Prague, Viničná 5, 128 44 Praha 2, Czech Republic. 3Department of
et Développement, Station Biologique de Roscoff, 29682, Roscoff, Plant Physiology and Anatomy, Faculty of Science, University of South
France. 2UPMC Univ Paris 06, CNRS, UMR 7139, Végétaux marins Bohemia, Branišovská 31, 370 05 České Budějovice, Czech Republic.
et biomolécules, Station Biologique de Roscoff, 29682, Roscoff,
France.3Current address: Department of Molecular and Cell Biology and Haploid male gametophyte plays a key role in plant fertility and crop
Biochemistry Brown University, Providence RI 02912, USA. production. Despite of signifiant progress in recent years, we still
have very limited understanding of the regulatory mechanisms that
Control of translation is a critical step in the regulation of gene expression have evolved to specify the gametophytic developmental programs.
involved in embryonic development, and mechanisms responsible for Therefore, it is necessary to identify transcription factors that are part of
human pathologies. The eukaryotic Initiation Factor 4E (eIF4E) interacts with such haploid regulatory networks. In our studies, we have focused on
the 5’ cap structure (m7GTP) and controls the cap-dependant translation.
The three eIF4E family members (eIF4E1, 2, 3) have been identified in basic-leucine zipper and heat shock transcription factors knowing to be
deuterostomes and interact specifically with different partners, extending involved in stress response and developmental processes. We report the
the biological impact of this translation factor. Only one isoform of each functional characterization of members of these gene families that are
actor has been found in sea urchin, making this organism a strong model expressed in both gametophytic and surrounding sporophytic tissues
to study the regulation of translation. Release of eIF4E from his repressor during flower development. The respective T-DNA insertion mutants
4E-Binding protein (4E-BP) is a key process in translation initiation and showed reduced transmission through male and female gametophytes,
is triggered by the phosphorylation of 4E-BP. The understanding of the pollen morphological defects, lower pollen germination efficiency and
interactions between these different actors, with functional and structural slower pollen tube growth both in vitro and in vivo conditions. Transient
approaches, is a very important step for cellular biology. Classical structural expression revealed the subcellular localization analysed proteins and
approaches suggest that 4E-BP is mostly or completely unstructured in in addition to other data, the nucleolar localisation of the heat shock
both free and bound states, and only the central domain of 4E-BP is known
to interact with eIF4E during the complex formation. These data are not transcription factor suggested its possible involvement in regulation
sufficient to explain the sharp phosphorylation mechanisms that control of mRNA/rRNA transcription. Acknowledgment: Authors gratefully
the association of 4EBP and eIF4E. Using an original structural technic acknowledge the financial support from the Czech Science Foundation
that consists in measurement of small angles X-ray scattering (SAXS), (grant 522/09/0858) and Ministry of Education, Youth and Sports of the
we show for the first time that 4E-BP adopts a folded structure upon the Czech Republic (grants LC06004 and OC10054).
binding to eIF4E but also that this inhibitor has a transitory structure when
it’s free in solution. SAXS allows us to see the rest of 4E-BP chain that is
missing in the crystallography complex structure, and reveals a « fuzzy
complex », involving a larger surface of interaction between these two
actors. The results that we obtained with sea urchins proteins, adopting
this new dynamic view of 4E-BP structure, open new perspectives for the
understanding of the sharp mechanisms of gene regulation by translation.

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POS-MON-53 POS-TUE-54
IN SILICO SEARCH OF POTENTIAL FUNCTIONAL DEMONSTRATION OF AUTOPHOSPHORYLATION OF
SITES WITHIN PROMOTERS OF MOUSE O6- THE LYN PROTEIN TYROSINE KINASE A NEW SITE
METHYLGUANINE-DNA METHYLTRANSFERASE GENE IN THE UNIQUE DOMAIN – THE ROLE OF THE NEW
AUTOPHOSPHORYLATION IN LYN KINASE FUNCTION
Iatsyshyna A.P., Pidpala O.V. and Lukash L.L.
Institute of Molecular Biology and Genetics, 150 Zabolotnogo Str., Jarasrassamee B., Chong Y.P., Williamson N.A., Purcell A.W. and
03680, Kyiv, Ukraine. Heung-Chin C.
Department of Biochemistry and Molecular Biology, Bio21 Molecular
The modulation of expression and activity of O6-methylguanine-DNA Science and Biology, University of Melbourne.
methyltransferase, MGMT, in tumors and normal tissues is currently
being investigated as possible strategies for improving cancer therapy. Lyn is a member of the Src family of protein kinases (SFKs). Since SFKs
At present little is known about organization and expression of mouse are important signaling enzymes participating in a variety of cellular
Mgmt gene, so the aim of our work was to search potential regulatory processes, activation of SFKs is transient and tightly regulated. It is
sequences within the promoters of this gene. Sequences of the mouse well known that autophosphorylation of Lyn at the conserved tyrosine
Mgmt gene promoters were taken from the database TRED. Identification (YA) in activation loop of the kinase domain results in activation. In
of functional sites was performed by using program TFSEARCH. addition to autophosphorylation at this site, we discovered a novel
There are 3 promoters of mouse Mgmt gene in TRED database: 77428 autophosphorylation site (Y32) in the unique domain of Lyn. In this study,
(known), 77429 (refseq, predicted), 77430 (refseq). In sequences of mutants of Lyn carrying mutations in one or both autophosphorylation
given promoters we found gomology with functional sites for 15, 9 and sites including Y32F Lyn, Y32, 397F Lyn, and Y397F Lyn were generated.
19 transcription factors (TF) respectively. Common for all promoters Biochemical analysis revealed that unlike autophosphorylation in
were such TFs: Nkx-2.5, Gata-1, SRY, USF. In the known promoter, most protein kinases, autophosphorylation of Lyn at this site occurs
except characterized by others AP-1 and E2F TFs, we detected binding intramolecularly. Using a phosphospecific antibody against Tyr-32 of Lyn,
sites (BS) for such TFs: CRE-BP, CREB, C/EBP, SRF, Oct-1, Tst-1, NF- we were able to demonstrate that Lyn undergoes autophosphorylation
E2, Lyf-1, c-Myc. Such exclusivity of revealed sites can be an evidence at Tyr-32 in rat tissues, confirming the physiological relevance of our
of tissue specificity of the Mgmt expression regulation. The predicted findings. To investigate the role of Tyr-32 autophosphorylation in the
promoter contains BSs for such TFs: LYF-1, HSF2, c-Ets-1, C/EBPβ, Ik- oncogenic action of Lyn, we set out to study the regulation and function of
2, except common TFs for all studied promoters. In the refseq promoter Lyn in Chronic myelogenous leukaemia (CML). Aberrant activation and
sequence we revealed unique BSs for TFs: CRE-BP, CREB, C/EBP, expression of Lyn has been reported to contribute to the development of
AP-1, c-Ets-1, Ik-2, Oct-1, YY1, Sox-5, TATA, STATx, c-Rel, Ik-1, NF-κB, drug resistance in CML – some CML patients fail to sustain hematologic
N-Myc. Thereby, by using in silico analysis of three promoters of the remission with the drug Gleevec. We have initiated studies of the role of
mouse Mgmt gene we revealed individual BSs that can be associated the Tyr-32 autophosphorylation in the oncogenic action and regulation
with the tissue-specific regulation of gene expression, and common of the kinase activity of Lyn in both the Gleevec-sensitive and Gleevec-
sites which could play a key role in regulation of this gene. resistant CML cells. Results of our studies will shed light on how Lyn
contribute to the development of drug-resistant CML.

POS-MON-55 POS-TUE-56
COPPER CHLORIDE INDUCED EXPRESSION OF INVESTIGATING THE CONTRIBUTION OF GENOMIC
CYTOGLOBIN AND HIF-1α IN VIVO INSTABILITY TO ALTERED mircoRNA EXPRESSION IN
OVARIAN CANCER CELL LINES
Jusman S.W.A.1, 2, Prijanti A.R.1, 2, Iswanti F.C.1, Ferdinal F.3, Suyatna F.D.2, 4,
Wanandi S.I.1, 2 and Sadikin M.1, 2 Kan C.W.S.1, Hahn M.A.1, Huh J.Y.1, Dykema K.2, Howell V.M.1 and
1
Department of Biochemistry & Molecular Biology, Faculty of Medicine, Marsh D.J.1
University of Indonesia. 2Biomedical Science Program, Faculty of 1
Functional Genomics Laboratory, Hormones and Cancer Group,
Medicine, University of Indonesia. 3School of Medicine, Tarumanegara Kolling Institute of Medical Research. 2Van Andel Research Institute,
University. 4Department of Pharmacology, Faculty of Medicine, Grand Rapids, MI, USA.
University of Indonesia.
Ovarian cancer is the most lethal gynaecological malignancy and
Copper is known to stabilize HIF-1α under normoxic condition, resulting the sixth most common cause of cancer death in Australian women.
in induction of HIF-1α target genes. Cytoglobin, the novel globin microRNAs (miRNAs) are small non-coding RNAs that regulate gene
from vertebrate is suggested as one of HIF-1α regulated genes. This expression and are often aberrantly expressed in cancer. This project
study observed the liver tissue response to copper chloride induction investigated whether chromosomal losses or gains may contribute to
in vivo. Male Sprague-Dawley rats were given 1.25 μmol of copper changes in expression of miRNA in ovarian cancer cell lines. Gene and
chloride solution intraperitoneally. The observation are made at 2, 6, miRNA expression microarrays were performed on four ovarian cancer
24, 48 and 72 hours after treatment and compared to control group. cell lines and a cell line model of normal ovarian surface epithelial cells
Liver tissue were analyzed for HIF-1α protein using ELISA technique, (OSEs). Predicted regions of chromosomal loss or amplification were
cytoglobin mRNA with real time RT-PCR and cytoglobin protein with identified by comparative genomic microarray analysis (CGMA) of gene
ELISA. It is showed that cytoglobin protein was up-regulated 48 hours expression data. CGMA predicted chromosomal loss at 5q in all four
after treatment compared to control group, while the HIF-1α protein cancer cell lines but not in OSEs. Loss of heterozygosity in chromosome
and cytoglobin mRNA showed tendencies to be stimulated 24 and 48 5q has been reported to be associated with early development of
hours respectively after treatment. It is concluded that copper chloride ovarian cancer. Investigation of miRNA located on chromosome 5q
stimulated expression of cytoglobin protein, which might be mediated revealed miR-146a to be significantly decreased (ANOVA, P<0.01)
by stabilization of HIF-1α protein due to inhibition of its degradation by in expression across all four cell lines compared to OSEs. Predicted
prolil hydroxylase. targets of miR-146a include CCBP2, chemokine-binding protein 2.
CCBP2 is reported to be over-expressed in vascular tumours and may
drive tumour development and growth. In summary, we have shown
that ovarian cancer cell lines have gene and miRNA expression profiles
that are distinct to those of OSEs. These results suggest that genomic
instability may contribute to the altered expression of a subset of miRNA
and their target genes in ovarian cancer. Supported by an Australian
Postgraduate Award, University of Sydney Cancer Research Fund and
the Cancer Institute NSW.

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POS-MON-57 POS-TUE-58
EXTENSION OF DROSOPHILA LIFESPAN BY THE CELLULAR SIGNALING PATHWAY MEDIATING
OVEREXPRESSION OF HUMAN CLUSTERIN INDUCTION OF HBV REPLICATION AND GENE
EXPRESSION BY ETHANOL
Kang B.H.1, Lee Y.N.2, Park J.J.2 and Min B.H.1
1
Department of Pharmacology and BK21 program in Biomedical Kim Y.H.1, 2 , Kim B.K.1, 2 and Park Y.G.1, 2
Sciences, College of Medicine, Korea University, 136-705, Seoul, Korea. 1
Dept. of Biochemistry. 2Division of Brain Korea 21 Program for
2
Department of physiology and BK21 program in Biomedical Sciences, Biomedical Science, Korea University College of Medicine, Anam-
College of Medicine, Korea University, 136-705, Seoul, Korea. dong, Seongbuk-Gu, Seoul, 136-170, Korea.

Clusterin (CLU) is a disulfide-linked heterodimeric glycoprotein Even though alcohol intake has been implicated in the etiology of HBV-
implicated in diverse biological processes. Expression levels of CLU related diseases, neither ethanol effect on HBV replication nor the
increase during cellular senescence or normal aging, but it is uncertain cellular signaling pathway mediating the effect are known. This study is
whether this is protective against aging, or is a consequence of aging. designed to elucidate the effect of alcohol on HBV replication and gene
To better understand the role of clusterin in organismal aging, we expression. Ethanol treatment increased the HBV promoter/enhancer
generated transgenic Drosophila alleles to induce expression of the activity and the levels of HBV transcripts, DNA and viral antigens. In
secretory form of human clusterin (hCluS) by using the Gal4/UAS contrast, acetaldehyde, a product of hepatic ethanol metabolism, had no
system. The hCluS protein sized 50-60 kDa was detected in both effect on the synthesis of HBV mRNA. Moreover, the ethanol-induced
adult homogenate and larval hemolymph of the flies over-expressing increase in HBV mRNA synthesis was not affected by pretreatment with
hCluS ubiquitously (da-Gal4>UAS-hCluS) or in motoneurons (D42- inhibitors of alcohol dehydrogenase or acetaldehyde dehydrogenase.
Gal4>UAS-hCluS). Life spans of these hCluS-over-expressing flies However, CYP2E1 did not make an effect on ethanol-induced synthesis
were significantly extended (39 days) than control flies that presented of HBV mRNA even though ethanol-induced ROS generation is well
no hCluS induction. The mean life-span of CS10 (+/+), D42-Gal4/+, known to be mainly dependent upon CYP2E1. Ethanol-induced
and UAS-hCluS/+ was 31, 33, and 35 days, respectively. In addition, synthesis of HBV mRNA was abolished by pretreatment with DPI and
the hCluS-over-expressing flies showed enhanced tolerance to heat Trolox. Ethanol increased the synthesis of IL-6 mRNA and the release
shock, wet starvation, and oxidative stress. Furthermore, the amount of IL-6 protein via ROS generation, and these events are necessary for
of reactive oxygen species (ROS) in the whole body was significantly ethanol-enhanced synthesis of HBV mRNA and DNA as well as increase
reduced in hCluS-over-expressing flies, compared to control flies. Over- of HBV promoter/enhancer activity. Ethanol-induced HBV replication
expression of hCluS in a group of neurons was sufficient to recapitulate and gene expression was dependent upon IL-6/JAK2 signaling but not
its effects of whole body expression, implying that hCluS works cell upon STAT1 and 3. This study indicates that the enhancement of HBV
nonautonomously. However, the patterns of their feeding behavior were replication by ethanol treatment requires IL-6 production and JAK2
not affected by hCluS expression. Taken together, these results suggest activation through ROS generation.
that hCluS may function as an antioxidant to reduce ROS levels and
delay the organismal aging in fruit flies. [This work was supported by the
Korea Science and Engineering Foundation (KOSEF) grant funded by
the Korea government (MEST) (No. 2009-009-1418) and BAERI (Basic
Atomic Energy Research Institute) grant from the National Nuclear R&D
Program (20090078713) funded all by the Korean Ministry of Education,
Science and Technology (MEST)].

POS-MON-59 POS-TUE-60
SUPPRESSOR OF CYTOKINE SIGNALING (SOCS)5 IS FUNCTIONAL ANALYSIS OF TRANSCRIPTION
A POTENTIAL REGULATOR OF THE IL-4-JAK/STAT FACTORS REGULATING FRUCTOSYLTRANSFERASES
PATHWAY INVOLVED IN THE FRUCTAN SYNTHETIC PATHWAY IN
WHEAT
Kolesnik T.B., Colombus R.E., Chakravorty A., Wilson T.A., Sprigg N.S.,
Carter W., Zhang J.-G., Babon J.J., Nicola N.A. and Nicholson S.E. Kooiker M., Xue G.P. and McIntyre C.L.
The Walter & Eliza Hall Institute of Medical Research, Parkville, CSIRO, 306 Carmody road, St. Lucia, Australia.
Victoria, Australia.
Water soluble carbohydrates (WSCs) are accumulated in the stems
SOCS5 has been implicated in regulation of the Th1/Th2 balance by and leaf sheath of several cereals like wheat, barley and oats.
inhibition of IL-4 signaling in Th1 cells through interaction with the IL-4 The main WSCs found in wheat stems at the grain filling stage are
receptor alpha chain (IL-4Rα)1. However, in SOCS5-deficient mice, Th1/ fructans, which are linear or branched oligosachharides, synthesised
Th2 cells differentiate normally and the mice mount normal B and T cell from sucrose in the vacuole. For the synthesis the enzymes
responses to mitogenic stimuli2. Analogous to the Th1/Th2 paradigm, sucrose:fructan 6-fructosyltranferase (6-SFT) and sucrose:sucrose
macrophages differentiate into classically activated or alternate in 1-fructosyltransferase (1-SST) are essential. Fructans are an important
response to IFNγ or IL-4, respectively. To dissect the role of SOCS5 in temporary carbon reserve and are hydrolysed by fructan exohydrolases
IL-4 signaling in macrophages, we initially examined regulation of the when the grain needs sugars for grain filling. Under normal conditions
SOCS proteins. Socs5 mRNA as well as Socs1, Socs2 and Cis, was stem WSCs can contribute to up to 20% of the grain yield, but under
rapidly induced in response to IL-4, however, only SOCS1, SOCS5 and stress conditions (like drought stress) this percentage can increase to
CIS could inhibit IL-4 signaling in a STAT6-reporter assay. We further greater than 50%. Under water limited conditions a positive correlation
demonstrated that, like SOCS1, SOCS5 was able to directly inhibit between grain yield and WSC content in the stem at anthesis is often
JAK1 enzymatic activity. Extensive mutagenesis analysis showed that observed. Some enzymes that play an important role in stem fructan
the SOCS5 N-terminus, SH2 domain and SOCS box were required for accumulation are sucrose:fructan 6-fructosyltranferase (6-SFT) and
inhibition of JAK1 activation, whereas the N-terminus was essential for sucrose:sucrose 1-fructosyltransferase (1-SST), which are positively
interaction with the IL-4Rα. Western-blot analysis revealed no differences associated with genotypic variation in stem WSCs in recombinant
in the level of IL-4-stimulated JAK1 and STAT6 phosphorylation in inbred lines Seri/Babax (SB). We have recently identified a number of
SOCS5-deficient macrophages compared to wild-type cells. To address candidate transcription factors that are potentially involved in controlling
possible redundancy between SOCS1 and SOCS5, we analyzed IL-4 the expression of these fructosyltransferases in wheat and have been
signaling in macrophages lacking both SOCS proteins. JAK1 and STAT6 evaluating the regulatory role of these candidate genes in transgenic
phosphorylation were up-regulated in SOCS1-deficient cells to the same wheat.
extend as in double knock-out cells indicating that, at least in primary
macrophages, SOCS1 and SOCS5 are not functionally redundant. The
biological role of SOCS5 in IL-4 signaling remains to be elucidated.
1
Seki et al., PNAS 2002, 99(20): 13003-8; 2Brender et al., 2004 MCB,
24(13): 6094-6103.

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POS-MON-61 POS-TUE-62
TLR REGULATION OF SPSB1 REGULATES INOS GENERATION OF A GRANZYME B/EGFP REPORTER
INDUCTION FOR LIVE-CELL IMAGING
Lewis R.S., Kuang Z., Kolesnik T.B., D’Cruz A., Low A., Norton R.S. Bird C.H.1, Prescott M.1, Harper I.2 and Bird P.I.1
and Nicholson S.E. 1
Department of Biochemistry and Molecular Biology, Monash
Walter and Eliza Hall Institute. University, Australia. 2Monash Micro Imaging, Monash University,
Australia.
The mammalian innate immune system has evolved to recognise foreign
molecules derived from pathogens via the Toll-like receptors (TLRs). Granzyme B (GrB) is a serine proteinase found in the cytolytic granules
TLR3 and TLR4 can signal via the TIR domain-containing adapter of cytotoxic T lymphocytes (CTLs) and natural killer cells (NK cells). Upon
inducing interferon (IFN) β (TRIF), which results in the transcription target cell engagement, GrB and other granule proteins are released
of a small array of genes, including IFNβ. iNOS is an enzyme that is into the intercellular space. It is thought that perforin facilitates the entry
rapidly induced by a range of stimuli, including cytokines and microbes, of GrB into the target cell cytosol where GrB activates the apoptotic
and catalyses the production of nitric oxide (NO). NO is a potent source pathway. However, there is currently no direct evidence of GrB release
of reactive nitrogen species that play an important role in the killing into the cytosol of target cells, and there is increasing evidence that it is
of intracellular pathogens and forms a crucial component of the host involved in extracellular remodelling. Here we describe the generation
defence. We have recently identified iNOS as a target of the mammalian and characterisation of a GrB/GFP fusion protein which is expressed
SPRY domain-containing SOCS box (SPSB) -2 protein. The SOCS box stably in NK cells and trafficks correctly to the granule. The GrB is
is a peptide motif, which in conjunction with elongins B and C recruits inactive so will not induce cell death, but rather act as a tracer. Using
cullin-5 and Rbx-2 to form an active E3 ubiquitin complex. Here we this fusion protein we wish to follow GrB during target cell engagement
show that SPSB1 is the only SPSB family member to be regulated by and lymphocyte migration.
the same TLR pathways that induce iNOS expression and characterise
the interaction between SPSB1 and iNOS. Through the use of SPSB-1
transgenic macrophages and shRNA knockdown of SPSB-1 we have
shown that SPSB-1 controls the induction of iNOS and the subsequent
production of NO downstream of TLR3 and 4. Further, we demonstrate
that regulation of iNOS by SPSB-1 is dependent on the proteasome
via the SPSB-1 C-terminal SOCS box. These data suggest that SPSB-
1 acts through a negative-feedback loop that together with SPSB2
controls the extent of iNOS induction and NO production.

POS-MON-63 POS-TUE-64
INTRACELLULAR TRAFFICKING OF RICIN TOXIN: SIDEROPHORE CONJUGATES AS ANTIMICROBIAL
COMPUTATIONAL MODELLING AND KINEMATICS AND IRON-OVERLOAD AGENTS
Chong D., Walker A. and Skvortsov A. Obando D., Brites L.J., Shi C., Liu J. and Codd R.
Defence Science and Technology Organisation, 506 Lorimer St., School of Medical Sciences (Pharmacology), University of Sydney,
Fishermans Bend, VIC 3207, Australia. NSW 2006, Australia.

To explain the efficacy of inhibitors against toxins in various cell types, Under iron-deprived conditions, pathogenic and non-pathogenic
the intracellular trafficking of the inhibitors and indeed, the agent itself bacteria produce low-molecular-weight molecules called siderophores
must be understood. In this study, live cell 3D/4D confocal microscopy to solubilise Fe(III), which is sparingly soluble under the oxic, aqueous
was used to visualise ricin toxin transport in human small airway and pH neutral conditions in the environment and in the mammalian host.
epithelial cells to develop a computational model of toxin trafficking. In An avid recognition event between the Fe(III)-loaded siderophore and
order to predict the rate at which the toxin will reach various subcellular receptors at the bacterial cell surface is followed by the complex traversing
compartments, such as the juxtanuclear Golgi, the trafficking route of the the cell to ultimately deliver iron to the cytoplasm for incorporation into
toxin is modelled as a diffusive transport process. An analytic solution key Fe-containing molecules, such as cytochromes and ribonucleotide
of the diffusion equations is then constructed by treating the boundaries reductase. The competition between bacteria for Fe is reflected in the
of the cell wall and the nucleus as conformally invariant fractals. The chemical diversity of native siderophores: most bacteria produce a
driving parameters of the subcellular trafficking of ricin (i.e. mean unique siderophore that is recognisable only by its cognate cell surface
squared displacement) were derived from confocal microscope image receptor. Molecules that thwart regular Fe(III)-siderophore mediated
analyses and were used to calibrate the transport model. This work uptake present a platform for the design of new narrow spectrum
ultimately aims to establish a computational model of the interactions antibiotics. In our group, we have modified the structure of a siderophore
between inhibitor, agent and target cell. native to a non-pathogenic gram positive bacterium to produce a suite
of potential antibiotic compounds. Of the four compounds we prepared,
one of these prevented growth of our target bacterium at an MIC of 60
nmol/cm2 lawn. The chemical differences in the family of compounds
we prepared is subtle, which presents some intrigue with regard to fully
understanding the mechanism of action. These results will shed new
light on the potential of tackling antibiotic design via iron deprivation
and the possibilities of designing siderophore-based compounds
against mammalian pathogens, including Pseudomonas aeruginosa,
Bordetella pertussis and Mycobacterium tuberculosis. In other work, we
have prepared new siderophore conjugates that show promise as new
therapeutics for the treatment of iron-overload disease in humans. In
this paper, details of each of these programs will be discussed.

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POS-MON-65 POS-TUE-66
STRUCTURAL REARRANGEMENTS OF THE RAB2A PARTICIPATES IN THE TRAFFICKING AND
P. FALCIPARUM GAMETOCYTE: AN ESSENTIAL SECRETION OF APOLIPOPROTEIN E
PROCESS FOR PARASITE TRANSMISSION
Dinnes D.L.1, Kockx M.1, Jessup W.1 and Kritharides L.1, 2
Dearnley M.K.1, 2 , Dixon M.W.A.1, 2, Yeoman J.1, 2, Hanssen E.3 and
1
Macrophage Biology Group, Centre for Vascular Research, UNSW,
Tilley L.1, 2 Sydney, Australia. 2Concord Repatriation General Hospital, University
1
La Trobe Institute for Molecular Science, La Trobe University, VIC of Sydney, Sydney, Australia.
3086, Australia. 2ARC Centre of Excellence for Coherent X-ray
Science, La Trobe University, VIC 3086, Australia. 3Bio21 Institute, Apolipoprotein E (apoE) is a ~34kDa glycoprotein that is secreted from
University of Melbourne, VIC 3000, Australia. several cells, including macrophages, hepatocytes and astrocytes, with
known roles in immunoregulation, protection from atherosclerosis and
Transmission of the malaria parasite Plasmodium falciparum depends Alzheimer’s disease. We have previously shown that apoE is transported
on the production of the specialised sexual blood stage parasites called in vesicles along the microtubular network and is regulated by PKA,
gametocytes. Despite the fact that the first forms of the malaria parasite PP2B (calcineurin) and intracellular Ca2+ (Kockx M et al, Circ Res (2007)
identified were the sexual stages or gametocytes, many questions 101:608; Kockx M et al, J Biol Chem (2009) 284:24144). However, the
remain about the fundamental biology of this lifecycle stage. Maturation transport proteins regulating its vesicular movement are unknown. The
of the gametocyte to the transmissible stage V form is dependent on aim of this study was to identify if any proteins of the Rab GTPase family
the parasites ability to remodel its host RBC. During this remodelling, play a role in this process. HEK293 cells stably transfected with human
parasite derived structures are formed within the RBC cytoplasm apoE or apoE tagged with GFP (apoE-GFP) were subjected to Rab1A,
called Maurer’s clefts which function as protein-sorting organelles 2A, 6A, 8A, 10 or 27B siRNA using non-silencing sequences as a
for parasite proteins en route to the RBC membrane. In this study we control. Silencing of Rab2A inhibited the rate of secretion 5-fold relative
assess the genesis and maintenance of the Maurer’s clefts and the to control, whereas silencing of other Rab family members had no
resident Maurer’s cleft protein the Ring Exported Protein 1 (REX1), effect. Rab2A silencing had no effect on apoE mRNA levels, increased
across gametocyte development. We also investigate the formation and apoE protein levels within cells and pulse chase metabolic labeling
composition of the inner membrane complex (IMC) of the gametocyte confirmed a direct inhibition of apoE secretion. Confocal microscopy
across several developmental stages. Previous studies have identified demonstrated accumulation of apoE in a perinuclear compartment after
this complex within the gametocyte; however few have drawn parallels Rab2A silencing, and this was supported with a loss of highly sialylated
to the IMC found in the merozoite. We aim to identify novel constituents apoE isoforms (determined by 2-dimensional electrophoresis) implying
of the IMC and investigate its resemblance to the IMC in the asexual a block of transport through the Golgi network. We have demonstrated
merozoite. Purified gametocytes from differing developmental stages for the first time a major role for the GTPase Rab2A in regulating apoE
were examined by electron and fluorescence microscopy to map the transport and secretion, the manipulation of which may provide new
morphology of the developing gametocyte with particular emphasis avenues for modulating apoE secretion.
on the Maurer’s cleft using resident protein REX1 and the IMC protein
GAP50. An understanding of the structures and remodelling mediating
gametocyte maturation will prove invaluable in the development of novel
transmission blocking approaches.

POS-MON-67 POS-TUE-68
DETERMINING THE INTRACELLULAR LOCALISATION BINDING OF P110 RETINOBLASTOMA PROTEIN
OF ATNHX5 AND ATNHX6 AND THEIR ROLE IN INHIBITS NUCLEAR IMPORT OF SV40 LARGE TUMOUR
CELLULAR TRAFFICKING AND SALT TOLERANCE ANTIGEN THROUGH A PHOSPHOREGULATED
MECHANISM
Ford B.A. and Gendall A.R.
Depratment of Botany, La trobe University, Victoria, Australia. Fulcher A.J.1, Dias M.M.1 and Jans D.A.1, 2
1
Monash University, Clayton, Victoria, Australia. 2ARC centre of
AtNHX5 and AtNHX6, like AtSOS1 and AtNHX1-4 are part of the Excellent for Biotechnology and Development.
large CPA1 monovalent cation/proton antiporter sub group of the
sodium hydrogen exchanger super family. AtNHX5 has been shown Nuclear import of the simian virus SV40 large tumour antigen (T-ag) is
to suppress the Na+ sensitive phenotype of the nhx1 yeast mutant dependent on its nuclear localisation signal (NLS) within amino acids
and is up-regulated in response to NaCl. Arabidopsis plants over 126-132 that is recognised by the importin α/β1 heterodimer, as well
expressing the tomato AtNHX5/6 ortholog LeNHX2 showed increased as a protein kinase CK2 site at serine 112 upstream of the NLS, which
tolerance to high Na+ concentrations. The intracellular localisation of enhances the interaction c. 50-fold. Here we show for the first time
AtNHX5 and AtNHX6 in Arabidopsis has been determined by studying that T-ag nuclear import is negatively regulated by further N-terminal
the expression of a 35S::NHX5ORF:CFP, a 35S::NHX6ORF:YFP and sequences (amino acids 102-110) which represent the binding site (BS)
a 35S::YFP:NHX6ORF. Our results show that AtNHX5 and AtNHX6 for the retinoblastoma (Rb) tumour suppressor protein. Quantitative
co-localise to the same intracellular location and are not localised to confocal laser scanning microscopic analysis of the transport
either the tonoplast or the plasma membrane, and may be localized to properties of T-ag constructs with or without Rb binding site mutations
endosomes. Endosomal compartments are the main transport vesicles in living transfected cells or in a reconstituted nuclear transport system
in the secretory and endocytic pathways. It has been proposed that the indicate that the presence of the RbBS significantly reduces nuclear
endosomal localized NHXs regulate endosomal pH and are essential accumulation of T-ag. A number of approaches, including the analysis
for protein sorting in the endocytic and secretory pathways. Preliminary of T-ag nuclear import in an isogenic cell pair with and without functional
analysis of root tip cells from the nhx5 nhx6 double mutant shows a p110Rb implicate p110Rb binding as being responsible for the reduced
disruption to the normal development of lateral roots. It may be the case nuclear accumulation, with the serine106 phosphorylation site within the
that in the nhx5 nhx6 double mutant the endosomal traffic required to RbBS appearing to enhance the inhibitory effect. The involvement of
deliver auxin influx carriers AUX1 and LAX3 to their ultimate destination p110Rb in modulating T-ag nuclear transport has implications for the
is disrupted preventing the normal auxin signaling required for lateral regulation of nuclear import of the other proteins from the various other
root development. viruses of medical significance that interact with p110Rb, and how this
may relate to transformation.

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REGULATORY ROLE OF PRIP IN EXOCYTOSIS ARL5B IS A GOLGI-LOCALISED SMALL G PROTEIN
THROUGH THE INTERACTION WITH PROTEIN INVOLVED IN REGULATION OF RETROGRADE
PHOSPHATASE TRANSPORT
Gao J., Takeuchi H., Zhang Z. and Hirata M. Houghton F.J. and Gleeson P.A.
Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Bio21 Molecular Science and Biotechnology Institute, University of
Science, Kyushu University. Melbourne.

Exocytosis is one of the most basal and important cellular events. The Small G proteins play an essential role in the regulation of membrane
minimal machinery for the final step, membrane fusion, is SNARE traffic. Small proteins (~20kD) include members of the Rab, Arf and Arl
complex consists of syntaxin, SNAP-25 and VAMP. Phospho-state of families. The Arf/Arl family members were identified initially by sequence
these proteins are known to be involved in the regulation of exocytosis. homology (Kahn et al., 2006). In the GTP-bound form, the Arfs/Arls
We have shown that a protein phosphatase-1 anchoring protein, PRIP are able to bind specific effectors. Although some Arf-like or Arl family
(phospholipase C-related but catalytically inactive protein), has some members have been well characterised, such as Arl1 (Lu et al., 2001)
inhibitory role in regulating exocytosis. In the present study, we examine and ARFRP1/Arl3 (Panic et al., 2003; Setty et al., 2003), the function of
the role of PRIP in phospho-dependent regulation of exocytosis using many Arls remains undefined. An important role of small G proteins is
pheochromocytoma cell line, PC12 cells that secrete noradrenalin in the regulation of membrane traffic at the trans-Golgi network (TGN)
(NA). Pretreatment of the cells with forskolin enhanced NA secretion (Derby et al, 2004; Lu and Hong, 2003). The cDNA encoding a number
and the enhancement was gradually diminished by removing forskolin. of Arls were amplified by RT-PCR from HeLa cells, mutated to the GTP
Exogenous expression of PRIP accelerated the decreasing process of or GDP bound forms by PCR mutagenesis and cloned into GFP vectors.
NA secretion. Correlatively with the NA secretion, SNAP-25 was strongly Using confocal microscopy analysis of transfected HeLa cells, we
phosphorylated by forskolin treatment of the cells and dephosphorylated identified Arls that localise to the Golgi. Arl5bWT and Arl5bQ71L localise
gradually after removing forskolin. In addition, exogenous expression to the Golgi in close proximity to TGN golgins, and Arl5bQ71L-GFP is
of PRIP accelerated the dephosphorylation process. We also showed observed on tubules associated with the TGN. Functional analysis was
that dephosphorylation of SNAP-25 at Thr138, phosphorylated by cAMP- conducted using siRNA depletion and membrane transport was studied
dependent protein kinase, was mainly catalyzed by PP-1. These results using antibody internalisation assays. Depletion of Arl5b by RNAi in
suggest that PRIP is involved in phospho-dependent regulation of cultured cells results in disruption of the intracellular localisation of
SNARE proteins through modulating the activity of protein phosphates-1, mannose 6-phosphate receptor, and alters the transport dynamics of
thus regulating exocytosis. . the membrane cargo TGN38 and the shiga toxin B fragment between
endosomes and TGN. Therefore, Arl5b is a trans-Golgi small G protein
which regulates endosome-TGN transport. Kahn, R.A., et al. J Cell Biol
(2006), Lu,L. and Hong,W. Mol Biol Cell (2003), Derby, M C. et al. J
Cell Sci.(2004)., Lu,L., et al. J Cell Sci (2001), Panic,B., et al. Curr Biol
(2003), Setty, S.R., et al Curr Biol (2003).

POS-MON-71 POS-TUE-72
TOXICITY OF AMYLOID TRANSTHYRETINS TO DOWN LONG-TERM MEMORY AND LEARNING IMPAIRMENT
SYNDROME FIBROBLAST IN NEDD4 HETEROZYGOUS MICE
Annanon S., Kaewmeechai S. and Prapunpoj P. Bongiorno D.1, 2 , Boase N.3, Kumar S.3 and Poronnik P.1, 2
Department of Biochemistry, Faculty of Science, Prince of Songkla 1
School of Medical Science. 2Health Innovations Research Insitute
University, Hat Yai, Songkhla 90112, Thailand (HIRi), RMIT University, Bundoora. 3Centre for Cancer Biology, SA
Pathology, Frome Road, Adelaide.
Amyloidosis is a group of human diseases characterized by the intra-
or extracellular deposition of aggregated amyloid proteins, leading to Nedd4 (Neural precursor cell Expressed Developmentally Down-
progressive disruption of the normal tissue architecture and consequently regulated 4) a ubiquitin ligase (E3) has been implicated in neuronal
impairing organ function. Among of these proteins, amyloid β (Aβ) which development. Although numerous potential substrates of Nedd4 have
is the major constituent of amyloid plaques in brain cortex of Alzheimer been uncovered, the exact physiological function(s) of Nedd4 remain
‘s disease (AD) and Down syndrome (DS), and transthyretin (TTR) are unclear (Yang and Kumar 2010). As Nedd4 knockout mice die at birth
of interest. Inter-relationship in particular the protective function of TTR due to growth retardation and vascular defects, we investigated the
on cytotoxicity of Aβ has been demonstrated. Among peripheral tissues, impact of Nedd4 in memory and learning using Nedd4 heterozygous
fibroblasts from DS patients were shown carrying Aβ protein precursor mice. Nedd4 heterozygous (n=13) and wild-type (n=8) littermate controls
(APP) and having endocytic dysfunction similar to neurons in AD. In were assessed for short-term memory using Y-maze. Long-term spatial
this study, the cytotoxicity and the protective effect on Aβ of TTR were memory and learning was also assessed using Morris Water Maze
explored in greater details in normal and DS fibroblasts. Human wild- (MWM). Time taken to locate a hidden platform corresponds to the
type TTR and TTR variants including V30M and L55P were synthesized learning phase. On the final day of testing, the platform is removed and
using the heterologous gene expression system of Pichia pastoris. Cell duration to enter the platform quadrant corresponds to the memory
viability and apoptosis were examined and compared. The preliminary phase. We found that Nedd4 heterozygote mice took significantly longer
results showed that not only Aβ but also amyloidogenic TTR V30M to find the hidden platform than wild-type mice in MWM test (Day 6,
and L55P were toxic to both normal and DS fibroblasts, however with 65.3±9.5 and 27.0±4.6 sec). Furthermore, when the hidden platform
different sensitivity. was removed, Nedd4 heterozygote mice also took significantly longer
to enter the platform quadrant compared to wild-type mice (23.0±5.4
and 6.4±1.3 sec). However, there was no significant difference in time
spent in the novel arm of the Y-maze between Nedd4 heterozygote
and wild-type mice (150.4±5.4 and 158.3±8.7 sec). These data show
that in Nedd4 heterozygous mice, where ~50% of the ubiquitin ligase is
removed is sufficient to produce long-term spatial memory and learning
deficits, with spared short-term memory. Yang B, Kumar S. 2010. Nedd4
and Nedd4-2: closely related ubiquitin-protein ligases with distinct
physiological functions. Cell Death Differ 17:68-77.

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THE INVOLVEMENT OF EXOSOMES IN EVALUATION OF SRU BIND LABEL-FREE
INTERCELLULAR PRION TRANSMISSION TECHNOLOGY FOR ASSESSMENT OF KAPPA OPIOID
RECEPTOR LIGANDS IN CELL-BASED ASSAYS
Coleman B.M.1, 2, 3, Hanssen E.3, 5, Masters C.L.4, Lawson V.A.2, 4 and
Hill A.F.1, 3, 4 Deuis J.1, Vetter I.2, Lewis R.2 and Cabot P.1
1
Department of Biochemistry and Molecular Biology, The University of 1
School of Pharmacy, University of Queensland, Australia. 2Institute for
Melbourne. 2Department of Pathology, The University of Melbourne. Molecular Bioscience, University of Queensland, Australia.
3
Bio21 Molecular Science and Biotechnology Institute, The University
of Melbourne. 4Mental Health Research Institute, The University of Opioid receptors have long been known to be effective targets for the
Melbourne. 5Electron Microscopy Unit, Bio21 Molecular Science and production of analgesia. Recent interest has developed in targeting the
Biotechnology Institute, The University of Melbourne. kappa opioid receptor (KOR) subtype, which couples to inhibition of
adenylate cyclase through Gαi. Label-free technology is now available
Prion diseases are invariable fatal neurodegenerative disorders, which to assess ligand-receptor interactions in cell-based assays, replacing
are associated with an abnormal isoform (PrPSc) of the host encoded more conventional measurement of second messenger signalling. The
cellular prion protein (PrPC). The diseases are also transmissible, with the aim of this study was to assess the ability of BIND label-free technology
infectious agent postulated to be composed principally of PrPSc. However, (SRU Biosystems) to detect the activation of the KOR. HEK-293 cells
the conditions, cofactors, conformations and aggregates necessary for transfected with the rat KOR were exposed to varying concentrations
transmission remain to be defined. PrPSc and prion infectivity are present of U-50488, a KOR agonist, with or without naloxone, an opioid
within exosome preparations. Exosomes are 40-100 nm membrane receptor antagonist. Addition of U-50488 caused a naloxone-sensitive,
vesicles of endocytic origin released by most cell types in vitro and concentration-dependent increase in peak wavelength shift with an
recent studies have also identified them in vivo in body fluids. However, EC50 of 2.7 nM, consistent with literature values. Therefore BIND label-
due to limitations associated with standard sucrose density gradients it free technology appears to be an effective technique to assess activity
is not clear whether prion infectivity localises within exosomes or with of KOR agonists in cell-based assays, and may be a useful tool in the
an alternate, potentially unique vesicle. Recent developments in virus future to identify new KOR agonists.
purification techniques have enabled the separation of virus particles
(such as HIV-1 and influenza) from exosomes and other membrane
vesicles. This was previously unobtainable using standard sucrose
density gradients. We have demonstrated the application of a novel,
rate-zonal gradient separation technique to show that prion infectivity is
indeed associated with exosomes; a finding that is further validated with
biochemical techniques. A benefit of this technique is that it produces
highly purified exosomes without disturbing their native structure, making
them amenable to a variety of downstream analytical techniques. Using
this technique coupled with transmission electron microscopy we
have found that the ultra-structure of exosomes is far more intricate
than previously thought. This could potentially provide insight into the
currently unknown mechanism by which exosomes interact with cells.

POS-MON-75 POS-TUE-76
DYNEIN/COPII INTERACTION WITH MUTANT SOD1 IN ASCENDING GABAERGIC/PEPTIDERGIC CONTROL
SOD1G93A TRANSGENIC MICE AND NSC34 CELLS OF AROUSAL, STRESS AND MOTIVATED BEHAVIOUR:
FOCUS ON NUCLEUS INCERTUS AND RELAXIN-3
Farg M.1, Walker A.1, Turner B.2, Horne M.2 and Atkin J.1, 2 SIGNALLING
1
1 Department of Biochemistry, La Trobe University | Bundoora,
Victoria. 2Howard Florey Institute, University of Melbourne, Australia. Gundlach A.L.1, Ma S.1, 2, Smith C.M.1, Ryan P.J.1, Hossain M.A.1,
Wade J.D.1, Bathgate R.A.D.1, Verberne A.J.M.2, Blasiak A.3 and
Recent evidence suggests that disruption of the dynein/dynactin Olucha-Bordonau F.E.4
transport machinery occurs in ALS. Mutation in the dynactin subunit 1
Florey Neuroscience Institutes, The University of Melbourne,
p150 Glued causes human ALS and mice overexpressing mutant dynein Australia. 2Department of Medicine, Austin Health, The University of
heavy chain or Tgdynamitin mouse display slowly progressive motor Melbourne, Australia. 3Institute of Zoology, Jagiellonian University,
neuron degeneration. Also, mutant but not wildtype SOD1 physically Krakow, Poland. 4Department of Anatomy, University of València,
interacts with dynein and dynein co-localises with mutant SOD1 València, Spain.
inclusions. Impairment of dynein-dynactin function by p50 dynamitin over-
expression interferes with endosome trafficking and results in Golgi The nucleus incertus (NI) in the ventromedial pontine grey is a major
fragmentation. Fragmentation of the neuronal Golgi apparatus has source of ascending GABAergic projections that innervate limbic and
been observed in both sporadic and SOD-mediated familial ALS as hypothalamic regions involved in arousal, sleep/wakefulness and
well as in pre-symptomatic transgenic SOD1G93Aanimals. Bidirectional related autonomic and neuroendocrine functions. NI neurons and other
transport between the endoplasmic reticulum (ER) and Golgi apparatus small midbrain populations express the neuropeptide, relaxin-3; and
is mediated by the dynein transport machinery. Proteins exiting the existing anatomical and functional studies suggest relaxin-3 receptor
ER are transported in vesicles coated by coat protein complex II (RXFP3) signalling should modulate ‘behavioural state’, arousal and
(COPII). Recently there has been a surge of publications describing the responses to stress, and related behaviours. The precise nature of
importance of ER stress in ALS. We described induction of the whole these actions and the circuits and transmitters/peptides involved are
unfolded protein response in both transgenic SOD1G93A animals and in not known, however. We have conducted studies in rats and mice that
reveal: (i) NI neurons in the rat are activated by behavioural activity
human sporadic ALS patients. We hypothesised that disruption in ER to
and neurogenic stressors, and by CRF and orexin; (ii) relaxin-3 neurons
Golgi trafficking may contribute to ER stress in ALS. Anterograde and in rat and mouse innervate networks controlling circadian activity and
retrograde transport proteins were examined in transgenic SOD1G93A metabolic balance, and the emotional and cognitive circuits of the
mice and in NSC34 cell lines transfected with mutant and wt SOD1. We amygdala and septohippocampal system; (iii) central RXFP3 activation
demonstrated a physical interaction between mutant and not wildtype increases feeding in satiated rats; (iv) RXFP3 activation/inhibition in
SOD1 and both COPII and dynein in transgenic SOD1G93A animals at medial septum modulates spatial working memory and hippocampal
p10, 60 days before the onset of symptoms and before the onset of theta rhythm in the rat; (v) RXFP3 activation in central amygdala can
ER stress. This was confirmed; in NSC34 stable cell lines express block fear expression and may enhance fear extinction in rats; and (vi)
human SOD1 A4V/ G85R/ G37R and G93A. Also, COPII/ COPI found relaxin-3 knockout mice are hypoactive and have altered sleep patterns
to co-localise with mutant SOD1 inclusions. These data suggest that during the dark/active phase of the circadian cycle. These studies
alteration in dynein mediated transport is a very early event in disease have increased our knowledge of the role of the ascending NI GABA/
and preceeds ER stress in ALS. relaxin-3 network in homeostatic and complex behaviours and identified
its potential as a therapeutic target in neuropsychiatric disease.

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OXIDATIVE STRESS INDUCES MULTIPLE CASPASE- THE C-SRC PROTEIN TYROSINE KINASE IS
INDEPENDENT CELL DEATH PATHWAYS IN PRIMARY REGULATED BY CALPAIN MEDIATED CLEAVAGE
CORTICAL NEURONS UPON OVER STIMULATION OF GLUTAMATE
RECEPTOR IN CULTURED PRIMARY NEURONS
Higgins G.C.1, Devenish R.J.1, Beart P.M.2 and Nagley P.1
1
Department of Biochemistry and Molecular Biology, Monash Hossain M.I. and Cheng H.C.
University, Wellington Rd, Clayton 3800, Victoria, Australia. 2Florey Department of Biochemistry and Molecular Biology, Bio21 Institute,
Neuroscience Institutes, University of Melbourne, Parkville, Victoria University of Melbourne.
3010, Australia.
Glutamate-induced excitotoxicity is one of the main features of ischemic
Neuronal cells can undergo a diverse range of death responses. These stroke and neurodegenerative diseases. Excess glutamate causes over-
death outcomes were previously thought to be limited to either apoptosis stimulation of NMDA receptor leading to excessive influx of extracellular
(involving caspases and energy-dependent) or unregulated necrosis calcium ion into neurons. The excessive influx of calcium ion activates
(independent of both caspases and the need for ATP). We have recently calpains which cleave many functional proteins and in turns enhances
shown that primary cortical neurons from mice exposed to an acute insult neuronal death. c-Src, an important member of non-receptor Src family
of hydrogen peroxide (H2O2) undergo caspase-independent cell death protein kinases (SFKs), binds to scaffolding proteins in the NMDA
that is highly regulated, predominantly manifesting autophagic cell death receptor to form a complex to regulate activity of the receptor. In this way
and programmed necrosis. In this current work we show these neurons c-Src plays crucial role in glutamate-induced neuronal death. Herein we
undergo caspase-independent cell death when exposed to a chronic report that under glutamate-induced excitotoxicity, c-Src is cleaved by
dose of superoxide (provided by exogenous xanthine oxidase in the calpain in cultured primary cortical neurons. The same phenomenon
presence of catalase, which acts as a sink for H2O2). We found that while was also found when recombinant c-Src was digested with calpain
key mitochondrial intermembrane space proteins (cytochrome c, Smac, in vitro. Using an N-terminal directed c-Src antibody we mapped the
AIF and Endonuclease G) are redistributed to cytosol, downstream cleavage site to the N-terminal region of c-Src. We also monitored
caspases (-3, -7, -9) were not activated. While this outcome was similar kinase activity of the protein under excitotixic condition using specific
to that of our previous work done with H2O2, we found that knockdown Src optimal peptide and phosphospecific antibodies. Kinase activity
of either Endo G (programmed necrosis) or Atg7 (autophagic cell death) assay demonstrated that c-Src activity undergoes a biphasic change
using siRNA was much less potent in blocking cell death. We conclude – it increases shortly after glutamate treatment but decreases once the
that superoxide invokes a diverse death response that involves some calpain-mediated cleavage starts. In summary results of our studies
autophagic cell death and programmed necrosis, but is predominantly demonstrate the involvement of c-Src in excitotoxic neuronal death. We
unregulated necrosis. This work highlights the significance of the type demonstrate for the first time that calpain mediated cleavage of c-Src
of oxidative stress exposure, in relation to the specificity of the neuronal as regulatory mechanism in neurons. Further investigation on the effect
cell death outcomes. of calpain mediated cleavage of c-Src on its activity and regulation may
provide insights into the mechanism of excitotoxic neuronal death.

POS-MON-79 POS-TUE-80
INVESTIGATING THE ROLE OF COPPER IN THE THE EXPRESSION OF TNF AND ITS RECEPTORS IN
NERVOUS SYSTEM SCHIZOPHRENIA AND MOOD DISORDERS
Hwang E.C.J. and Burke R. Jeon W.J.1, 2 , Gibbons A.S.1, 2 and Dean B.1, 2
School of Biological Sciences, Monash University, Wellington Rd, 1
Rebecca L. Cooper Laboratories, the Mental Health Research
Clayton, VIC, 3800, Australia. Institute, Parkville, Victoria, Australia. 2Department of Psychiatry, the
University of Melbourne, Parkville, Victoria, Australia.
Copper is an important trace metal required in balanced amounts
for proper cellular function and development. Strict control of copper Abberant expression of cytokines involved in pro-inflammatory pathways
levels is required as copper dyshomeostasis results in multiple adverse have been proposed to underlie the pathology of schizophrenia and
effects, such as cellular damage due to the generation of reactive mood disorders. We have recently reported an increase in the level of
oxygen species. Proper copper homeostasis is achieved via the action tumor necrosis factor (TNF) protein in the dorsolateral prefrontal cortex
of copper transport proteins which serve to either import, sequester or (Brodmann’s area (BA) 46), but not the anterior cingulate cortex (BA 24),
export copper. While the overall role of copper in the body has been from subjects with major depression. We sought to determine whether
investigated quite well, less is known about the specific role of copper in the mRNA expression of TNF and its receptors is altered in the cortex
the nervous system. Previous studies have shown that copper is likely to of post-mortem subjects with schizophrenia and mood disorders. Real-
play a role in both the function and development of the nervous system, time PCR was used to measure the levels of TNF, TNFR1 and TNFR2
however, further study is require to elucidate the specific mechanisms mRNA in BA 24 and BA 46 from subjects with schizophrenia (n=20),
by which this occurs. Using Drosophila Melanogaster as a model, I have major depression (n=10), bipolar disorder (n=10) and matched control
created a system within which to study the effects of both copper excess subjects. The level of TNF mRNA was significantly increased in BA 24,
and scarcity on the nervous system. This has been achieved through but not BA 46, from subjects with schizophrenia compared to controls
the targeted misexpression of copper transport proteins in the nervous (p<0.05). The level of TNFR1 mRNA was increased in BA 24 (p<0.05)
system via use of the GAL4-UAS system, thereby generating an in vivo and BA 46 (p<0.0001) in subjects with schizophrenia. TNFR2 mRNA
model within which to study the effects of copper dyshomeostasis. I expression was not altered in schizophrenia (p>0.05). Furthermore, there
have shown that both copper excess and scarcity result in the disruption was no change in the mRNA levels of TNF and either receptor in BA 24
of nervous system function, and have investigated some of the and BA 46 from subjects with major depression and bipolar disorder
mechanisms by which this occurs. compared to controls (p>0.05). Our findings suggest that abnormal
TNF signalling may be involved in the pathology of schizophrenia.
Furthermore, the increased TNF protein expression in BA 46 that we
previously reported in subjects with major depression does not appear
to result from increased gene expression.

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TARGETED GENE DELIVERY TO MICROGLIA VIA THE TOWARDS ISOFORM-SELECTIVE DYNAMIN
SCAVENGER RECEPTOR CLASS B, TYPE I INHIBITORS
Malmevik J.M.K.1, Rogers M.-L.1, Nilsson M.2, Nakanishi Y.3, Rush R.A.1, Mariana A.1, Hill T.A.2, McGeachie A.B.1, Chau N.1, Daniel J.1, Gorgani N.N.1,
Sims N.R.1 and Muyderman H.1 McCluskey A.2 and Robinson P.J.1
1
Centre for Neuroscience, Flinders Medical Science and Technology, 1
Cell Signalling Unit, Children’s Medical Research Institute, The
Flinders University, Australia. 2Institute for Neuroscience, Goteborg University of Sydney, Westmead, NSW 2145, Australia. 2Department
University, Sweden. 3Faculty of Pharmaceutical Sciences, Kanazawa of Chemistry, University of Newcastle, NSW 2308, Australia.
University, Japan.
The GTPase enzyme dynamin I is predominant in brain; dynamin II is
Microglia are the major contributors to inflammatory responses in the ubiquitously expressed; and dynamin III is found in brain and testes.
brain. Investigations of microglia are limited by the difficulty of selectively Dynamin I and dynamin II are required for synaptic vesicle endocytosis
targeting these cells within the complex environment of the mature brain. (SVE) and receptor-mediated endocytosis (RME), respectively. Dynamin
Receptor-mediated gene delivery constitutes a non-viral approach II has a second function in the abscission that completes mitosis.
which also avoids immune responses that can be produced even by Therefore, dynamin I-specific inhibitors might be useful in diseases
highly modified viral vectors. In this study, we tested the possibility that involve SVE (epilepsy), while dynamin II-specific inhibitors might
of selectively targeting microglia in vivo utilising receptor-mediated be effective as anticancer drugs with minimal side effects. Our team
gene delivery via the scavenger receptor class B type I (SR-BI). SR- has developed various classes of dynamin inhibitors which have been
BI expression in mixed glial primary cultures and in purified microglial discovered using a malachite green dynamin I-based GTPase assay
cultures was confined to microglia. Incubations of an antibody targeted at (dynamin I being activated by liposomes) and dynamin II-mediated
the extra-cellular domain of SR-BI resulted in microglia-specific uptake. RME cell-based assay. We have now developed a dynamin II-based
This SR-BI antibody was linked to the polycation polyethylenimine GTPase assay, using dynamin II produced by transient expression
(PEI) and bound to a plasmid encoding green fluorescent protein in Sf9 insect cells, which retains high specific activity comparable to
(GFP) to form an ‘immunogene’. GFP expression was seen in microglia endogenous brain dynamin I, as well as dynamin I cell-based assay
following incubations of this construct in mixed glial primary cultures. using synaptosome. When we tested our classes of dynamin inhibitor
Intrahippocampal infusions of the ‘immunogene’ resulted in a substantial that have been published (MiTMAB, pyrimidynes) in our 4 assays, they
GFP expression in CD11b- and ED1-positive microglia around the appear to be non-selective (PAN inhibitors). We recently published new
infusion site (n=5). GFP-positive microglia were found up to 4.2 mm class of dynamin inhibitors (iminodyns). These drugs inhibit dynamin
from the site of infusion. No colocalisation with astrocytic or neuronal GTPase activity, as well as endocytosis. By utilising our 4 type of assays,
markers was found. Control infusions with PEI and the GFP plasmid the iminodyns do not show any isoform-selectivity for dynamin I or II
alone produced no expression of the reporter gene (n=3). This research GTPase activity. Despite this, the iminodyns include three nanomolar
demonstrates for the first time the use of a non-viral transfection system potent dynamin I and II inhibitors, which also block endocytosis.
to selectively target the microglial cell population in vitro and in vivo. Nevertheless, our screening system allows further development
towards isoform-selective dynamin inhibitors from the iminodyns as well
as other classes.

POS-MON-83 POS-TUE-84
CLINICAL SIGNIFICANCE OF URINE SURVIVIN MRNA IDENTIFYING THE ROLE OF SPECIFIC NON-
IN PROSTATE CANCER DETECTION CONSERVED RESIDUES ON PI3K IN THE DISCOVERY
OF ISOFORM-SELECTIVE PI3K INHIBITORS
Almaghrebi M., Kehinde E.O. and Kapila K.
Kuwait University - Faculty of Medicine. Amran S.I.
Monash Institute of Pharmaceutical Science, 381 Royal Parade, Vic
Survivin is one of the most tumor-specific molecules, which antagonizes 3052 Australia.
apoptosis and promotes tumor associated angiogenesis. Thus, it
comes to no surprise that it is overexpressed in many types of cancers. Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase that catalyzes the
In prostate cancer, PSA levels alone have a low overall efficiency of biosynthesis of PI(3)P, PI(3,4)P2 and PI(3,4,5)P3 - second messengers
accurate diagnosis. Survivin was evaluated as an alternative molecular that trigger a wide range of downstream signalling cascades involved in
marker in patient urine samples. Expression levels were measured by cell survival, growth, adhesion and proliferation. The heterodimeric class
quantitative reverse transcription-real time polymerase chain reaction 1 PI3K proteins are composed of a regulatory subunit (p85) complexed
(RT-PCR) and correlated with clinicopathological data in urine samples with either one of 4 different isoforms of catalytic subunit (p110α, p110β,
from patients with BPH, BPH & Prostatitis, and prostate cancer. Survivin p110δ and p110γ). The PI3KCA gene encoding the α-isoform has been
levels were significantly elevated in urine from prostate cancer patients found to be frequently mutated in cancers such as breast, prostate,
compared with healthy controls (P <.001) and with noncancerous colon, liver and brain. The overall aim of this program is to elucidate the
prostate disorders: BPH (P <.05) and BPH with Prostatitis (p <0.05). molecular mechanism of binding of isoform selective small molecule
An optimal cutoff value of 25 pg was determined. Accordingly, 21% of PI3K inhibitors. Previous studies have shown that specific regions within
patient samples had survivin levels above 25 pg, of which only 3% were the catalytic subunit contain non-conserved residues that contribute
healthy individuals. In contrast, 55% of individuals with survivin levels to isoform selectivity. In this study mutant P13Kα isoforms were
above 25 pg were prostate cancer patients. The results indicate that generated by swapping the residues of the α-isoform for corresponding
urine survivin levels are elevated during prostate cancer developmnt, residues of other isoforms using site-directed mutagenesis followed
demonstrating its strength as a potential marker for discriminating by recombination of the mutant sequences into the baculovirus vector
benign from malignant disease. system. The resultant proteins were expressed in insect cells and
purified. The purified enzymes have been characterised using western
blot analysis and kinase assays. With retained enzyme activity these
mutant isoforms can be used to identify the role of non-conserved
residues in inhibitor binding and contribute to rational design of novel
PI3K inhibitors.

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POS-MON-85 POS-TUE-86
EVASION STRATEGIES OF ECHINOCOCCUS ETHYLENE MEDIATED DEFENCE AGAINST THE ROOT
GRANULOSUS TO TH1 HOST PROTECTIVE RESPONSE INFECTING FUNGAL PATHOGEN RHIZOCTONIA
DURING HUMAN INFECTION SOLANI IN MEDICAGO TRUNCATULA
Amri M. and Touil-Boukoffa C. Anderson J.P.1, Lichtenzveig J.1, 2, Oliver R.P.2 and Singh K.1
Team: “Cytokines and NOSynthases”, Laboratory of Cellular and 1
CSIRO Plant Industry, Floreat, Western Australia. 2Australian Centre
Molecular Biology, Faculty of Biological Science, USTHB, PB 32, El- for Necrotrophic Fungal Pathogens, Murdoch, Western Australia.
Alia, 16111, Algiers, Algeria.
The fungal necrotrophic pathogen Rhizoctonia solani (Kühn) is a
Background: Human echinococcosis is one of the world’s major significant constraint to the production of a range of crops as diverse
zoonotic infections. It usually manifests as unilocular cyst(s) mainly as cereals, canola and legumes, including causing the second largest
located in the liver. More recently, we have highlighted an evident role disease constraint to rice production in China and India. No strong
of IFN-gamma (Th1 cytokine) in parasite killing by NOS2 (Nitric oxide genetic resistance has been identified in these crops despite wide
Synthase2) pathway. Moreover, IL-10 (Treg cytokine) production seems ranging primary and secondary germplasm screens, suggesting
to be an evasive mechanism taken by the parasite to establish in the alternative strategies to improve resistance are required. In this study
host by Arginase pathway (Amri et al., 2007 & 2009). Of note, NOS2 and we characterise moderate resistance to R. solani AG8 identified in
Arginase are known to compete for the common substrate, L-Arginine. the model legume Medicago truncatula. The activity of an ethylene
Moreover, IL-10 downregulates IFN-gamma production. Indeed, more and jasmonate responsive promoter element was associated with
researches are required to identify factors present in parasite cyst the moderate resistance as was the induction of specific ethylene/
which affect protective Th1 response in Echinococcus granulosus jasmonate response transcription factors. Over-expression of some
human infection. Method: We investigate the effect of laminated- of these transcription factors in transgenic Medicago “hairy roots”
layer (accelullar layer of hydatic cyst) extract (LLs) on Th1/Treg and increased resistance to R. solani as well as to another destructive root
NOS2/Arginase balance in culture performed with mononuclear cells pathogen, the oomycete Phytophthora medicaginis. Over-expression of
(PBMC) of hydatid patients and healthy donors. Furthermore, we have these genes had no apparent impact on symbiosis with nitrogen fixing
investigated the effect of LLs on parasite viability in PBMC-parasite bacteria as the transgenic roots formed root nodules at comparable rates
cocultures. Results: Our results demonstrated that LLs reduced IFN- to wild type. This suggests that enhanced resistance to root diseases
gamma/NO production and enhanced IL-10 production and Arginase can be uncoupled from symbiotic plant-microbe interactions in the same
activity. In addition, LLs enhanced parasite survival in vitro. Similar tissue and ethylene dependent control of nodule number is distinct from
findings are observed in cultures and cocultures performed with PBMC ethylene dependent defenses.
of patients and healthy donors. Moreover, the major antigenic fraction
in LLs: the fraction 4 (12kDa, purified by chromatography) has the same
effect as LLs. Conclusion: Collectively, the present study provides
evidence that Echinococcus granulosus laminated layer impairs Th1
protective response and allow the parasite to survive. Inhibition of these
mechanisms seems to be important issue to address during the design
of anti-hydatic treatment.

POS-MON-87 POS-TUE-88
ATRX IS A SERTOLI CELL SURVIVAL FACTOR POLYMORPHISM OF SOD2 GENE IN PATIENTS OF
AND REGULATOR OF SPERMATOGENESIS VIA BRONCHIAL ASTHMA
INTERACTION WITH ANDROGEN RECEPTOR
Bhadoria D.P.2 , Bhadoria K.3, Dutta K.2, Kumar M.1, Singh B.1, Singh
Bagheri-Fam S.1, Argentaro A.1, Svingen T.2, Combes A.2, Koopman P.2 S.1, Kumar R.1, Bhadoria P.2, Anand R.2 and Sharma G.L.1
and Harley V.1
1
Institute of Genomics and Integrative Biology, University Campus Mall
1
Prince Henry’s Institute of Medical Research, Melbourne, Victoria, Road, Delhi- 110007, India. 2Maulana Azad Medical College and Lok
Australia. 2Institute of Molecular Bioscience, Brisbane, Queensland, Nayak (Irwin) Hospital, New Delhi 110002, India. 3Graduate School of
Australia. Science, University of Melbourne, Victoria 3010 Australia.

The ATR-X syndrome, (α-thalassemia mental retardation, X-linked) is Superoxide dismutase (SOD2) is an antioxidant protein and
a developmental disorder affecting males. The testicular abnormalities polymorphism of its gene may contribute to susceptibility to bronchial
of ATR-X patients comprise small testes, few seminiferous tubules, and asthma. There are no studies on polymorphism in SOD2 gene from the
a lack of germ cells. ATRX protein remodels chromatin in vitro though Indian subcontinent. The present study was conducted to investigate
its bona fide target genes are unidentified. To understand the gonadal association between SOD2 gene polymorphisms and pathogenesis of
role of ATRX, we inactivated Atrx specifically in Sertoli cells (ScAtrxKO bronchial asthma in Indian patients. Fifty patients of asthma diagnosed
mice) from embryonic day 14.5. Fluorescence-based three-dimensional as per ATS guideline 1987 and 50 normal controls were included in this
modeling at E17.5 revealed that testis cord volume in ScAtrxKO mice is study for drawing blood. Genomic DNA was isolated from peripheral
~30% of wildtype, with discontinuous or isolated testis cords apparent. blood leucocytes and used for the amplification of SOD2 gene. PCR
While Sertoli cell apoptosis is increased by 10-fold when compared to product was subjected to sequencing by Applied Biosystems 3730 DNA
wildtype, no difference in Sertoli cell proliferation was observed. These sequence analyzer. The sequencing data thus obtained was analyzed
data suggest that ATRX is required for fetal Sertoli cell survival and, in using SISA and Epi Info statistical softwares. Screening of sequencing
turn, for elongation of fetal testis cords. Adult ScAtrxKO testes weigh data of the subjects in either group identified an intronic mutation at
~25% of wildtype suggesting that seminiferous tubule hypoplasia is base number 14788 (rs2842980. Twenty eight subjects of asthma (56%)
primarily established during fetal life. Histological examination showed and 9 from the control group (18%) were heterozygous (A/T) for this
that a third of tubules contain germ cells arrested in late meiosis or SNP. One patient as well as one control was found to be homozygous
at the round spermatid stage. We found that the Androgen Receptor mutant. Wild type allele T was present in 70% of asthmatics and 89%
(AR)-dependent genes, Rhox5 and Claudin3, were significantly down- of controls showing the role of A/T genotype susceptibility of asthmatic
regulated in Sertoli cells of ScAtrxKO testes. Moreover, we show that patients to the disease.The occurrence of T to A mutation at 14788 base
ATRX and AR proteins interact in the TM4 Sertoli cell line and co- position was higher in asthmatics than in controls (p<0.001) indicating
operatively activate the Rhox5 promoter. In summary, ATRX plays an its involvement in asthma and it could be an important risk factor the
important role in Sertoli cell survival during fetal testis development and disease.
in adult testis function, where ATRX protein interacts with AR to regulate
the transcription of AR-dependent genes that control spermatogenesis.

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POSTERS MONDAY & TUESDAY

POS-MON-89 POS-TUE-90
STATUS OF ANTIOXIDANTS IN BRONCHIAL ASTHMA EFFECT OF ANTIOXIDANTS SUPPLEMENTATIONS IN
SALT-INDUCED DYSLIPIDAEMIA IN ALBINO RATS
Bhadoria P.2 , Bhadoria K.3, Dutta K.2, Kumar M.1, Singh B.1, Singh S.1,
Kumar R.1, Bhadoria D.P.2, Anand R.2 and Sharma G.L.1 Bilbis L.S., Saidu Y., Ahmad K., Abbas A.Y., Baba A.U. and Shuaib A.M.
1
Institute of Genomics and Integrative Biology, University Campus Mall Usmanu Danfodiyo University Sokoto, Nigeria PMB 2346 Sokoto,
Road, Delhi- 110007, India. 2Maulana Azad Medical College and Lok Nigeria.
Nayak (Irwin) Hospital, New Delhi 110002, India. 3Graduate School of
Science, University of Melbourne, Victoria 3010 Australia. Cardiovascular disease (CVD) is associated with many risk factors
including oxidative stress and dyslipidaemia. The current work
There is an increased production of reactive oxygen species (ROS) evaluated the effects of antioxidants supplementation on salt-induced
by inflammatory cells in the airways of patients of bronchial asthma, dyslipidaemia in albino rats. Rats were grouped into 11 groups of 7 rats
resulting into oxidative stress. In order to combat and neutralize the each. Groups 2-11 were fed 8% salt diets for 4 weeks while group 1
deleterious effects of ROS, various endogenous antioxidant processes served as control and were fed normal rat feed. Water was provided
occur in the hosts involving enzymatic and non-enzymatic mechanisms. to all the groups ad libitum. The animals in groups 3-11 were then
The present study was undertaken to investigate the status of supplemented with vitamin A; vitamin C; vitamin E; Cu; Mn; Zn; vitamins
antioxidant markers in Indian asthmatic patients. Fifty asthmatics A,C and E combined; Cu, Mn and Zn combined; and all the vitamins and
diagnosed as per ATS guidelines (1987), and 50 normal controls were minerals combined respectively for additional 4 weeks simultaneously
recruited for drawing blood. Sera separated from whole blood was used with salt loading. Group 2 was not supplemented and served as the
for estimation of antioxidant markers namely, Superoxide dismutase negative control. The body weight changes, pulse rate of the rats were
(SOD), Catalase, Ascorbic acid and lipid peroxidation. Total SOD was monitored. Serum levels of Total cholesterol, triacylglyceride, low
estimated by the method of Kono et al. (1978). The activity of catalase density lipoprotein cholesterol, very low density lipoprotein cholesterol,
enzyme was assayed by the method of Beers and Sizer (1952). Ascorbic high density lipoprotein cholesterol and glucose were estimated. The
acid assay was performed by the method of Natelson el al. (1971). The results indicated that the vitamins reduced significantly serum lipid
assay for microsomal lipid peroxidation byproduct, Malondialdehyde, profiles and the atherogenic index by up to 80 %. The serum glucose
was performed by the method of Wright et al 1981. Results revealed that levels of the rats supplemented with antioxidant vitamins and minerals
there was marked decrease in total SOD (p<.01) and Catalase (p<.01) in were also significantly (P<0.05) lowered compared with the negative
asthmatics as compared to controls. However, there was no significant control group. These results suggest that the reduction of serum lipid
difference in ascorbic acid levels between patients and controls (p>.05). profile and glucose level may be due to regulation of cholesterol and
There was increased lipid peroxidation and antioxidant levels were found lipoprotein metabolism and increased insulin sensitivity as a result of
to be reduced in asthmatics. It seems that their level was not sufficient the supplementations. It may thus suggest that the antioxidants may
to scavenge free radicals produced during oxidative stress and tissue provide protection against CVDs and metabolic syndrome in salt
damage. induced dyslipidaemia in rats.

POS-MON-91 POS-TUE-92
TARGETING THE TROPOMYOSIN ISOFORM TM5NM1 INVESTIGATION OF MITOCHONDRIAL FUNCTION IN A
IMPAIRS TUMOUR SURVIVAL, PROLIFERATION AND METHYLMALONIC ACIDURIA MOUSE MODEL
MIGRATION
Buck N.E.1, Siddiqui T.1, Laskowski A.2, Frazier A.E.2, Pitt J.J.3,
Bonello T.1, Stehn J.1, Schevzov G.1, Coombes J.1, McCluskey A.2, Thorburn D.R.2, 3 and Peters H.L.1
Haass N.3, Dixon I.4 and Gunning P.1
1
Cell & Gene Therapy Group, Murdoch Childrens Research Institute
1
Department of Pharmacology, School of Medical Sciences, University and Department of Paediatrics, University of Melbourne, Royal
of New South Wales, NSW, Australia. 2School of Chemistry, University Children’s Hospital, Parkville, Victoria, Australia. 2Mitochondrial
of Newcastle, NSW, Australia. 3Centenary Institute of Cancer Medicine Research, Murdoch Childrens Research Institute and Department
and Cell Biology, University of Sydney, NSW, Australia. 4Genscreen of Paediatrics, University of Melbourne, Royal Children’s Hospital,
Pty Ltd, Melbourne, VIC, Australia. Parkville, Victoria, Australia. 3VCGS Pathology, Murdoch Childrens
Research Institute, Royal Children’s Hospital, Parkville, Victoria,
The actin cytoskeleton is a fundamental regulator of key cellular Australia.
functions including proliferation, motility and apoptosis. Aberrations Methylmalonic aciduria (MMA) is an organic acid disorder resulting from
in these processes are hallmarks of tumorigenesis, making the actin a defect in the mitochondrial enzyme methylmalonyl-CoA mutase. We
cytoskeleton an attractive chemotherapeutic target. The function of have developed an MMA mouse model that recapitulates key aspects
actin filament populations is regulated by their association with distinct of the intermediate form of MMA: elevated methylmalonic acid levels
tropomyosin isoforms. As cells transform they demonstrate an increased in urine, blood and various organs and survival into adulthood. We
reliance on a subset of low molecular weight (LMW) tropomyosins. The have investigated the impact of deficient methylmalonyl-CoA mutase
LMW isoform, TM5NM1, has been shown in over-expression and knock- on mitochondrial enzyme activities. Respiratory chain complexes (I, II,
down models to impart a proliferative advantage on the cell and regulate III, IV), citrate synthase and aconitase activities were assayed. There
directed cell movement. Objective: We have developed a novel class was no difference between Co II, aconitase and citrate synthase activity
of compounds designed to target TM5NM1 containing filaments, and between MMA and control mice, suggesting that the tissues were not
aim to characterise their effect on cellular function. Summary of affected by significant oxidative stress. Comparison of MMA to control
Results: The lead compound, TR100, reduced viability in a panel of mouse samples show reduced levels (up to 50%) of the complexes with
neuroblastoma and melanoma cell lines (average LC50 ~2-3μM) and subunits encoded by mitochondrial DNA (I, III and IV) in the liver and
inhibited survival and growth in a 3D melanoma model which simulates muscle, and Co III, IV in the brain (kidney enzyme activity unchanged).
the tumour microenvironment. To dissect the role of TR100 in cell Fluorescent microscopy showed no difference in the number and
death, FACS analysis was performed with the human melanoma cell distribution of mitochondria in kidney, muscle, lung or skin from control
line SK-N-MEL28. Cells treated with 5μM TR100 underwent G0/G1 and MMA mice. Mitochondrial DNA levels were determined using
cell cycle arrest, while programmed cell death was induced at higher real-time PCR by comparing Mtco1 to β-actin. The amount of mtDNA
concentrations of TR100. Finally, migration towards a directional cue, present in MMA mouse kidney, heart and muscle were 60±20% the
measured by in vitro scratch wound assay, was significantly impaired at amount present in controls. There was little change in the amount of
non-lethal concentrations of TR100. Conclusions: We have described mitochondrial DNA in MMA mouse liver and brain (85±23% of controls).
a novel class of chemotherapeutic compounds which specifically target These data support the concept that pathogenesis of MMA may be
an actin filament population required for tumour survival, proliferation mediated by an affect on expression of respiratory chain complexes
and migration. with subunits encoded by mitochondrial DNA.

OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 123


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POS-MON-93 POS-TUE-94
THE ROLE OF UBIQUITIN IN THE REPLICATION OF THE MITOCHONDRIAL CITRATE CARRIER IS
HEPATITIS C VIRUS REQUIRED FOR CHROMOSOME INTEGRITY IN
DROSOPHILA AND HUMAN CELLS
Cardeira S.S. and Bishop N.E.
Microbiology Department, La Trobe University, Bundoora, Vic, Carrisi C.1, Morciano P.2, Cestra G.3, De Benedetto G.E.1, Corona
Australia. D.F.V.4, Musio A.5, Cenci G.2 and Capobianco L.1
1
University of Salento. 2University of L’Aquila. 3Sapienza University.
Hepatitis C Virus (HCV) infects 170 million people worldwide. Its high 4
University of Palermo. 5ITB Pisa.
prevalence, rate of chronic infection and the major risk of developing
liver cirrhosis and hepatocellular carcinoma make hepatitis C a Chromosomal aberrations are key events in the initiation and progression
prominent global problem. The HCV genome encodes 10 proteins, of cancer. We isolated a P-element induced mutation in Drosophila that
four structural proteins -core, E1, E2 and p7- and six non-structural causes chromosome fragmentation. We named sea the gene specified
proteins -NS2, NS3, NS4a, NS4b, NS5a and NS5b. Current treatment by this mutation. The P-element is inserted into the 5’UTR of CG6782
for hepatitis C is effective in only approximately 50% of cases. By which encodes a protein orthologous to the mammalian citrate carrier
increasing our knowledge regarding the non-structural proteins of HCV, SLC25A1. It exports citrate from mitochondria supplying the cytosol
and their role in replication, new targets for antiviral treatments may with acetyl units. Sea shows biochemical properties similar to those of
be revealed. Ubiquitin is a 76 residue protein that becomes covalently SLC25A1 indicating a functional conservation of this carrier. We found
bound to specific lysine residues of target proteins. The ubiquitination of that Sea is reduced in mitochondria of sea mutants which also exhibit
proteins is a regulatory post-translational modification, much like protein a reduced citrate transport activity and low levels of citrate in cytosolic
phosphorylation. Ubiquitin is known to be important for the budding of extracts as revealed by LC/MS. Interestingly, western-blot of nuclear
many enveloped viruses from cellular membranes, a crucial step in viral protein extracts and immunostaining of polytene chromosomes with
replication. My work is focused on characterizing the role of ubiquitin anti-acetylated histone antibodies revealed a reduction of AcH3 and
in the replication of HCV. The NS5a protein has been implicated in AcH4 in sea mutants. Therefore, the chromosome breakage phenotype
establishment of persistent HCV infections by its ability to modulate observed in sea mutants seems to be a consequence of reduced levels
intracellular signalling pathways. For example NS5a expression inhibits of histone-acetylation. Notably, SLC25A1 siRNA-treated human primary
the degradation of epidermal growth factor receptor, a process highly fibroblasts exhibited a phenotype similar to Drosophila sea mutants and
dependent on ubiquitination, but the exact mechanism is unknown. low levels of AcH4. These results suggest an unexpected evolutionary
HCV core protein is also known to be ubiquitinated, which leads to its conserved role for Sea/SLC25A1 in the chromosome integrity providing
subsequent degradation. I am screening HCV proteins for interactions an intriguing link between cellular metabolism and epigenetics.
with proteins involved in cellular ubiquitination processes. I have
preliminary evidence for the direct interaction of ubiquitin with one of
the HCV non-structural proteins, and am investigating the wider role for
ubiquitin in HCV replication.

POS-MON-95 POS-TUE-96
DETECTION OF PROGRP-DERIVED PEPTIDES β-ARRESTIN1 PROMOTE CHRONIC MYELOCYTIC
IN HUMAN COLORECTAL CANCER CELLS AND LEUKEMIA PROGRESS DEPENDENT ON JNK PATHWAYS †
ASSESSMENT OF THEIR ROLE IN CELLULAR Zhang P.1, Long J.2, Li K.1, Liu H.2, Tan J.1, Tu Z.2 and Zou L.1
PROLIFERATION. 1
Center for Clinical Molecular Medicine, Children’s Hospital, Chongqing Medical
University, Chongqing 400014, China;. 2Department of Clinical Biochemistry
Patel O.1, Chang M.1, Nordlund M.2, Shulkes A.1 and Baldwin G.1 and the Key Laboratory of Laboratory Medical Diagnostics in the Ministry of
1
Department of Surgery, University of Melbourne, Austin Health, Education, Chongqing Medical University, Chongqing 400016, China.
Melbourne, Australia. 2Department of Medical Biochemistry, Arrestins (Arrs) are scaffold proteins consisting of four members: β-arrestin1
Radiumhospitalet, Rikshospitalet University Hospital, Oslo. (β-Arr1), β-arrestin2 (β-Arr2), x-arrestin, and s-arrestin. Only β-Arr1 and
β-Arr2 are ubiquitously expressed. The traditional functions of β-arrestins are
to mediate desensitization, sequestration, and recycling of G protein-coupled
Oneel Patel1, Mike Chang1, Marianne S. Nordlund2, Arthur Shulkes1 and receptors (GPCRs). Mounting evidence suggests that, in addition to regulation of
Graham Baldwin1. Background and aim: Amidated gastrin-releasing GPCR signals, β-arrestins also serve as modulators in a number of intracellular
peptide (GRP) is the prototypical autocrine growth factor. We have signaling pathways, including ERK, JNK and ASK1, which play important roles
previously demonstrated that non-amidated peptides derived from the in the regulation of various cellular functions in both normal and malignant cells.
C-terminus of proGRP are also biologically active in colorectal cancer Some reports have showed that β-Arrs participate in tumor-related signaling
(CRC) cell lines in vitro, via a receptor distinct from the GRP receptor pathways such as p53/MDM2, TGF-β1, IGF1R pathways, which function vitally
[1]
. This study investigates the quantities of proGRP-derived peptides as cell antiapoptosis, cell growth, and proliferation in tumor cells. We have
Methods: proGRP-derived peptides obtained from boiling water previously disclosed that rapid xenograft tumor progression in β-Arr1 transgenic
extracts of the human CRC cell lines DLD-1, SW1222, HCT 15, HT-29 mice by enhancing tumor angiogenesis and PI3K inhibitors suppressed the
and HCT 116 were quantitated by region-specific ELISA. Proliferation of β-Arr1-elevated MMP9 activity and VEGF. However, little is known about β-Arrs
function in leukemia, especially in chronic myelocytic leukemia (CML). Here
DLD-1 cells after reduction of proGRP-derived peptide concentrations we demonstrated that the expression of β-Arrs apparently increased in the
by transfection with proGRP shRNA was measured by [3H]-thymidine bone marrow (BM) and periphery blood (PB) samples from leukemia samples
incorporation. Results: In CRC cell extracts ELISA assays for proGRP- compared with benign hematological disease patients. However, there was
derived peptides containing residues 48-61, 56-88 or 48-88 detected no significant difference of β-Arr expressions among the diverse subtypes of
3-15, 10-60 and 20-152 fmol/106 cells, respectively. Little or no GRP18- leukemia (AML, ALL and CML) samples (P>0.05). But the relative expression
27amide or GRP1-27amide was detected. Stable transfection of DLD-1 of β-Arr1 was always higher than β-Arr2 in the same patient of leukemia. We
cells with proGRP shRNA significantly reduced propresent in a panel of further found that over-expression of β-Arr1 could promote leukemia cells to
5 CRC cell lines as well as the effect of such peptides on proliferation in proliferate, and vice versa in CML K562 cells in vitro. Moreover, K562 cells
vitro. liferation. Conclusions: These results indicate that non-amidated knocking out β-arrestin1could activate JNK signal pathways, and JNK inhibitors
peptides derived from the C-terminus of proGRP are present in CRC could block the JNK activation by β-arrestin1 in cells. The results from CML
xenograft mice further showed that the survival rate was much higher in K562-
cells and stimulate the proliferation of CRC cells in vitro. Such peptides siRNA-β-arrestin1 mice than in K562-β-arrestin1 mice, which was closely
are attractive targets for novel CRC therapies. Reference: 1. Patel O., related to JNK activation. Furthermore, JNK inhibitors could affect the tumor
Dumesny C., Shulkes A. and Baldwin G.S. (2007) C-terminal fragments growth of K562-siRNA-β-arrestin1 mice. In conclusion, β-arrestin1 could
of the gastrin-releasing peptide precursor stimulate cell proliferation via promote chronic myelocytic leukemia progression dependent on JNK signal
a novel receptor. Endocrinology. 148: 1330-1339. pathways, which disclosed the pathogenesis mechanism and signal pathways
of β-arrestin1 regulating CML. [† This wok was financial supported by Natural
Science Fund of China (30871103, 90919013), Chongqing Natural Science
Fund CQCBST 20082207 and 2008 New Century Excellent Talent Program
from Education Ministry of China.].
Page 124 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
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POS-MON-97 POS-TUE-98
THE SEROTONIN 5-HT4, RECEPTOR SPLICE VARIANTS EFFECTS OF HPV ONCOPROTEINS ON HLA-A2
INTERACTS WITH SPECIFIC PDZ DOMAIN PROTEINS PROMOTER IN CERVICAL CARCINOMA CELL LINE
VELI 1-3/LIN7A, B, C HOMOLOGUES: MECHANISMS IN
RECEPTOR TARGETING Mohd Habib S.H.1, 3, Abdullah A.1, 3, Othman S.1, 3, Karsani S.A.2, 3 and
Yusof R.1, 3
Chinkwo K.A., Coupar I.M. and Irving H.R. 1
Department of Molecular Medicine, Faculty of Medicine, University
Medicinal Chemistry and Drug Action, Monash Institute of of Malaya, 50603 Kuala Lumpur Malaysia. 2Biochemistry Division,
Pharmaceutical Sciences, Monash University, Parkville VIC 3052, Institute of Biological science, Faculty of Science, University
Australia. of Malaya, 50603 Kuala Lumpur, Malaysia. 3Drug Design and
Development Research Group, University of Malaya, 50603 Kuala
Several disorders of the gastrointestinal tract are associated with Lumpur, Malaysia.
abnormal serotonin metabolism and / or signalling such as irritable bowel
syndrome (IBS). The largest quantity of 5-HT4 receptors is present in Human papillomavirus (HPV) infection, particularly type 16, is
the intestine of mammals and have modulatory and prokinetic functions causally associated with the development of cervical cancer. The key
and form a clinical target for IBS. Alternative splicing results in 11 splice transforming proteins of high risk HPV are E6 and E7, which block
variants of the 5-HT4 receptor, most differing in their C-terminal (Coupar cell cycle exit in epithelial cells committed to differentiation, thereby
et al., 2007) and the 5-HT4 (a, d, e, f and g) receptor splice variants allowing viral replication. The major cellular target of E6 and E7 are
possess canonical type 1 or type 2 PDZ domains. Mouse 5-HT4a p53 and retinoblastoma cell proteins respectively, which play a pivotal
receptor interacts at the C-terminus with Veli3 (Joubert et al, 2004). The role in negative regulation of cell growth. E6 and E7 are constitutively
Veli or Lin7A, B or C homologue proteins are involved in post synaptic expressed in cervical cancer and their continuous expression is
vesicle function and localization (Jo et al., 1999). We hypothesise that required for maintenance of the transformed phenotype. The activities
the human 5-HT4 receptors containing canonical PDZ domains may of E6 and E7 are regulated by viral E2, a transcription regulator protein
interact with Lin7A, B or C homologues and that this interaction may be that is characteristically non-functioning in cancer cells. In this study
important in contributing to receptor function (including down-regulation) we are prospecting the ability of E2 in mitigating human MHC class 1
in the gastrointestinal tract. We have shown that the 5-HT4 a, b, c and d gene, specifically on HLA-A2 promoter region using a dual luciferase
transcripts are expressed relatively strongly in the human sigmoid colon assay system. We demonstrate that introduction of viral E2 via transient
and that the Lin7 A, B and C homologue transcripts are also expressed transfection can markedly up regulate the activity of HLA-A2 promoter
in the colon (Chetty et al, 2009). To follow up these observations, we are by as much as 40%. This can be taken as direct evidence that the E6/E7
investigating the interaction between 5-HT4a and 5-HT4d receptor splice are responsible for suppressing that activity of HLA-A2, as the ectopic
variants and Lin 7 homologues in COS-7 and colonic cell lines. Here we re-introduction of E2 would inhibit the expression of E6/E7.We are
report on the generation of N-terminal FLAG tagged 5-HT4 receptor and currently investigating the underlying mechanisms behind this uptrend
Lin 7 homologues with C-terminal V5, c-Myc and HA constructs and and the possible role of E2 in up-regulating the effect of interferon.
their expression in COS-7 cells. Protein interactions will be detected
using immunoprecipitation and immunofluorescence techniques will be
used to visualise cellular co-localisation and receptor recycling. Chetty
et al., 2009 Neurogastroenterol Motil 21:551–558.e15; Coupar et al.,
2007 Curr Neuropharmacol 5: 224-31; Jo et al., 1999 J Neuroscience
19:4186-4199; Joubert et al, 2004 J Cell Sci 117:536-5379.

POS-MON-99 POS-TUE-100
A MOLECULAR BASIS FOR THE DIFFERENTIAL BIOCHEMICAL CHARACTERIZATION OF
EXTRACELLULAR FUNCTION OF PAI-1 AND PAI-2 IN ABNORMAL FIBRINOGENS CAUSING HEREDITARY
CANCER DYSFIBRINOGENEMIA
Cochran B.J.1, Croucher D.R.2 and Ranson M.1 Kotlin R., Suttnar J. and Dyr J.E.
1
Illawarra Health and Medical Research Institute, University of Institute of Hematology and Blood Transfusion, U nemocnice 1, 128 20
Wollongong. 2Cancer Research Program, Garvan Institute of Medical Praha 2, Czech Republic.
Research.
Fibrinogen, a 340 kDa glycoprotein, consists of three different pairs of
Plasminogen activator inhibitors type-1 (PAI-1, SERPINE1) and type-2 polypeptide chains (Aα, Bβ, and γ) each encoded by a distinct gene
(PAI-2, SERPINB2) are potent irreversible inhibitors of the urokinase- (FGA, FGB, and FGG), clustered on chromosome 4q32.1. The molecule
type plasminogen activator (uPA). The strong prognostic value of tumour is posttranslationally modified - phosphorylated, glycosylated and
expression of uPA and PAI-1 in metastasis and poor patient outcome is sulphated. Fibrinogen is synthesized by hepatocytes and is secreted
well established. Conversely, tumour expression of PAI-2 is associated to the circulation, where it plays a crucial role in the hemostasis,
with favourable outcome and increased relapse free survival. We angiogenesis, platelet aggregation, cell migration and inflammation.
have previously demonstrated that the interaction between uPA:PAI-2 Inherited defects in fibrinogen are very rare and may cause life
and receptors of the low density lipoprotein receptor (LDLR) family is threatening complications like thromboses or serious bleeding.
markedly different to that of uPA:PAI-1, offering a molecular basis for Dysfibrinogenemia is a disease characterized by inherited abnormality
the contrasting prognostic data. Herein, we validate this hypothesis by in the fibrinogen molecule, resulting in functional defects. We have
replacing residues of PAI-2 with those corresponding to LDLR binding biochemically characterized five new cases of dysfibrinogenemia with
sites in PAI-1, thereby introducing LDLR binding. This has profound abnormal coagulation test results. Genomic DNA was amplified by PCR
impacts on the physiological functionality of PAI-2; not only increasing and dideoxysequencing was performed. Functional examinations were
the clearance rate of uPA from the cell surface, but altering non- carried out using fibrin polymerization, fibrinolysis, and measurement
proteolytic functions via the activation of pro-mitogenic cell signalling of kinetics of fibrinopeptide release. Abnormal fibrinogens were studied
pathways. This work provides the first definitive explanation of what, on by SDS-PAGE and mass spectroscopy. We have found two unrelated
the surface, appears to be paradoxical biological functionalities. families with substitution Aα Arg16Cys and two unrelated families with
substitution Aα Arg16His. Mutant chains were found to be present in
the patient plasmas; abnormal fibrinopeptides release and impaired
fibrin polymerization were obtained. Mutations are situated at the site
of fibrinopeptide release catalyzed by thrombin. The last case is a novel
mutation γ Tyr363Asn impairing fibrin polymerization. Tyr 363 is situated
in the fibrinogen polymerization site and substitution γ 363Asn changes
the conformation of the interacting site. This work was supported by
a grant of the IGA of the Ministry of Health, nr. NS 9636-3/2008; by
a grant of The Ministry of Health, nr. 2373601; and by a grant of The
Academy of Sciences of the Czech Republic, nr. KAN200670701.

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POS-MON-101 POS-TUE-102
REDUCED FERROPORTIN EXPRESSION EXPLAINS CONTRIBUTION OF METABOLIC PERTURBATIONS
WHY INTESTINAL IRON ABSORPTION IN THE TO THE NEURODEGENERATIVE EVOLUTION OF
NEONATAL RAT IS HYPORESPONSIVE TO STIMULI ALZHEIMER’S DISEASE
THAT REDUCE ABSORPTION IN ADULTS
Daulatzai M.A.
University of Melbourne, Parkville, Vic 3010, Australia.
Darshan D., Wilkins S.J., Frazer D.M. and Anderson G.J.
Queensland Institute of Medical Research, Brisbane, Australia.
Mak Adam Daulatzai Sleep Disorders Group/EEE, University of
Melbourne, Victoria, Australia Alzheimer’s disease (AD) is relentlessly
Neonatal mammals have a particularly high rate of intestinal iron
progressive with significant burden on medical and financial resources.
absorption to ensure an adequate supply of iron for growth. In adults,
Our longevity is increasing and aging is the most important risk factor.
iron absorption responds to a range of systemic signals, but whether the
AD is diagnosed only when considerable brain damage is done. The goal
neonatal gut is similarly responsive remains unclear. We thus examined
of this paper is to address important senescent stigmata, and highlight
in rats whether elevated body iron (i.p. iron dextran; 0.3mg/g) and
metabolic disturbances that exacerbate AD neuropathogenesis. The
inflammation (LPS; 100ng/g), treatments that reduce iron absorption
latter is correlated with cholinergic hypofunction and neuronal losses in
in adults, have similar effects in neonates. Gene expression was
several key neocortical areas including hippocampus, basal forebrain
measured by qPCR and protein levels by western blotting. Intestinal iron
(BFB), entorhinal cortex (ERC), posterior cingulate cortex (PCC), and
absorption was determined by measuring the retention of an oral dose
retrosplenial parietal cortex (RPC) to name a few. These affected
of 59Fe. Neonatal rats (15 days old) absorbed 93.1±2.0% of an oral iron
regions possess amyloid plaques and neurofibrillary tangles (NFT).
dose compared to 39.1±2.6% in adults (7 weeks old). In response to iron
Neuroimaging has shown significant hypoperfusion of BFB and several
dextran and LPS, iron absorption in adults decreased by 53% and 72%
key brain regions in old rats and elderly humans. These perfusion
respectively, whereas the corresponding reductions in suckling animals
decreases occur prior to substantial brain atrophy in AD. Furthermore,
were only 14% and 9%. Despite this, expression of the iron-regulatory
there is significant evidence of decreased cerebral glucose metabolic
hormone hepcidin was strongly induced by both iron dextran and LPS
rate (CMRglc) in parietal and temporal cortices also years prior to clinical
at both ages. The hepcidin target, ferroportin (Fpn), was expressed at
AD diagnosis. Most consistently documented CMRglc hypometabolism
the mRNA level but not at the protein level in the duodenum of suckling
occurs in parietal cortex, particularly the most vulnerable RPC.
animals up to 17 days of age. Expression was robust in adults. The
Respiration is essential to life. Upper airway (UA) patency is due to
Fpn1A splice variant, which is under iron-dependent translational
motor activity of dilator muscles, particularly genioglossus innervated
control, predominated in neonates. In conclusion, iron absorption in
by hypoglossal nerve. Nocturnal hypofunction of genioglossus results
the neonate is largely refractory to systemic signals. This reflects the
in variable collapse of UA. Almost all elderly suffer from some kind of
limited expression of Fpn protein in the small intestine at this time and
sleep-disordered breathing condition including obstructive sleep apnoea
the dominance of an iron-dependent Fpn splice variant. This adaptation
which has reached an epidemic proportion. There is well documented
helps to ensure maximal iron supply to the growing pup.
involvement of brain stem nuclei including reticular, solitarius, ambiguous,
motor nucleus of vagus, and hypoglossal, in ventilatory regulation,
genioglossus function and UA patency. Hypofunction/atrophy of the
above nuclei in elderly, results in UA collapse, hypoxia, hypoxemia,
depressed CMRglc, and enhancement of AD neuropathology. Significant
data would be presented to substantiate this thesis.

POS-MON-103 POS-TUE-104
APOE POLYMORPHISM IN CORONARY HEART
DISEASE PATIENTS FROM PUNJAB, INDIA EFFECTS OF RHO GTPASES IN METASTASIS
Dinsdale B., Li S.F. and Parish R.W.
Dhiman S.R.1, Khanna D.2 and Balgir P.P.2 LaTrobe University.
1
Department of Human Biology, Punjabi University, Patiala-
147002,Punjab, India. 2Department of Biotechnology, Punjabi
University, Patiala-147002, India. Transition from a primary carcinoma to secondary tumours result in
higher mortality rates. The spread of cancer, is primarily dependant on
cell migration, cell invasion and metastasis. DNA arrays have defined
Although there is a decline in several forms of infectious diseases with a pattern of gene expression that correlates with the progression to
a general improvement in the health and socio-economic status, there metastasis, and among the genes found to play a role is a member
is an increase in various diseases like, hypertension, stroke, diabetes of the RAS-superfamily of small guanosine triphosphates (GTPases),
and coronary heart disease due to the changes in life style and dietary RhoC. RhoC is a known regulator of cellular migration through its effects
habits. Coronary Heart Disease (CHD) contributes significantly to the on the cytoskeleton and cellular adhesion. Changes in expression
morbidity and mortality in many countries and is becoming the leading and activation of RhoC can result in a metastatic phenotype. RhoC is
cause of death in developing countries too. It has been shown that there over-expressed in metastatic cells of many cancers, and as such we
is a high prevalence of CHD, in both, urban and rural populations of are interested in varying expression in three sublines from a murine
India.In the present study, Apolipoprotein E genotypes were determined mammary model (derived from spontaneously arising tumour in a
by Polymerase chain reaction at SNPs rs7412 and rs 429358 amongst Balbc/cfC3 mouse) that demonstrate varied metastatic potential. By
152 Coronary Heart Disease (CHD) patients and compared with ectopically expressing RhoC in different sublines, we have observed
300 normal healthy individuals (Controls) from North Western Indian distinct morphological changes that are indicative of a significant shift
population of Punjab, India. All the three ApoE alleles - ApoE2, ApoE3 in metastatic potential. We have carried out Matrigel invasion assays
and ApoE4 were observed in the CHD patients and normal controls. to further characterise these transgenic lines, in conjunction with
The frequency of ApoE3 allele was found to be the highest, followed by creating RhoC loss-of-function mutants to further investigate its role in
ApoE4 and ApoE2 alleles, both, in the CHD patient, as well as, in the metastasis.
Control series. Chi-square analysis reveals statistically non-significant
differences between the patient and control series (Chi-square =1.435,
d.f. = 2, P = 0.4879). ApoE being a prominent mediator in cholesterol
homeostasis pathway has a significant influence on the plasma lipid
levels. The influence of ApoE genotypes on plasma lipid and lipoprotein
levels will be discussed.

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POS-MON-105 POS-TUE-106
SEC14-DEPENDENT SECRETION OF THE FUNGAL STEAROYL-COA DESATURASE 1 DEFICIENCY
INVASIN, PHOSPHOLIPASE B1, IS ESSENTIAL FOR REDUCES APOPTOSIS IN THE HEART OF OBESE OB/
PATHOGENICITY OF CRYPTOCOCCUS NEOFORMANS OB MICE
Djordjevic J.T.1, 2, Chayakulkeeree M.1, 2, 3, Johnston S.4, Bijosono Oei J.1, 2, Dobrzyn P.1, Pyrkowska A.1, Miyazaki M.2, Ntambi J.M.3 and Dobrzyn A.1
May R.4, Williamson P.R.5 and Sorrell T.C.1, 2 1
Laboratory of Cell Signaling and Metabolic Disorders, Nencki
1
Faculty of Medicine, University of Sydney. 2Centre for Infectious Diseases Institute of Experimental Biology, Warsaw, Poland. 2Divisions of
and Microbiology, Westmead Millennium Institute, Westmead Hospital, Renal Diseases and Hypertension & Endocrinology, Metabolism and
Sydney. 3Faculty of Medicine, Siriraj Hospital, Mahidol University, Diabetes, University of Colorado, Denver, CO, USA. 3Departments
Bangkok, Thailand. 4School of Biosciences, University of Birmingham, of Biochemistry and Nutritional Sciences, University of Wisconsin-
Edgbaston, Birmingham, United Kingdom. 5National Institute of Allergy Madison, Madison, WI, USA.
and Infectious Diseases, NIH Bethesda, MD, USA.
Rationale: Stearoyl-CoA desaturase (SCD), the rate limiting enzyme
The secretory pathway of the AIDS-related pathogen, Cryptococcus in the synthesis of monounsaturated fatty acids, was recently shown
neoformans, exports phospholipase B1 (Plb1), an enzyme essential to be involved in regulation of cardiac substrate utilization. Objective:
for fungal invasion and dissemination throughout the host. Using gene In the present study, using ob/ob;SCD1-/- mouse model, we tested the
deletion analysis, secretion of Plb1, but not other major virulence hypothesis that lack of SCD1 could improve steatosis and left ventricle
determinants, is shown to be dependent on CnSEC14-1, which encodes function in leptin deficiency. Methods and Results: We show that
a putative phosphatidylinositol transfer protein (PITP). Similar to the disruption of SCD1 gene significantly improves cardiac function in ob/
Plb1 deletion mutant (CnΔplb1), the Plb1 secretion-deficient CnSEC14-1 ob mice by correcting systolic and diastolic dysfunction and decreasing
deletion mutant (CnΔsec14-1) displayed reduced lung and brain left ventricle mass and diameter. The improvement is associated with
burdens and attenuated pathogenicity in an animal model. Furthermore, reduced expression of genes involved in fatty acids (FA) transport and
expulsion of live C. neoformans from macrophages was attenuated lipid synthesis, and reduction in cardiac free FA (by 39%), diacylglycerol
in both Plb1 secretion-deficient strains. CnSEC14-1 restored high- (by 20%), TG (by 33%), and ceramide (by 34%) levels. The rate of FA
temperature growth to an S. cerevisiae SEC14TS mutant confirming beta-oxidation is also significantly lower in the heart of ob/ob;SCD1-/-
it to be the orthologue of the secretion-essential Golgi PITP-encoding mice compared to ob/ob controls. Moreover, SCD1 deficiency reduces
gene, ScSEC14. Deletion of CnSEC14-2, a closely-related homologue cardiac apoptosis in ob/ob mice due to increased expression of
of CnSEC14-1, and CnSFH5, the more distantly-related SEC fourteen- antiapoptotic factor Bcl-2 and inhibition of inducible nitric oxide synthase
like homologue, singly or in combination (CnΔsec14-2, CnΔsfh5 and and caspase-3 activities. Conclusions: Reduction in myocardial
CnΔsec14-2/CnΔsfh5, respectively), abrogated neither Plb1 secretion nor lipid accumulation and inhibition of apoptosis appear to be the main
pathogenicity. However, Plb1 secretion and pathogenicity were restored mechanism responsible for improved left ventricle function in ob/ob
in CnΔsec14-1 following genetic reconstitution with either CnSEC14-1 mice caused by SCD1 deficiency. These results suggest that inhibition
or CnSEC14-2, despite CnΔSEC14-2 retaining a wildtype phenotype, of SCD could be a potential therapeutic strategy for the treatment of
confirming that CnSEC14-2 over-expression functionally compensates obesity-related cardiomyopathies. This work was supported by Polish
for CnSEC14-1. C. neoformans therefore uses distinct mechanisms to Ministry of Science and Higher Education grant N N301 0129 33.
export virulence determinants, with CnSEC14-1 constituting part of a
Plb1-specific secretion pathway essential for pathogenicity.

POS-MON-107 POS-TUE-108
DOES DYSFERLIN DO MORE THAN JUST REPAIR ABILITY OF α-CASEIN IN PREVENTING OF AMYLOID
MEMBRANES? FIBRIL FORMATION OF STRESSED α-LACTALBUMIN
IN CROWDED SYSTEM
Evesson F.J.1, 2 , Peat R.A.1, Lek A.1, 2, Lemckert F.A.1, 2, North K.N.1, 2
and Cooper S.T.1, 2 Ghahghaei A. and Faridi N.
1
Institute for Neuroscience and Muscle Research, The Children’s Department of Biology, Faculty of Science, University of Sistan and
Hospital at Westmead, Westmead, 2145, NSW, Australia. 2Discipline of Baluchestan, Zahedan, Iran.
Pediatrics and Child Health, The University of Sydney, Sydney, 2000,
NSW, Australia. Amyloid fibrils arise from the slow aggregation of intermediately folded
protein states. In this study the kinetics of the protein fibril formation
Dysferlin is a muscle membrane protein which is mutated in a form of of α-lactalbumin and its prevention by αs-casein in the presence and
late onset muscular dystrophy. Dysferlin belongs to the ferlin family of absence of crowding agent, dextran (68 kDa) have been compared
proteins which have ancient origins in eukaryotic biology, and emerging by thioflavin T binding assay. It was found that αs-casein, a molecular
roles in a variety of vital cellular processes. Deficiency of dysferlin is chaperone found in bovine milk, is a potent in vitro inhibitor of
characterised by an impairment of rapid calcium-activated resealing of α-lactalbumin fibrillization. The effect of αs-casein in preventing fibril
damaged muscle fibres, although its precise role in this process remains formation was significant, although reduced in comparison with the
poorly understood. We have created a panel of dysferlin expression absence of crowding agent, dextran. Interaction between chaperone
constructs bearing domain deletions or targeted patient mutations for and α-lactalbumin and structural change in target protein are also
use in cell biology assays studying protein lifetime, plasma membrane indicated by intrinsic fluorescence intensity, ANS binding assay, CD
expression and trafficking. We have used co-expression models and co- spectroscopy and size-exclusion HPLC. In summary, α-casein interact
immunoprecipitation to study associations and interactions of dysferlin with α-lactalbumin and prevent amyloid formation but not as well as in
with other proteins involved in cellular trafficking and membrane repair. the presence of crowding agent, dextran.
Our studies demonstrate that dysferlin is a dynamic transmembrane
protein, showing relatively transient expression at the plasma membrane
of cultured muscle cells (half-life of ~ 3 hours). This plasma membrane
expression is attenuated in domain deletion mutants, and in several
patient missense mutants, resulting in a significant acceleration of
endocytosis and protein degradation. Our efforts are now focused on
defining the constituents and cargo of dysferlin’s endocytic pathway.
Recent results demonstrate that syntaxin-4 demarks the dysferlin
endocytic route. We are now defining the relationship between dysferlin
and syntaxin-4, with potential relevance to the regulated exocytosis of
membrane resealing, or the altered immune function often observed in
patients with dysferlin muscular dystrophy.

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POS-MON-109 POS-TUE-110
A HIGH-THROUGHPUT METHOD FOR THE DETECTION UNTANGLING THE WEB OF HEPATITIS C VIRUS NON-
OF HOMOEOLOGOUS GENE DELETIONS IN STRUCTURAL PROTEIN 4B FUNCTION
HEXAPLOID WHEAT
Fitzsimmons N.M. and Bishop N.E.
Fitzgerald T.L.1, Kazan K.1, Li Z.2, 3, Morell M.K.2, 3 and Manners J.M.1 La Trobe University, Bundoora.
1
CSIRO Plant Industry, 306 Carmody Road St Lucia QLD 4067
Australia. 2CSIRO Plant Industry, GPO Box 1600, Canberra ACT 2601, Hepatitis C virus (HCV) causes chronic hepatitis, liver cirrhosis and/or
Australia. 3CSIRO Food Futures National Research Flagship, PO Box hepatocellular carcinoma, in an estimated three percent of the human
93, North Ryde 1670, NSW, Australia. population. HCV is a single-stranded, positive-sense RNA virus,
classified in its own genus Hepacivirus within the Flaviviridae family.
Plant lines featuring full or partial gene deletions provide a useful resource The virus encodes ten proteins: Core, E1, E2, p7, NS2, NS3, NS4A,
for the study of gene function by reverse genetics and also have the NS4B, NS5A, and NS5B. The Core, E1 and E2 proteins are structural
potential to be exploited for crop improvement. The use of deletion lines proteins, while the remainders are non-structural proteins. My project
in polyploid plant species has been impeded by the presence of multiple focuses on the role of the non-structural protein NS4B, which has a
copies of homoeologous genes corresponding to each of the ancestral poorly-characterized role in viral replication. A number of positive-
genomes. Such homoeologous gene copies usually show high DNA sense RNA viruses replicate their genome on the surface of intracellular
sequence homology, and may have either redundant or homoeologue- membranes. Some viruses also rearrange the intracellular membranes.
specific functions. Hexaploid (‘bread’) wheat (Triticum aestivum) is an The purpose of rearranging membranes is not fully understood however
economically significant polyploid crop species, and is an allopolyploid some hypotheses are that they could be used as a physical support
possessing three progenitor genomes designated as ‘A’, ‘B’, and ‘D’. for the viral replication complex, allow compartmentalization of viral
To facilitate the screening for specific homoeologous gene deletions in products, may permit viral RNA tethering, or perhaps protect the RNA
hexaploid wheat, we have developed a TaqMan qPCR-based method from host defenses. For HCV these rearranged membranes are termed
that allows high-throughput and large-scale detection of homoeologous membranous webs or membrane-associated foci (MAFs). Evidence to
copies in any gene of interest. To test the system, we chose the date suggests NS4B plays a role in formation of these MAFs. Current
TaPFT1 gene, an orthologue of the disease susceptibility gene PFT1 in data indicate the membranes rearranged by HCV are derived from both
Arabidopsis, where mutants lacking a functional PFT1 gene have been the endoplasmic reticulum and early endosomes. However, both the
shown to possess increased resistance to a Fusarium pathogen. We molecular mechanisms involved, and the impact on host cell function
applied this method to the screening of 4500 M2 mutant wheat lines due to the MAF formation, is poorly characterized. I will present my
generated by Heavy Ion Irradiation in order to identify knockouts of each latest data on the characteristics and formation of HCV-induced MAFs,
homoeologous copy of TaPFT1. We detected multiple lines featuring and data on the impact of the membrane rearrangements on normal
deletions of each TaPFT1 homoeologue, and confirmed these deletions host cell function. My results are based on findings in cultured cells over-
using a CAPS method. Mutants in TaPFT1 and other wheat disease expressing HCV NS4B.
susceptibility genes are now being further developed for phenotypic
testing for potential changes in response to fungal pathogens.

POS-MON-111 POS-TUE-112
INDUCED SPLICE-SWITCHING TO STUDY DYSTROPHIN MODULATION OF MHC CLASS I GENE EXPRESSION
ISOFORM FUNCTION AND EXPRESSION BY DENGUE VIRUS
Fletcher S., Adams A., Greer K., Johnsen R.J. and Wilton S.D. Othman S., Abdul Rahman N. and Yusof R.
University of Western Australia, 35 Stirling Highway, Crawley WA Drug Design and Development Research Group, University of Malaya,
6009. 50603 Kuala Lumpur, Malaysia.

Duchenne muscular dystrophy (DMD) is an X-linked, relentlessly Mosquito-borne dengue fever (DF) and dengue haemorrhagic fever
progressive muscle wasting disorder with a predictable course and (DHF) are caused by dengue viruses belonging to the Flaviviridae family
fatal outcome. DMD is caused by mutations in the dystrophin gene that that comprised four closely related but antigenically distinct serotypes,
prematurely terminate the protein, and affects 1 in 3500 male births. DV1 to DV4. There are over 100 million cases of dengue infections, half
Although treatment options have been limited, advances in clinical care a million cases of DHF and 24000 deaths worldwide every year, with
have doubled the life expectancy of affected boys over the last 2-3 more than 2.5 billion people at risk for endemic transmission. Despite
decades, but do not address the primary etiology of DMD, the absence aggressive efforts in dengue research, the control of dengue diseases
of dystrophin. Biological therapeutics are now becoming available, and awaits complete elucidation of its complex immunopathogenesis,
antisense oligomer-mediated splice manipulation to exclude selected many theories of which showed participation of both vector and host
exons can overcome disease-causing mutations and restore dystrophin responses, involving both innate and adaptive components of the
expression. We show that peptide-conjugated phosphorodiamidate human immune response. In the adaptive immune response, the
morpholino oligomer induced splice-switching can also be used to alter major histocompatibility complex (MHC) class I molecules are critical
isoform expression, or induce a frame-shift and prevent translation for antigen-specific immune recognition, expressed on the surface
of functional products. Exclusion of selected exon blocks to yield in- membrane of virtually all mammalian cells to display fragmented pieces
frame transcripts can allow mapping of functional protein domains, of foreign antigen. In contrast to many viruses that escape this host
based upon exon boundaries, and permits physiological evaluation of response by suppressing the MHC Class I pathway, infection by other
dystrophin isoforms to aid in the design of splice-manipulation therapies. Flaviviruses has been shown to up-regulate the cell surface expression
It is also possible to efficiently disrupt the normal dystrophin mRNA of MHC Class I complex. In dengue infection, the mechanisms by
reading frame and ablate dystrophin expression in wild-type mouse which the virus regulates the MHC Class I remain elusive. Hence, this
muscle. Total suppression of dystrophin gene expression was induced study elucidates comprehensively and systematically the effect of all
by selected exon removal and maintained for several weeks in vivo, serotypes of dengue virus infection on the expression and regulation of
resulting in severe dystrophic pathology in diaphragm within 4 weeks transcriptional activities of the human MHC class I HLA-A2 gene. This
of commencing treatment in wild-type neonatal mice. Manipulating adds further understanding to the role of dengue virus in manipulating
expression through altered splicing patterns could be applied to many the MHC antigen presentation pathway in this immune-pathogenic
different genes, offering the opportunity to induce transient mouse disease that has been posing great economic and social burden on
models to study the consequences of gene suppression or isoform affected many nations, Malaysians included.
selection in vivo.

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POS-MON-113 POS-TUE-114
D-AMINO ACID OXIDASE: PATHOPHYSIOLOGICAL IRON HOMEOSTASIS IN WHOLE BODY AND
BASIS AND MOLECULAR TARGET FOR INTESTINE-SPECIFIC HEPHAESTIN KNOCKOUT MICE
SCHIZOPHRENIA
Fuqua B.K.1, Wolkow N.2, Bell A.1, Hsu J.1, Nguyen M.P.1, Yu C.C.1,
Fukui K., Ono K., Abou El-Magd R.M., Iwana S., Kawazoe T., Chung S.P., Dunaief J.L.2, Anderson G.J.3 and Vulpe C.D.1
Song Y., Shishido Y., Yorita K. and Sakai T.
1
Department of Nutritional Science and Toxicology, University
Division of Enzyme Pathophysiology, The Institute for Enzyme of California, Berkeley, USA. 2FM Kirby Center for Molecular
Research, The University of Tokushima, Tokushima, JAPAN 770-8503. Ophthalmology, Scheie Eye Institute, University of Pennsylvania, USA.
3
Queensland Institute of Medical Research, Brisbane, Australia.
D-Amino acid oxidase (DAO) has been proposed to be involved in
the decreased glutamatergic neurotransmission in schizophrenia and Hephaestin (Heph) is a vertebrate multicopper ferroxidase (MCF)
the modulation of the enzyme activity is expected to be therapeutical. important for the transfer of dietary iron from intestinal cells to the
Here we show the inhibitory effect of a classical antipsychotic drug, blood. The gene is mutated in the sex-linked anaemia (sla) mouse and
chlorpromazine, and an atypical drug, risperidone, on human DAO results in severe iron deficiency. However, sla mice still retain some
activity. As for chlorpromazine, human DAO was inhibited with Ki hephaestin ferroxidase activity. They survive, breed and their anaemia
value of 0.7 mM. We also found that the oligomerized chlorpromazine improves with age. Heph is also expressed at low levels in other tissues,
produced by irradiation, showed an enhanced inhibition (Ki =7 μM). including the brain, pancreas, and placenta, but its role in these tissues
Risperidone was also shown to have a hyperbolic mixed-type inhibition is less clear. To gain a better understanding of the role of Heph in iron
with Ki value of 41 μM. The effect of risperidone was then tested using homeostasis, we used the cre-lox system to generate knockout mouse
rat C6 cells and stable C6 transformant over-expressing mouse DAO models with whole body and intestine-specific (villin promoter) ablation
(designated as C6/DAO) as well as pig kidney epithelial cell line (LLC- of Heph. Both types of mice are small, yet viable, indicating that Heph
PK1). Risperidone exhibited a protective effect from D-amino acid is not essential and that other mechanisms exist, MCF-dependent or
and oxidative stress induced cell death in both C6/DAO and LLC-PK1 not, to compensate for Heph deficiency. At 8 weeks of age the knockout
cells. In search of the in vivo pathophysiological role of DAO, we then mice remain small, but their rate of growth is comparable to that of wild-
analyzed the distribution of DAO mRNA and protein in the brain. In rat, type littermates. Both strains also develop a microcytic, hypochromic
the distribution of DAO mRNA was newly detected in choroid plexus anaemia, a picture consistent with severe iron deficiency, confirming that
(CP) epithelial cells in addition to glial cells of pons, medulla oblongata, Heph plays an important role in iron acquisition. In addition, mice born
and especially Bergmann glia of cerebellum. In agreement with the to knockout mothers, regardless of pup genotype, exhibit truncal hair
results in rat, the immunoreactivity for DAO was detected in glial cells loss that improves after weaning, also consistent with iron deficiency.
of rhombencephalon and in CP in the human brain. Furthermore, higher These mouse models will serve as valuable tools to study the role of
level of DAO expression was observed in schizophrenic CP epithelial Heph and associated proteins in iron transport in the small intestine and
cells than that in non-schizophrenic cases. These results suggest that other tissues.
DAO expression level is altered in schizophrenia and the increase in
DAO expression is involved in aberrant D-amino acid metabolism. In
particular, gene expression of DAO in CP suggests that DAO may regulate
D-amino acid concentration by modulating the cerebrospinal fluid, and
may be regarded as a potential therapeutic target for schizophrenia.

POS-MON-115 POS-TUE-116
POLYMORPHISM DEPENDANT SIGNALING BIAS IN ANTI-HELICOBACTER PYLORI ACTIVITY OF EDIBLE
THE HUMAN CALCITONIN RECEPTOR PLANT FROM NORTHEASTERN THAILAND
Furness S.G.B., Khreish M., Chrisopoulos A. and Sexton P.M. Gamnarai P.1, Rojpibulstit P.1, Kangsadalampai S.1, Boonsiri P.2,
Drug Discovery Biology Laboratory, Monash Institute of Daduang J.3 and Vilaichone R.K.1
Pharmaceutical Sciences, Parkville Victoria 3052, Australia. 1
Faculty of Medicine, Thammasat University (Rangsit Campus),
Pathumthani, 12121. Thailand. 2Faculty of Medicine, Khon Kaen
The calcitonin receptor is a Family-B G protein-coupled receptor that University, Khon. Kaen, 40002, Thailand. 3Faculty of Associated
mediates responses to the peptide hormone calcitonin. Calcitonin is Medical Sciences, Khon Kaen University, Khon. Kaen, 40002,
involved in calcium homeostasis by inhibition of osteoclast-mediated Thailand.
bone resorbtion and potentiation of renal calcium secretion. Calcitonin
also exerts anorectic and analgesic effects in the central nervous ABSTRACT Helicobacter pylori is a common bacterial pathogen in
system as well as suppressing gastric secretion and intestinal mobility. human. It is found in association with peptic ulcer diseases and gastric
A number of single nucleotide polymorphisms have been reported cancer. In Thailand, the infection has been reported to be higher in
for the calcitonin receptor. Of these, a coding polymorphism resulting northeastern part than in any other regions of the country. Surprisingly,
in either Leucine or Proline at position 447 in the carboxy terminal the prevalence of peptic cancer among people in this region was very
tail has been reported to be associated with osteoperotic risk. We low. The explanation for this finding was not clear. It was thought that
hypothesized that these polymorphisms may result in altered signaling a variety of local plants in their daily foods may play an indirect role in
profiles providing a molecular basis for apparent calcitonin receptor the cancer protection by reducing the severity of the H. pylori infection
dependant clinical changes in calcium homeostasis. Since both salmon and, consequently, lower the rate of peptic cancer. In this study, we
and human calcitonin are used clinically, we also hypothesized that assessed the anti-H. pylori activity of 21 edible plants from northeastern
these ligands may show different signaling profiles with respect to this Thailand to look into the potential nutraceuticals against the H. pylori
polymorphism. A single previous report using a transient transfection ATCC 43504. Minimum inhibitory concentration (MIC) was determined
model demonstrated no difference in calcitonin stimulated adenylate by agar dilution method for ethanol and ethyl acetate plant extracts. Of
cyclase activity at either polymorphic variant of the receptor (Wolfe et. all 21 plant extracts, ethanol extracts of Polygonum adoratum Lour.,
al. Mutat Res. 2003; 522:93-105.). We have used several model cell Limnocharis flava (L.) Buchen., Limnophila aromatica Merr, Tiliacora
backgrounds, COS-7, 3T3 and HEK-293, to analyse differences in triandra Diels, and Spilanthis acmella Murr. (MIC 125 microgram/ml) and
signaling arising from these polymorphs. We have shown that there are ethyl acetate extracts of Polygonum adoratum Lour., Justicia gangetica,
a number of polymorphism-dependant changes in signaling efficacy Limnophila aromatica Merr, Cratoxylum formosum subsp. pruniflorum
toward adenylate cyclase, extracellular signal-regulated kinase (ERK) (Kurz) Gogel., Cratoxylum formosum (Jack) Dyer ssp, Tiliacora triandra
phosphorylation and intracellular calcium release. Further, we have Diels, Ottelia alismoides (L.) Pers., Hydrocharis morsus-ranae Linn.,
demonstrated that these signaling biases are dependent on cellular Tacca chantrieri Andre and Spilanthis acmella Murr. (MIC 62.5 to
background. We have also observed that there are no apparent 125 microgram/ml) exhibited the highest anti-H. pylori activity. This
polymorphism dependent differences in signaling profiles in response information provides more insights into another role of edible plant as
to salmon and human calcitonin in these model cell systems. an anti-bacterial neutraceutical food.

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POS-MON-117 POS-TUE-118
SUBCELLULAR TRAFFICKING OF VIRAL PROTEINS COMPARING THE CHAPERONE ACTIVITY OF
IS CENTRAL TO VIRAL IMMUNE EVASION AND GLYCEROL AND β-CASEIN IN PREVENTING
PATHOGENICITY AGGREGATION OF PROTEINS IN CROWDED SYSTEM
Moseley G.W.1, Wiltzer L.1, Oksayan S.1, Rowe C.L.1, Marsh G.2, Wang L.F.2, Ghahghaei A. and Rahmany Asgarabad F.
Blondel D.3, Ito N.4 and Jans D.A.1 Department of Biology, Faculty of Science, University of Sistan and
1
Monash University, Clayton, Victoria, Australia. 2AAHL, Geelong, Baluchestan, Zahedan, Iran.
Victoria, Australia. 3Gif-sur-Yvette, France. 4Gifu Univeristy, Gifu,
Japan; CNRS.
Aggregation of proteins is a frequent occurrence during their transition
from random coil to native structure. The influence of glycerol and
The principal host response to viral infection is the production of β-casein in the refolding of ovotrnasferrin, insulin, α-lactalbumin and
interferon (IFN), which induces a potent antiviral state in host cells. catalase under aggregating conditions was studied by visible absorbtion
Productive infection therefore requires evasion/subversion of the IFN spectroscopy, intrinsic fluorescence, ANS binding assay and HPLC.
system, which is achieved by viral proteins with IFN-antagonist activity. Both chaperone effectively prevented aggregation of ovotransferrin, and
These IFN-antagonists often undergo regulated subcellular trafficking via α-lactalbumin during renaturation of proteins in the absence of dextran.
interaction with host cellular transport machinery (nuclear import/export As dextran increased the rate of aggregation, β-cacein and glycerol was
proteins and cytoskeletal factors), enabling the targeting of multiple found less efficient in preventing them. It was observed that dextran
stages of intracellular IFN signaling pathways in specific subcellular sites. accelerated insulin aggregation but it had little effect on the time to the
IFN-antagonist trafficking thus represents a potential target for antiviral onset of insulin aggregation. β-Casein and glycerol were found equally
therapy. To demonstrate the importance of IFN-antagonist trafficking to effective in preventing aggregation of insulin both in the presence and
viral pathogenicity, we have examined the archetypal IFN-antagonist of absence of dextran. Glycerol effectively suppressed thermally induced
rabies (P-protein), finding that it undergoes intricately regulated targeting aggregation of catalase whereas β-casein co precipitated of proteins.
via several nuclear export (NESs) and nuclear localization (NLSs) In conclusion glycerol is a more effective chaperone than β-casein to
sequences, as well as sequences for association with microtubules protect protein from aggregation and precipitation. The more enhanced
(MTs) and the MT motor, dynein. This appears to be central to P-protein’s protein reactivation by glycerol may be due to their more ability to bind
targeting and inactivation of the vital IFN signaling factors IRF3 and to hydrophobic sites in protein folding intermediate(s) followed by their
STAT1 within different cellular compartments. Our recent data indicate subsequent removal as the protein refolds. In addition structural change
that NES-mediated P-protein nuclear export is essential to its inhibition in β-casein at higher temperature may explain the different effects of
of IFN-dependent STAT1 signaling, and is a key pathogenicity factor glycerol and β-casein in protecting catalase.
for infectious rabies virus, the first such demonstration for any virus.
Our data further indicate that P-protein nuclear import makes similar
crucial contributions to IFN-antagonist function, and that P-protein-MT
interaction provides additional, novel mechanisms of STAT1 inhibition.
These data indicate that efficient viral immune evasion requires viral
protein interaction with diverse cellular factors in multiple subcellular
sites, with implications for the development of therapies for incurable
human-pathogenic viruses.

POS-MON-119 POS-TUE-120
THE EXPRESSION OF TRANSMEMBRANE TUMOUR MITOCHONDRIAL ABNORMALITIES IN THE
NECROSIS FACTOR IS INCREASED IN THE ANTERIOR MECP2TM1TAM MOUSE MODEL OF RETT SYNDROME
CINGULATE OF SUBJECTS WITH BIPOLAR DISORDER
Gold W.A.1, Williamson S.L.1, Pelka G.J.2, Tam P.P.L.2, Gibson J.1 and
Gibbons A.S., Tawadros N., Scarr E. and Dean B. Christodoulou J.1, 3
Rebecca L. Cooper Laboratories, Mental Health Research Institute of
1
NSW Centre for Rett Syndrome Research, Western Sydney Genetics
Victoria, Parkville, Victoria 3052, Australia. Program, Children’s Hospital at Westmead, Sydney, Australia.
2
Embryology Unit, Children’s Medical Research Institute, Sydney,
Altered pro-inflammatory cytokine signalling has been implicated in the Australia. 3Disciplines of Paediatrics & Child Health and Genetic
pathologies of mood disorders and schizophrenia. We have previously Medicine, University of Sydney, Australia.
reported an increase in the expression of the transmembrane isoform of
tumour necrosis factor (tmTNF) in Brodmann’s area (BA) 46 but not BA Rett Syndrome (RTT) is a severe childhood neurodevelopmental
24 from subjects with major depression, while the expression of soluble disorder, primarily caused by mutations in the X-linked gene methyl-
isoform of TNF (sTNF) was not altered in either region. To determine CpG-binding protein 2 (MECP2). RTT occurs predominately in females
whether TNF is involved in other psychiatric disorders, we examined with an incidence of 1 in 8-10,000. There is considerable phenotypic
TNF expression in post-mortem subjects with bipolar disorder (n=10) variability, with ‘classical’ RTT patients exhibiting developmental arrest
and schizophrenia (n=20) and matched control subjects. tmTNF in infancy, stereotypic hand movements, loss of communicative skills,
and sTNF protein expression was measured using Western blotting. progressive loss of intellectual functioning, and the deceleration of head
Contrasting our previous observations in major depression, the level growth. Phenotypic similarities between RTT and patients with primary
of tmTNF was significantly increased in BA 24 (mean ratio of internal mitochondrial respiratory chain disorders prompted investigations into
standard ± SE: CON = 2.78 ± 1.03; BD = 7.15 ± 1.75; P<0.05) but not abnormalities of the respiratory chain function as well as mitochondrial
BA46 (CON = 0.88 ± 0.10; BD = 1.84 ± 0.47; P>0.05) from subjects ultrastructure in RTT patients as well as in mouse models. The aim of this
with bipolar disorder. The level of sTNF protein was not changed in study was to investigate the mitochondrial function of the brain and other
subjects with bipolar disorder in either region examined (BA 24: CON organs in the Mecp2tm1Tam mouse model, by investigating the individual
= 1.31 ± 0.17; BD = 1.49 ± 0.26; P>0.05; BA 46: CON = 1.35 ± 0.15; BD respiratory chain complexes (COI, COII, COIII, COII+III and COIV)
= 1.44 ± 0.17; P>0.05). The expression of tmTNF and sTNF protein was using spectrophotometric techniques. When we compared symptomatic
not altered in subjects with schizophrenia in BA 24 or BA 46 (P>0.05). mice to their wild type litter mates we observed an increase in COIII
While our findings suggest increased tmTNF expression is associated activity (wt=171.3±58.9; null=256.6±75.8, p=0.0302) in the heart, and
with mood disorders, the affected brain regions differ between bipolar a decrease in COII+III (wt=111.2±46.59; null=61.19±20.89, p=0.003)
disorder and major depression. Furthermore, aberrant TNF signalling and COIV (wt=151.6±53.75;null=79.56±32.49, p=0.0007) activity in
in mood disorders may not involve the classical pro-inflammatory skeletal muscle. Interestingly, we found no changes in activity in the
signalling pathways mediated by sTNF. brain of symptomatic mice. Furthermore, no differences were observed
in mitochondrial activity in pre-symptomatic mice. This data imply that
perturbations in mitochondrial function appear to occur only at disease
onset and may contribute to the clinical phenotype in RTT patients.

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POS-MON-121 POS-TUE-122
THE ROLE OF ENDOPLASMIC RETICULUM STRESS IN SHARK ANTIBODIES AND NCAMS AS NOVEL
TYPE 1 DIABETES DIAGNOSTICS
Gorasia D.G., Dudek N.L., Wee S., Mangum J.E., Hubbard M.J. and Griffiths K., Casey J., Parisi K. and Foley M.
Purcell A.W. Biochemistry Department, Latrobe University, Australia.
The University of Melbourne.
Variable domains from the shark immunoglobulin new antigen receptor
Type I Diabetes (T1D) is an autoimmune disease which results (IgNARs) and homologous domains from human neural cell adhesion
from the specific destruction of pancreatic beta cells. Proinsulin is molecules (NCAMs) are small, robust molecules which can be engineered
a target of infiltrating T cells but it’s unclear why it’s trageted. A role to bind antigens of interest with high affinity and specificity. IgNARs
of misfolded proinsulin in diabetes is seen Akita mice which harbors have superior physicochemical properties to conventional mammalian
a mutated proinsulin II (Cys96Tyr) gene and develops diabetes due to antibodies, showing greater stability to extreme temperature, urea
endoplasmic reticulum (ER) stress. Recently, protein misfolding and concentration, and pH. There is significant potential for their application
associated cellular stress is emerging as a key factor in beta cell death as diagnostic reagents, particularly in point of care situations with high
mediated by apoptosis. Proinsulin folding takes place in the ER but ambient temperature and humidity. Similarly, NCAMs have widespread
little is known about how ER chaperones guide proinsulin production, potential as therapeutic and diagnostic reagents. We used a phage
particularly under conditions of ER oxidative stress. In this study we display approach to select high affinity binders from IgNAR and NCAM
have characterized a number of chaperones that assist in the folding libraries, and detail and compare here their various properties.
of proinsulin. The tools to study proinsulin folding were generated by
expressing flag tagged proinsulin in a pancreatic beta cell line (NIT-1).
Immunoprecipitation studies were performed using flag antibody. We
have identified a number of chaperones such as BiP (GRP 78), PDI and
DnaJ homolog subfamily B member 11 (DnaJ11B or HSP 40), that interact
with proinsulin. It has been shown that BiP interact with proinsulin, to
our knowledge this will be the first study to show that BiP together with
Dnaj 11 B interact with proinsulin. Our study on proteomic analysis of
islets from NOD mice showed that islets undergo oxidative stress during
development of diabetes as chaperones like BiP were upregulated.
Further understanding of the role, each identified chaperone plays in
proinsulin folding may provide insights into how proinsulin is targeted
during development of T1D and how oxidative stress affects folding of
proinsulin.

POS-MON-123 POS-TUE-124
PROPAGATION OF THE PRION PROTEIN VIA THE FIELD TRIALS OF TRANSGENIC COTTON
EXOSOMAL PATHWAY EXPRESSING A DEFENSIN GENE FOR FUNGAL
CONTROL
Guo B.B.1, 2 , Vella L.J.2, 3, Cappai R.2, 3, 4, Bellingham S.A.1, 2, 4 and
Hill A.F.1, 2, 4 Hinch J.M.1, Gaspar Y.M.1, McGinness B.S.1, McKenna J.A.1, Anderson
1
Department of Biochemistry and Molecular Biology, The University M.A.2 and Heath R.L.1
of Melbourne, Victoria 3010, Australia. 2Bio21 Molecular Science and 1
Hexima Limited c/o School of Botany, The University of Melbourne Vic
Biotechnology Institute, The University of Melbourne, Victoria 3010, 3010. 2Dept of Biochemistry, La Trobe University Vic 3086, Australia.
Australia. 3Department of Pathology, The University of Melbourne,
Victoria 3010, Australia. 4Mental Health Research Institute of Victoria, Cotton plants (Gossypium hirsutum) expressing a gene encoding the
Melbourne, Victoria, Australia. defensin NaD1 from the female sexual tissues of Nicotiana alata have
been tested for enhanced resistance to fungal pathogens in the field.
Prion diseases are a group of fatal neurodegenerative diseases Transgenic cotton plants were tested in Queensland and in New South
characterised by conversion of the host encoded prion protein, PrPC, Wales, Australia in fields naturally infested with Fusarium wilt fungus
into the infectious isoform, PrPSc. Unlike other neurodegenerative (Fusarium oxysporum f. sp. vasinfectum) or Verticillium wilt fungus
diseases, prion diseases are transmissible. According to the protein (Verticillium dahliae). Three trials against Fusarium wilt in the growing
only hypothesis, the misfolded variant acts as an infectious agent, seasons of 2006-2007, 2007-2008 and 2008-2009 demonstrated that
instigating the conversion of PrPC to PrPSc. Despite extensive research, the defensin technology gave very clear advantages to cotton plants,
the pathway by which this infection is transmitted between cells and with almost three times the survival rate in a high disease year compared
tissues remains elusive. We have identified a role for exosomes in with plants that lack the defensin gene. The surviving plants had higher
transmitting prion infectivity between different cell types. Exosomes numbers of bolls per plant and consequently a higher yield per plant.
are small membranous vesicles released by cells, and are derived Two trials against Verticillium wilt in 2007-2008 and 2008-2009 also
from the endosomal system. Both PrPC and PrPSc has been detected demonstrated a yield increase associated with defensin technology. No
in exosomes isolated from two cell models: the rabbit epithelial (RK13) adverse phenotypic effects or phytotoxicity were observed in transgenic
cell line overexpressing mouse PrP; and the mouse hypothalamic GT1-7 defensin cotton throughout the trials.
cell line expressing endogenous levels of PrP. Exosomes allow efficient
intercellular spread of prion infectivity, however the factors that govern
the sorting of PrP into exosomes is unknown. A difference between the
N-terminal immunoreactivity and glycosylation patterns of exosomal and
cellular PrP was observed, prompting the hypothesis that PrP isoforms
undergo preferential packaging into exosomes. Previous studies have
identified mono-ubiquitination as a tag for exosomal sorting and we
aimed to investigate if exosomal PrP is ubiquitinated. This modification
could have important implications for intercellular transmission of prion
infectivity as it could be a pre-requisite or consequence of exosomal
sorting.

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POS-MON-125 POS-TUE-126
PROTEOMIC STUDY ON RAPAMYCIN TREATED MINCLE IS A NOVEL REGULATOR OF THE
HUMAN CANCER CELL LINE INNATE IMMUNE RESPONSE TO TREHALOSE-6,6-
DIMYCOLATE
Rajagopalan S.1, Guha N.1, Reddy Y.1, Joseph S.1, Lateef S.1 and
Hennessy T.2 Hitchens K.J., Manzanero S. and Wells C.A.
1
Agilent Technologies, India Pvt.Ltd, Bangalore, India. 2Agilent Eskitis Institute for Cell and Molecular Therapies, Griffith University.
Technologies, Singapore Pte Ltd, Yishun, Singapore.
Macrophage inducible C-type lectin, Mincle, plays a non-redundant
Rapamycin is an immunosuppressant drug that specifically inhibits role in the innate immune systems recognition and response to the
mTOR activity and cellular hyper-proliferation in many cell types opportunistic pathogen Candida albicans. Recently, Mincle has been
resulting in G1 growth arrest. In the current study, we aim to understand demonstrated to be a key receptor for Trehalose-6,6-dimycolate
the effect of rapamycin treatment on various biological pathways using (TDM). TDM is a highly inflammatory component of the Mycobacterium
genomic, proteomic and metabolomic analysis of rapamycin treated tuberculosis cell wall. TDM alone can induce granuloma formation and
human cancer cell line HEK293. Human cell line HEK293 was treated is a component of the tuberculosis subunit vaccine. Absence of Mincle
with rapamycin and cells were harvested at 15min, 45min, 2hrs and results in poor induction of T cell immunity to the vaccine, making it
16hrs after rapamycin treatment. Control samples without rapamycin important for generating a memory immune response. In this study
treatment were likewise harvested at similar time points. Proteins significant differences were found in the inflammatory profile of Mincle
were extracted from the cells using PPS silent extraction kit, reduced, knockout (in comparison to wild type) macrophages responding to TDM.
alkylated and digested using trypsin. Peptides were analyzed in five The absence of Mincle resulted in the ablation of some inflammatory
replicates on a Q-TOF coupled to an HPLC chip system. Differentially responses (i.e. TNFα) or in the partial reduction of others (ie. IL-1β).
expressed features between the rapamycin treated and non-treated Surprisingly, the response to TDM’s building blocks, the molecules
samples were identified. Concentration of rapamycin was optimized Trehalose or Mycolate alone, appeared to be independent of Mincle.
with respect to cell toxicity and the optimum concentration was found to Our results establish that Mincle works both independently of and in
be 40nM. Proteins were extracted from one million cells of treated and collaboration with other receptors in the macrophage response to TDM,
control samples respectively at the specified time points. Experimental and is only involved the response to the whole TDM molecule. Therefore
procedures for protein extraction, digestion and LC-MS analysis were Mincle plays a key role directing part of the inflammatory response to
optimized. Approximately 3000 features were obtained in each LC-MS TDM.
analysis. The reproducibility in the replicate LC-MS analyses of the
digested protein mixture was tested and found to be good. Statistically
significant differentially expressed features were analysed for control
and treated samples of corresponding time points and in a subsequent
experiment identified using targeted MS/MS analysis and database
search.

POS-MON-127 POS-TUE-128
EFFECTS OF MISSENSE MUTATIONS IN PAH ON G-QUADRUPLEX STABILIZER BMVC4 INDUCES
PROTEIN QUATERNARY STRUCTURE AND ENZYME SENESCENCE THROUGH BOTH INHIBITING
FUNCTION: IMPLICATIONS FOR DISEASE SEVERITY IN TELOMERASE ACTIVITY AND REPRESSING OF HTERT
PHENYLKETONURIA EXPRESSION IN CANCER CELLS
Ho G.1, 2 , Reichardt J.3 and Christodoulou J.1, 2 Huang F.-C.1, Chang T.-C.2 and Lin J.-J.1
1
Genetic Metabolic Disorders Research Unit, Children’s Hospital at 1
Institute of Biopharmaceutical Sciences, National Yang-Ming
Westmead, Sydney, Australia. 2Disciplines of Paediatrics & Child University, Taipei, Taiwan. 2Institute of Atomic and Molecular Sciences,
Health and Genetic Medicine, University of Sydney, Sydney, Australia. Academia Sinica, Taipei, Taiwan.
3
School of Medical Sciences, University of Sydney, Sydney, Australia.
Telomeres consist of tandem repeats of guanine-rich sequences that
Phenylketonuria (PKU) is a recessive inborn error of phenylalanine can fold into a variety of four-stranded structures, G-quadruplexes.
metabolism, predominantly caused by mutations in the phenylalanine Telomerase is the enzyme responsible for telomere replication and
hydroxylase (PAH) gene. There is a high level of genetic heterogeneity, represents a promising neoplasia therapeutic target. Inhibition of
with over 500 mutations reported to date. The majority of mutations telomere replication can be achieved by stabilization of G-quadruplex
are missense or non-synonymous amino acid changes, which are DNA structures. Here we designed and synthesized carbazole
postulated to destabilise the structure of the folded protein. In PAH, a derivatives that stabilized G-quadruplex DNA structure. We found 3,6-
homotetrameric protein, slight local changes can reduce the efficiency bis(4 -methyl-2-vinylpyrazinium iodine) carbazole (BMVC4) increased
of catalytic function by decreasing the amount of properly-folded the melting temperature of G-quadruplex and inhibited telomerase
tetramers, the sole functional form. To study the effects of different activity. The BMVC4-treated cells ceased to divide after a lag period.
missense mutations we transfected COS-7 cells with PAH of wild- Hallmarks of senescence including morphological changes, detection
type human sequence or containing a missense mutation previously of senescence-associated β-galactosidase activity were detected
identified in patients. Protein lysates were separated by native PAGE in BMVC4-treated cancer cells. The BMVC4-induced senescent
and analysed by Western blot. Quaternary structure (tetramer, dimer phenotype is accompanied by progressive telomere shortening and
or monomer) was deduced by size. The amount of intact tetrameric partial co-localization of the DNA damage foci and telomere association
protein roughly correlated with the predicted severity of the missense protein TRF2, indicating that BMVC4 caused telomere uncapping
mutations based on patient phenotype. Missense mutations known to after long-term treatments. Interestingly, we also found that BMVC4
cause the most severe form of PKU (e.g. p.F299C and p.R408W) led to repressed the expression of hTERT, the catalytic subunit of telomerase.
very low or undetectable levels of tetrameric PAH, whereas mutations Repression of hTERT appeared to be caused by the reduction of c-myc,
(p.V245A, p.E390G) causing the mild form of PKU had similar levels a major transcription factor, upon BMVC4 treatments. Since disruption
of tetrameric PAH compared to wild-type. Mutations of the latter group of the G-quadruplex-forming sequence located at the promoter of c-myc
showed decreased levels of enzyme activity, suggesting that mutations eliminated the repressing effects of BMVC4, the results indicated that
of amino acids involved in catalytic activity in PAH are likely to cause BMVC4 repressed c-myc through stabilization of the G-quadruplex
a less severe form of the disease compared to mutations causing structure located at the promoter of c-myc. All together, our results
structural instability. indicated that BMVC4 induced senescence through both inhibiting
telomerase and repressing telomerase expression.

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POS-MON-129 POS-TUE-130
CHARACTERIZATION OF TOXICITY OF ALS-LINKED HEPATOMA-DERIVED GROWTH FACTOR (HDGF)
SOD1 MUTANT PROTEINS IS CORRELATED WITH MALIGNANCY AND
INVASIVENESS OF BREAST CANCER
Jang J.Y.1, Yoon E.J.1, Cho H.M.1, Kim Y.M.1, Rhim H.S.2 and Kang S.M.1
1
School of Life Sciences and Biotechnology, Korea University, Chen S.C.1, Yeh M.H.4, Chen H.Y.3 and Tai M.H.1, 2, 3
Seoul 136-701, Korea. 2Research Institute of Molecular Genetics, 1
Institute of Biomedical Sciences, National Sun Yat-Sen University,
Department of Biomedical Sciences, College of Medicine, the Catholic Kaohsiung 804, Taiwan. 2Department of Medical Education and
University of Korea, Seoul 137-701, Korea. Research, Kaohsiung Veterans General Hospital, Kaohsiung 813,
Taiwan. 3Department of Biological Sciences, National Sun Yat-
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative Sen University, Kaohsiung 804, Taiwan. 4Department of Surgery,
disease caused by the degeneration of motor neurons. The disorder Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan.
causes muscle weakness and atrophy throughout the body. In many
cases there is a family history of the disease, in which case it is referred Hepatoma-derived growth factor (HDGF) is a novel growth factor that
to as familial ALS (FALS). Of these, 20% are mapped to the Cu/Zn participates in the pathogenesis of a variety of cancers. However, the
superoxide dismutase (SOD1) gene on chromosome 21. SOD1 is an role of HDGF in breast cancer progression remains unclear. This study
enzymatic antioxidant found in the cytosol, nucleus, peroxisomes, as aimed to elucidate the association of HDGF expression with prognosis
well as the mitochondrial intermembrane space of eukaryotic cells of breast cancer. A total 93 surgically resected breast cancer specimens
and in the periplasmic space of bacteria. The mechanism for mutant were collected for immunohistochemical analysis. It was found that
SOD1 toxicity remains unknown. However, two main hypotheses are HDGF was significantly elevated in breast cancer tissues compared with
the impairment of the functions of important proteins by interaction of non-tumor ones. Moreover, elevated HDGF expression was correlated
misfolded mutant SOD1 proteins and the toxic effect of mutant SOD1 with tumor grades, stages, Ki-67 proliferation index, and lymph node
aggregates. Therefore, an attractive therapeutic approach could metastasis. By analysis of HDGF expression in various human breast
be to reduce levels of mutant SOD1 expression in neuronal cells. cancer cell lines, it was found that HDGF was significantly up-regulated
Previously, we found that intracellular Aβ directly interacted with SOD1. at the protein level in the most invasive human breast cancer cell line,
Furthermore, we mapped the SOD1 binding region to Aβ amino acids MDA-MB-231. Moreover, exogenous supply of recombinant HDGF
26-42. Interestingly, intracellular Aβ bound to the SOD1 G93A mutant augmented the invasiveness of MDA-MB-231 cells in a dose-dependent
with greater affinity than to wild-type SOD1. Using a fragment (a.a. 26- manner. Thus, it is concluded that HDGF over-expression is correlated
42) of Aβ as a competitive inhibitor, we reduced the interaction between with malignancy and invasiveness of breast cancer. Besides, HDGF
mutant SOD1 and proteins that were reported to bind mutant SOD1. may represent a novel diagnostic and therapeutic target for breast
Furthermore, we mapped a domain in SOD1 that interacts with the cancer.
proteins. We also developed ways to disperse SOD1 mutant aggregates.
These studies will contribute to therapeutic approach of ALS.

POS-MON-131 POS-TUE-132
THE ROLE OF GLUTAREDOXIN1 IN CELLULAR TWO BAM COMPLEXES IN KLEBSIELLA PNEUMONIAE
COPPER HOMEOSTASIS
Alcock F.H., Clements A. and Lithgow T.J.
Ackland S.1, Singleton W.1, McInnes K.1, McKirdy R.1, Winnall W.2, Host-Pathogen Molecular Biology Unit, Department of Biochemistry
Mercer J.1 and La Fontaine S.1 and Molecular Biology, Monash University, Clayton, 3800, Australia.
1
Centre for Cellular and Molecular Biology, School of Life and
Environmental Sciences, Deakin University, Burwood, VIC. 3125. The cell envelope of Gram negative bacteria consists of the cytoplasmic
2
Centre for Reproduction and Development, Monash Institute of membrane, a peptidoglycan layer and an outer membrane, which
Medical Research, Clayton, VIC. 3125. contains lipopolysaccharide in the outermost leaflet. The majority of
clinical K. pneumoniae isolates also produce a mucoid polysaccharide
Copper (Cu) is an essential heavy metal that is required by all capsule. These two polysaccharide layers are primarily responsible
organisms for a wide range of critical enzymes that harness the CuII/ for the barrier quality of the outer membrane, which provides the cells’
CuI redox couple. Hence, it is important in many physiological and primary defence against fluctuating environmental conditions and
disease processes. Uncontrolled intracellular Cu levels can lead to the toxic small molecules. Different classes of outer membrane proteins
generation of excessive reactive oxygen species (ROS) and oxidative are required for uptake of sugars and cofactors essential for growth.
stress. The dire consequences of Cu dysregulation are evident in the Correct assembly of outer membrane proteins, LPS and capsular
inherited Cu transport disorders, Menkes and Wilson diseases, and in polysaccharide, and its regulation, is a complex task, mediated by
neurodegenerative diseases. Cells have developed systems to regulate a number of multi-component machines and pathways. The BAM
levels of metal ions and ROS to prevent their toxic accumulation. The complex is a multimeric machine found in the outer membrane of all
mammalian Cu-transporting P-type ATPases, ATP7A and ATP7B Gram-negative bacteria, which functions to assemble β-barrel proteins
regulate the Cu status of the body by transporting Cu into the secretory in the membrane. The central component of this complex is BamA,
pathway for incorporation into essential enzymes, and by relocating to the an essential, 90 kDa β-barrel protein, with homologues in the outer
cell periphery to efflux excess Cu. We have identified new key roles for membranes of mitochondria and chloroplasts. We analysed the genome
glutathione (GSH) and the thiol oxidoreductase, glutaredoxin 1 (GRX1), of Klebsiella pneumoniae, a Gram-negative bacterial pathogen, and
in regulating the activity of ATP7A and ATP7B. GSH is a major redox found that it encodes two BamA-like proteins: the canonical BamA,
buffer in cells and is the most abundant intracellular antioxidant. GRX1 identified as such by gene synteny, and a second isoform, which
has a protective role in oxidative stress-related conditions and plays we have called BamK. We are using a combination of microbiology,
a pivotal role in redox regulation of protein function. The Cu-ATPases microscopy, cell biology and biochemical techniques to investigate the
are glutathionylated. That is, they are postranslationally modified at localisation and protein partners of BamK, and its role in assembly of
their Cu-binding cysteines to form protein-SSG mixed disulfides. When the K. pneumoniae cell envelope.
Cu levels are elevated, GRX1 interacts with ATP7A and ATP7B to
deglutathionylate them, thus allowing them to bind Cu for subsequent
transport. The effects of GRX1 overexpression and knockdown on Cu-
ATPase activity, cellular Cu homeostasis and overall cell growth and
viability will be discussed.

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POS-MON-133 POS-TUE-134
INVESTIGATING THE ROLES OF KRüPPEL-LIKE EZRIN AND NHERF ARE REQUIRED FOR THE
FACTORS IN MEGAKARYOCYTE DIFFERENTIATION RECOMBINANT PODXL ENHANCING ADHESION,
MIGRATION AND CELL-CELL INTERACTIONS
Artuz C.M.1, Mak K.S.1, Funnell A.P.W.1, 2, Pearson R.C.M.1, 2 and
Crossley M.1, 2 Fernandez D.2, Nowakowski A.2, Vilar-Egea M.P.2, Parrilla R.1, 2 and
1
School of Biotechnology and Biomolecular Sciences, University Ayuso M.S.1, 2
of New South Wales, NSW 2052. 2School of Molecular Bioscience, 1
Centre of Biological Research, CSIC, Calle Ramiro de Maeztu 9,
University of Sydney, NSW 2006. 28040 Madrid, Spain. 2CIBER de Enfermedades Raras, ISCII.

The Krüppel-like factors (Klfs) are a family of transcription factors Podocalyxin (PODXL) is a type I transmembrane glycoprotein initially
that bind GC rich sites and CACCC-boxes present in the promoters described in the epithelial cells of the kidney glomeruli, postulated to
and enhancers of many genes. They have been shown to participate exert antiadhesive properties; however, we have previously observed
in a diverse range of biological processes including erythropoiesis, that heterologous expression of recombinant PODXL enhanced the
adipogenesis and stem cell biology. One of the family’s transcriptional adherence of cells to immobilized ligands, spreading, migration, as well
activators, erythroid Klf (Eklf or Klf1) is best known for its roles in red as the interaction with other cells. The enhancing effect of PODXL on
blood cell differentiation. Recently, it has also risen to prominence cell adhesion and migration decreased significantly in deletion mutants
as an inhibitor of the megakaryocyte program. Erythrocytes and of the cytosolic domain or the DTHL domain. Thus, the integrity of the
megakaryocytes differentiate from a common progenitor, termed the cytosolic carboxylic domain of PODXL, included the terminal DTHL
Megakaryocyte/Erythroid Progenitor (MEP). In order to elucidate the domain, is required for PODXL to exert its effect on cell adhesion and
mechanisms by which Klf1 influences differentiation decisions in the cell-cell interactions. In the podocytes of kidney glomeruli the DTHL
MEP, it is important to consider two highly related repressor Klfs, basic domain of PODXL interacts with ezrin and NHERF to establish a link
Klf (Bklf or Klf3) and Klf8. The three Klfs have been shown to function with the actin cytoskeleton, essential for the function of podocytes. To
in a tight regulatory circuit. Klf1 activates both Klf3 and Klf8, and Klf3 determine whether the recombinant PODXL expressed in CHO cells was
represses Klf8. Since these Klfs recognise similar DNA motifs, they may also linked to the cytoskeleton through ezrin and NHERF we performed
compete or act redundantly. Our aim is to understand the molecular immunoprecipitation studies of cell extracts. We detected ezrin in anti-
network of gene control through which Klf1, 3 and 8 influence cell fate PODXL immunoprecipitates and PODXL when the immunoprecipitation
during differentiation in the MEP, particularly in megakaryopoiesis. Given was performed with anti-ezrin or with anti-NHERF. Thus, recombinant
that Klf1 acts primarily as a transcriptional activator, we are investigating PODXL expressed in CHO cells enhance the cell adhesion properties
whether it indirectly represses megakaryopoiesis through driving either even though the signaling to the cytoskeleton are similar to the one
or both of the repressors Klf3 and Klf8. Our strategy is to establish the reported in cells like podocytes that show anti-adherent properties.
molecular contributions that Klf1, Klf3 and Klf8 make to the regulation of These observations suggest the cellular environment might be a crucial
megakaryopoiesis, using both cell lines and knockout mouse models. factor in defining the functional properties of PODXL.

POS-MON-135 POS-TUE-136
CELL BIOLOGY OF AUTISM: CHARACTERISATION OF MOLECULAR SIZES OF ACID-INSOLUBLE AND ACID-
THE DELETED IN AUTISM-1 GENE FAMILY SOLUBLE GLYCOGEN AND THEIR RESPONSES TO
EXERCISE AND RE-FEEDING
Aziz A. and Bishop N.
Department of Microbiology, La Trobe University, Bundoora. Barnes P.D.1, Clode P.2 and Fournier P.A.1
1
School of Sport Science, Exercise and Health, The University of
Autism spectrum disorder (ASD), first described by Leo Kanner in Western Australia. 2Centre for Microscopy, Characterisation and
1943, is among the most heritable of all neurological conditions, with Analysis, The University of Western Australia.
a prevalence of at least 1:100 being commonly accepted. Variation in,
or deletion of, many different genes appears causative of ASD, and an Muscle glycogen exists as acid soluble (ASG) and acid insoluble (AIG)
emerging common theme is a defect in the secretory pathway, secreted forms, with AIG reported to be the most responsive fraction to exercise
proteins and/or regulators of these. The Deleted In Autism 1 (DIA1) and re-feeding when glycogen is extracted using homogenisation-
gene was recently identified in a study designed to identify recessive free protocols, whereas ASG is the most responsive fraction using
autism genes. The normal cellular function of the DIA1 gene product is homogenisation-dependent protocols. It has been proposed that AIG
unknown. We have carried out detailed bioinformatics-based analyses corresponds to a population of lower molecular weight glycogen species
of DIA1 which has revealed a closely related human gene. We have that act as precursors for ASG synthesis. However, the molecular size
named this gene DIA1R, for DIA1-related. DIA1R was generated by a distributions of AIG and ASG in skeletal muscles and their responses
gene duplication event preceding the divergence of vertebrates. While to changes in glycogen levels have never been examined. Using
deletion of DIA1 is implicated in development of ASD, DIA1R localizes transmission electron microscopy analyses of AIG and ASG from
to a region of the X chromosome implicated in mental retardation and muscles extracted using a homogenisation-free and homogenisation-
syndromes with ASD-like features. We found both gene products to dependent protocols, we compared the molecular size distributions of
be ubiquitously expressed, including in brain tissue. At the cellular AIG and ASG from fed Wistar rats as well as the responses of these
level, both proteins have predicted signal peptides, indicating a role in fractions to 3-minutes of intense exhausting swimming with or without
the secretory pathway. We are currently investigating the subcellular a subsequent 24-hours re-feeding period in rats previously fasted
localisation and cellular role of both DIA1 and DIA1R. Characterising the for 24 hours. Contrary to the interpretations made in the literature,
role of genes involved in ASD will lead to a better understanding of the the molecular size distribution of both AIG and ASG were normally
biological basis of ASD, leading to opportunities for improved diagnosis distributed, with similar ranges and average molecular sizes irrespective
and therapy for those with ASD. of extraction protocol. Immediately after exercise, the ASG molecular
size distribution shifted markedly towards smaller glycogen molecules,
and AIG distribution changed relatively little. After 24 hours of recovery,
both the AIG and ASG molecular size distributions returned to those
found in fed rats. All changes in total glycogen concentrations were
accounted for by the ASG fraction. In conclusion, the different responses
of AIG and ASG suggest that these are physiologically distinct glycogen
populations.

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FUNCTIONAL STUDIES OF HOMEDOMAIN- SUMO MODIFICATION OF THE ZINC-FINGER PROTEIN
INTERACTING PROTEIN KINASE USING C. ELEGANS CTCFL/BORIS
AS A MODEL
Bernier-Latmani J. and Shaw P.
Berber S. and Nicholas H.R. Experimental Pathology Division, Institute of Pathology, University of
School of Molecular Bioscience, The University of Sydney, NSW 2006. Lausanne.

The Homeodomain-Interacting Protein Kinases (HIPK) are a family Post-translational modification by the small ubiquitin-like SUMO
of serine/threonine protein kinases shown to be critical in regulation proteins (SUMO-1 and SUMO-2/3) is recognized to play an important
of many cellular processes including cell survival, proliferation and role in target protein function by affecting DNA binding, sub-cellular
apoptosis. Since there are four HIPK family members in mammals that localization, protein stability and co-factor association. CTCFL is a
show some redundancy, functional studies of this protein family can be paralog of the ubiquitous genome regulator CTCF, but is expressed solely
simplified with the nematode Caenorhabditis elegans since it expresses in the testis and some cancers. Due to high homology in their respective
a single HIPK protein (HPK-1). Both HIPKs and HPK-1 localise to nuclear zinc-finger regions it has been proposed that CTCF and CTCFL bind
speckles suggesting that they are likely be involved in analogous cellular similar DNA sequences but perform different functions. CTCFL has
processes. To expand on this knowledge we performed phenotypic been implicated in the establishment of methylation at the Igf2/H19
analyses on a worm strain carrying a deletion mutation within hpk-1 with imprinting control region and transcriptional regulation of a number of
the global aim of discovering some of the in vivo functions of worm HPK- genes including BRCA1, c-myc, and BAG1. Recently CTCF has been
1. Additionally, to gain insights into molecular mechanisms of HPK-1 shown to be modified by SUMO, whereby enhancing repression of a
function, microarray analysis was performed on the hpk-1 mutant strain. c-myc promoter reporter construct. In the present work we show that
These analyses have revealed that various cellular processes may be CTCFL is efficiently SUMOylated in vitro and when over-expressed
regulated by HPK-1 including ageing, metabolism and expression of in 293T cells. Endogenous CTCFL is SUMOylated, as determined by
collagens. immunoprecipitation of SUMO-1 and SUMO-2/3 modified proteins in
K562 cells. Both SUMO-1 and SUMO-2/3 are attached at two residues,
K181 and K645, as shown by point mutation analysis. Characterization
of the functional consequences of CTCFL SUMOylation is ongoing.
Alteration of CTCFL sub-cellular localization by SUMOylation is being
analyzed by immunofluorescence confocal microscopy. Furthermore,
the effect of SUMOylation on CTCFL DNA-binding and gene regulation
activity is being assessed using EMSA and luciferase reporter assays.
Also, the consequence of SUMOylation on the stability of CTCFL is
being investigated. This work will help elucidate basic CTCFL function
and potentially provide a basis to understand its role in both testis and
cancer.

POS-MON-139 POS-TUE-140
REGULATION OF COPPER UPTAKE AND EFFLUX IN CHARACTERIZATION OF MAVI-P35, A BACULOVIRAL
DROSOPHILA INTESTINAL ENTEROCYTES MULTIPLE CASPASE INHIBITOR
Binks T. and Burke R. Brand I.L.1, George C.1, Lovric M.1, Pantaki D.1, Kitevska T.1, Clem
School of Biological Sciences, Monash University. Wellington Rd R.J.2 and Hawkins C.J.1
Clayton 3800 Victoria, Australia. 1
La Trobe University, Department of Biochemistry, Melbourne 3086,
Australia . 2Molecular, Cellular and Developmental Biology Program,
All animals require the essential biometal copper as an enzymatic Arthropod Genomics Center, Division of Biology, Kansas State
cofactor for processes as diverse as energy production, free radical University, Manhattan, KS 66506, USA.
detoxification and pigmentation. Most copper is absorbed via the
polarized enterocytes that line the intestinal lumen. Active transport P35 from Autographa californica (Ac) MNPV was the first identified
is required to transfer copper across the apical enterocyte membrane multiple caspase inhibitor expressed by a baculovirus to prevent the
from the lumen into the cells, then again across the opposite basolateral host cell from undergoing infection-induced apoptosis. A number of P35
enterocyte membrane and out into the bloodstream for distribution relatives have since been identified, including some less closely related
throughout the body. Therefore the intestinal enterocytes are an homologues belonging to the P49 sub-family. Ac-P35 has been studied
excellent system to study directional transport of copper through a extensively. It protects against mammalian, nematode and insect cell
polarized cell layer. We are studying this process in the Drosophila death and inhibits all caspases tested, with the exception of the initiator
larval midgut, using targeted over expression and suppression of known caspases DRONC, Sf-caspase-X and caspase-9. In contrast, the best
copper transport and copper chaperone genes together with two Green studied P49 sub-family member SlNPV-P49 seems to be a generally
Fluorescent Protein (GFP) reporter genes that respond to copper less potent caspase inhibitor, but it can inhibit Ac-P35-resistant initiator
depletion and copper accumulation respectively. We will also present caspases. A new member of the P35-family has been recently identified
protein localization studies in these midgut cells using GFP fusion by sequencing the genome of the baculovirus Maruca vitrata (Mavi)
proteins created for the major fly copper uptake and efflux proteins. MNPV. This P35-family member (Mavi-P35) shares 85% sequence
And using our Drosophila enterocytes model, we will demonstrate novel homology to Ac-P35, but its active loop possesses a different caspase
roles in copper homeostasis for genes previously implicated in cellular cleavage sequence, reminiscent of sequences efficiently cleaved by the
vesicle trafficking / transport pathways. insect initiator caspase DRONC. This suggested to us that, unlike other
members of the P35 sub-family, Mavi-P35 may inhibit initiator caspases
such as DRONC. In this study, Mavi-P35 was characterized by testing
its ability to inhibit caspase-induced yeast death and apoptosis of
mammalian and insect cells. Recombinant Mavi-P35 was tested in
vitro against a range of caspases and their susceptibility to inhibition
was quantitated. Our results show that Mavi-P35 is able to inhibit an
even broader range of caspases than Ac-P35, including the Drosophila
melanogaster caspase DRONC.

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THE GENETICS AND CELL BIOLOGY OF COPPER PEPTIDASE EXPRESSION IN LYMPHOCYTES
HOMEOSTASIS IN DROSOPHILA
Chen Y., Yao T.W., Chowdhury S., Gorrell M.D. and Yu D.M.T.
Lye J.1, Binks T.1, Hwang J.1, Camakaris J.2 and Burke R.1 Centenary Institute, Sydney Medical School, University of Sydney,
1
School of Biological Sciences, Monash University. Wellington Rd NSW, Australia.
Clayton, Victoria 3800. Australia. 2Department of Genetics, University
of Melbourne. Parkville, Victoria 3010. Australia. The dipeptidyl peptidase (DP) IV gene family is specialized in hydrolyzing
the prolyl bond, which is useful for degrading many peptide hormones.
Copper is an essential micronutrient required as a cofactor for enzymes DPIV (CD26) has roles in lymphocyte activation and proliferation. DPIV
such as Cytochrome C oxidase, Cu, Zn Superoxide dismutase and and its closest relative, fibroblast activation protein, also have roles
Tyrosinase. Disruption of copper homeostasis can result in serious in liver disease. We have associated DP8 and DP9 expression with
human conditions such as Menkes disease (copper deficiency) and disease pathogenesis [1] and observed DP8 and DP9 expression by
Wilson disease (copper toxicity). The major copper uptake (Ctr1) leukocytes and B and T cell lines while others have shown a role for
and efflux (ATP7) genes are highly conserved from yeast through to DP9 in processing antigenic peptides [Geiss-Friedlander R, et al., JBC
vertebrates. Drosophila has three Ctr1 orthologues and a single ATP7 2009]. A greater understanding of DP8 and DP9 in the immune system is
orthologue, DmATP7. We are using a combination of targeted gene needed. This study investigated DP8 and DP9 expression in stimulated
over expression and gene suppression to examine the in vivo role of lymphocytes and diseased human liver. DP8 and DP9 expression levels
the key copper transport genes in various Drosophila tissues. We find were measured by Western blot and quantitative PCR in Jurkat T cells
that reduction of copper uptake or increase in copper efflux in cuticle- and Raji B cells treated with mitogen, hydrogen peroxide, dithiothreitol,
producing cells results in a striking hypopigmentation phenotype due Mitomycin C or serum starvation. DP8 and DP9 protein levels were
to lack of activity of the Laccase cuproenzyme. Genetically-induced upregulated in mitogen stimulated Jurkat T cells but unaffected by the
copper deficiency in the Drosophila adult eye results in a severely other treatments. DP9 protein expression was similar in human primary
flattened eye phenotype while increased copper uptake in the eye biliary cirrhosis liver compared with normal liver. The novel finding that
causes a rough eye phenotype typical of increased cell death. We will DP8 and DP9 expression was upregulated in activated T cells suggests
present genetic interaction studies showing that the effects of increased that, like DPIV/CD26, DP8 and DP9 might have roles in T cell activation.
copper uptake can be compensated for by increasing copper efflux and Reference: [1] Yu, D.M.T., et al., The in vivo expression of dipeptidyl
are exacerbated by blocking copper efflux. We will also present data peptidases 8 and 9. J Histochem Cytochem 2009. 57:1025-1040.
showing how the cellular localization of these copper transport proteins
fits with their proposed cellular functions, and exciting recent data from
the Australian Synchrotron where we have used X ray Fluorescence
Microscopy to produce elemental maps of Drosophila tissues and prove
directly for the first time, that our genetic manipulations do indeed alter
copper levels in a targeted manner in vivo.

POS-MON143 POS-TUE-144
AUTOPHOSPHORYLATION OF ATM AT SERINE 1981
PURINE BIOSYNTHESIS AND ITS REGULATION IN MEDIATES THE INTERACTION WITH MDC1 AND
NODULES OF SUB-TROPICAL LEGUMES STABILIZES ATM AT SITES OF DNA DOUBLE-STRAND
Chen C.1, Bussell J.2, 3, Atkins C.2 and Smith P.1
BREAKS
1
School of Biological Sciences, The University of Sydney, NSW
2006, Australia. 2School of Plant Biology, The University of Western Davis A., Zheng C. and Chen D.J.
Australia, WA 6009, Australia. 3Umea Plant Sciences Center, Division of Molecular Biology, Department of Radiation Oncology,
Department of Forest Genetics and Plant Physiology, Swedish University of Texas Southwestern Medical Center, Dallas Texas, USA.
University of Agricultural Sciences (SLU), 901 83 Umea, Sweden.
Ataxia telangiectasia mutated (ATM) plays a critical role in the cellular
Sub-tropical legumes such as cowpea and soybean (mainly of the response to DNA damage. In response to DNA double strand breaks
tribe Phaseoleae) export fixed nitrogen as ureides. De novo synthesis (DSBs), ATM is autophosphorylated at serine 1981. Although this
of purine nucleotide is the major pathway for assimilation of fixed autophosphorylation is widely considered a sign of ATM activation, it
nitrogen in these legumes and the product, IMP, is oxidised to form is still not clear if autophosphorylation is required for ATM functions
ureides that are exported from the nodules. There are 10 enzymatic including localization to DSBs and activation of ATM kinase activity. In
steps in the purine biosynthetic pathway and the enzymes involved this study, we show that localization of ATM to DSBs is differentially
are encoded by nine genes (the pur genes). The role of the products regulated with the initial localization requiring the MRE11-RAD50-NBS1
of nitrogen fixation in regulation of these genes was investigated in complex and sustained retention requiring autophosphorylation of ATM
nodules of cowpea by growing plants with their root systems in an at serine 1981. Ablation of the autophosphorylation site affects the ability
atmosphere devoid of nitrogen (80% Ar: 20% O2). In the absence of ATM to phosphorylate its downstream targets after DNA damage
of N2 (plants grown in Ar:O2) expression of Vupur1, 4, 5, 6, 7 and 8 especially at later time point after irradiation and confer radioresistance.
is at a level required to maintain basic cellular processes only. In air Biochemical evidence shows that autophosphorylated ATM directly
the level of these transcripts increases dramatically after N2 fixation interacts with the FHA domain of MDC1. Knock-down of MDC1 protein
begins, suggesting transcription is regulated by the products of fixation. recapitulates the effects of S1981A mutation on the retention of ATM
Other transcripts studied (Vupur2, 3, 9, IMPDH and uricase) showed no at DSBs and phosphorylation of downstream substrates. Moreover,
change in the level of expression over time, however, the expression of ablation of the ATM autophosphorylation site and MDC1 depletion did
Vupur2 and 9 and IMPDH was reduced in Ar:O2 compared to air while not show any additive effect on radiosensitivity. Together, these data
that of Vupur3 and uricase was the same in both treatments. We have illustrate the importance of autophosphorylation at serine 1981 for the
investigated promoters of the two genes encoding the fifth enzyme in interaction of ATM with MDC1 which serves to stabilize ATM at DSBs
the pathway, AIR synthetase (Gmpur5-1 and Gmpur5-2), in soybean and thereby promote a full-scale response to DNA damage in human
using GUS/GFP fusions. The Gmpur5-1 promoter drives expression in cells.
all cells but the infected cells of the nodule while the Gmpur5-2 promoter
drives expression almost exclusively in infected cells. We are currently
investigating the regulation of these promoters in response to nitrogen
using these constructs. We are also investigating the targeting of the
enzymes to organelles in nodules.

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POS-MON-145 POS-TUE-146
SAME SIGNAL TRIGGERS DIFFERENT CELL FUNCTIONAL CHARACTERIZATION OF A NOVEL
RESPONSES IN VARIOUS CELL LINES AURORA-A BINDING PROTEIN, AIBP IN CENTROSOME
STRUCTURE AND SPINDLE FORMATION
Cheung T.W. and Lam Y.W.
City University of Hong Kong. Chou C.H1, 2 , Wu C.H. 2, Huang C.Y. 3, Hsu C.M. 1, Howng S.L.4 and
Hong Y.R.1, 2
Andrographolide (Andro), a diterpenoid lactone isolated from a traditional 1
Department of Biological Sciences, National Sun Yat-Sen University,
herbal medicine. Andrographis paniculata, is an abundant component of Kaohsiung,Taiwan. 2Department of Biochemistry, Faculty of Medicine,
the plant Andrographis that has been commonly used as a folk remedy College of Medicine, Kaohsiung Medical University, Kaohsiung,
for alleviation of inflammatory disorders in Asia for millennia. Also, it is Taiwan. 3Institute of Clinical Medicine, National Yang-Ming University,
known to possess potent anti-cancer activities. According to previous Taipei, Taiwan. 4Department of Neurosurgery, Kaohsiung Medical
studies, it can trigger most of cell lines to go through G1/S arrest University Hospital, Kaohsiung, Taiwan.
followed by apoptosis. Cell cycle checkpoints are regulatory pathways
that control the order and timing of cell cycle transitions and ensure that Aurora-A is involved in chromosome alignment, centrosome maturation,
critical events are completed with high fidelity. In addition, checkpoints mitotic spindle assembly and regards to an oncogene. Aurora-A is also
respond to damage by arresting the cell cycle to provide time for repair known to bind to several other proteins affecting its up-regulation or
and by inducing transcription of genes that facilitate repair. However, down-regulation and localization. However, how these different binding
a feature of tumor cells is the alteration of appropriate cell-cycle signals work together to regulate Aurora-A is not properly known. To
progressions which are closely related apoptotic process. Currently, explore more Aurora-A interacting proteins, the low-copy yeast two-
people mainly uses anti-mitotic drugs which work by perturbing spindle hybrid screening using Aurora A as bait protein was performed. One
assembly through the activation of the spindle assembly checkpoint for novel gene, AIBp, was demonstrated to associate with Aurora-A in
treating cancers. The drugs cause mitotic arrest, and triggers apoptosis. yeast two-hybrid method and in vitro GST pull-down assay. Molecular
Based on past study, the response of HepG2 cells is different from the characterization showed that AIBp possessed a binding site at the
other cell lines under Andro treatment. To clarify what the differences C-terminal with Aurora-A (kinase domain). Interestingly, AIBp also
are, we use live cell imaging and western blotting approaches in the interacts with hNinein at the N-terminal, which overlaps a previously
study. We showed that Andro induces both G2/M and G1 arrest in two reported hNinein and GSK3β binding site. In kinase assay, AIBp interacts
tested cell lines. Then, in HeLa cells, it quickly goes into apoptosis. But, with the Aurora-A kinase domain functions as a positive regulator,
HepG2 cells mainly stay long in cell cycle arrests until necrosis occurred. whereas AIBp binding to hNinein appears to block the phosphorylation
The differences may not relate to DNA damaging or increasing ROS of hNinein by both Aurora-A and GSK3β. siRNA-mediated elimination
production. And, many M-phase related proteins show some regulation of AIBp from HeLa cells, results in a donut-like shape, asymmetrical
to reason the longer G2/M arrest observation in HepG2 cells, not in spindle pole and multiple spindle pole formation. We also demonstrated
HeLa cells. that both AIBp and Aurora-A are co-overexpressed in various brain
tumors. These studies demonstrate that AIBp may not only be required
for the dynamic movement of Aurora-A at the centrosomes and spindle
apparatus during the cell cycle, and may also be important during brain
tumorigenesis.

POS-MON-147 POS-TUE-148
PROTEOMIC PROFILE OF THE SOYBEAN TROPOMYOSIN TM5NM1 MEDIATES ACTIN-
SYMBIOSOME MEMBRANE DEPENDENT CELLULAR PROLIFERATION
Clarke V.C.1, Loughlin P.C.1, Jacoby R.P.2, Taylor N.L.2, Millar A.H.2, Coombes J.D.1, 2 , Schevzov G.1, Stehn J.1, Creed S.3, Desouza M.1,
Day D.A.3 and Smith P.M.C.1 Bach C.T.2, 3, O’Neill G.2, 3, Musgrove E.4 and Gunning P.1
1
School of Biological Sciences, The University of Sydney, NSW, 1
Oncology Research Unit, School of Medical Sciences, University of
Australia. 2ARC Center of Excellence in Plant Energy Biology, The New South Wales. 2Sydney Medical School, University of Sydney.
University of Western Australia, WA, Australia. 3Flinders University, 3
Kids Research Institute, Children’s Hospital at Westmead. 4Cancer
SA, Australia. Research Program, Garvan Institute of Medical Research.

Soybeans are able to form a symbiotic association with soil bacteria, Progression through the cell cycle is associated with profound changes
Bradyrhizobium japonicum, whereby atmospheric nitrogen is fixed by the in the actin cytoskeleton. Disruption of the actin cytoskeleton with
bacteria and made available to the plant in exchange for nutrients. This pharmacological agents causes arrest in G1 phase and failure to enter
symbiotic relationship occurs within specialised root structures termed S-phase, indicating that an intact actin cytoskeleton is required for cell
nodules. Free-living Bradyrhizobium bacteria invade the soybean roots cycle progression. The lack of specificity of these agents has hindered
and become engulfed within the plant cell, surrounded by a membrane the analysis of the mechanisms responsible for this regulation. We have
of plant origin known as the symbiosome membrane (SM). It is this previously identified functionally distinct populations of actin filaments,
membrane that regulates the movement of solutes from plant to bacteroid each containing different isoforms of the actin filament-associated
(the symbiotic form of the rhizobium) and vice versa. The SM is a unique protein, tropomyosin (Gunning, O’Neill, Hardeman, Physiol Revs 2008).
structure containing an array of plant-derived proteins through which In the current study, we hypothesised that a specific population of Tm
the plant can regulate the symbiosis. Previous attempts to characterise isoform-containing actin filaments regulates cell proliferation. The role
the protein complement of this membrane have been hindered by its of Tm5NM1-containing actin filaments was investigated in knock-out,
hydrophobic nature and the absence of a complete soybean genome knock-down, and overexpression model systems. Our data show that
for reference. In this study, SM was isolated from mature nitrogen-fixing embryonic fibroblasts (MEFs) isolated from Tm5NM1/2-knockout mice
soybean root nodules and analysed using shotgun proteomic techniques. have an impaired rate of proliferation in response to growth factor
The recent release of the complete soybean genome has allowed for stimulation. This is accompanied by a dysregulation of MAPK signalling
the identification of peptide sequences. Our initial proteomic analysis of (phospho-ERK1/2) and decreased expression of the key G1-phase
soybean SM has identified forty putative SM-localised proteins, including mediator, Cyclin D1. Neuroblastoma SHEP cells treated with siRNA
ten previously localised to the SM such as nodulin-26, an aquaporin. against Tm5NM1/2 also have reduced proliferation rates and decreased
Bioinformatic analysis of novel matches has suggested that half may Cyclin D1 expression. In comparison, Tm5NM1-overexpressing
contain membrane-spanning domains, making them candidates for SM neuroblastoma-derived B35 cells display accelerated proliferation,
transporters. Further in-depth proteomic analysis of the membrane is and reduced propensity to withdraw from the cell cycle when treated
currently being undertaken and the function and localisation of putative with differentiation factors. These data suggest that actin-filaments
SM proteins will be investigated. containing Tm5NM1 mediate G1 phase progression, potentially via
controlling the fidelity of signalling through the ERK/MAPK pathway.
This indicates that Tm5NM1 has potential as a novel anti-proliferative
therapeutic target.

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DEXAMETHASONE INHIBITS INTESTINAL SECRETORY AGED-RELATED INCREASE IN XANTHINE OXIDASE
CELL ER STRESS BY UPREGULATING DEGRADATION EXPRESSION AND ACTIVITY IN SPLEEN FROM MICE
OF MISFOLDED PROTEIN ER STRESS (ERAD)
Vida C.1, Gonzalez E.1, Hernandez O.1, Rodriguez-Teres S.1, Hernanz
Das I.1, 2 , Png C.W.1, Tran T.1, 3, Eri R.1, Lourie R.1, 3, Adams R.1, Oancea A.2, Corpas I.1 and De la Fuente M.1
I.1, 3, Crane D.1, 2, Florin T.1, 3 and McGuckin M.1, 3
1
Department of Physiology. Faculty of Biology. Complutense University
1
Mater Medical Research Institute. 2Griffith University. 3University Of of Madrid (Spain). 2Department of Biochemistry. La Paz Hospital.
Queensland. Madrid. Spain.

Endoplasmic reticulum (ER) stress in intestinal secretory cells has been In mammalian cells, one of the major sources of reactive oxygen
linked with the pathogenesis of Inflammatory Bowel Diseases (IBD) species (ROS) is the enzyme xanthine oxidase (XO), which uses
which introduces ER stress, the unfolded protein response (UPR) and hypoxanthine and xanthine as reducing substrates producing ROS,
ER-associated degradation (ERAD) of misfolded proteins as pathways such as superoxide anion and hydrogen peroxide. XO has been
for therapeutic targeting in IBD. Glucocorticosteroids are an effective implicated in a variety of pathophysiological states and oxidation stress-
treatment for IBD, but little is known of their effect on ER stress. This related diseases. In addition, ageing is based on chronic stress, with an
study determined the effect of a glucocorticosteroid (dexamethasone, excessive production of ROS and a loss of the antioxidant defences, in
Dex) on ER stress in cultured LS174T colonic cells and in Winnie which the immune system has an important role. The aim of the present
mice with intestinal secretory cell ER stress caused by Muc2 mucin work was to determine the age-related changes in XO expression and
misfolding. mRNA expression of ER stress markers GRP78, spliced- activity in mouse spleen. ICR-CD1 adult (6 month-old), mature (13
XBP1, ATF6 and CHOP; glycoprotein chaperones CNX and CRT; and month-old), old (18 month-old) and long-lived (31 month-old) female
ERAD genes EDEM1, VCP and SEC61 were measured by qRT-PCR. mice were used. Animals were sacrificed and spleen was obtained,
Histological analysis was used to assess intestinal inflammation and which was homogeneized in order to determine XO activity using a
mucin biosynthesis. Upregulation of GRP78 (a key indicator of ER stress, comercial kit (Invitrogen). The XO protein expression was assayed by
15.1±1.3 and 14.3±1.0 fold increased by tunicamycin and thapsigargin in western-blot analysis. The results showed that both XO expression
LS174T cells, respectively) was completely inhibited by Dex (P<0.001). and activity increase with ageing. XO activity levels were significantly
All other ER stress markers followed the same pattern. Winnie mice increased in old (p≤0,001) and long-living (p≤0,001) mice with respect to
showed accumulation of misfolded Muc2 in secretory cells and a 40±3 adult mice. Finally, we analyzed the XO protein expression in this tissue,
fold increase in intestinal Grp78 compared to wild-type mice, and which showed a significant increase in old (p≤0,05) and long-living mice
elevated expression of all other ER stress/UPR markers. Four weeks (p≤0,01). Nevertheless, mature mice show similar XO activity levels
treatment with Dex significantly inhibited the increase in ER stress and XO protein expression as compared to the adults. In conclusion,
genes (P<0.001) and substantially increased production of correctly- the increase in the XO expression and activity in spleen from older
folded Muc2 (P<0.001). The ERAD genes Edem1 and Vcp were the mice may have a crucial role in the pathophysiological changes of
only ER stress-related genes that increased in Dex-treated mice. These the ageing process. MICINN(BFU2008-04336); Research-Group-
results suggest that Dex ameliorates ER stress by enhancing removal UCM(910379ENEROINN); RETICEF (RD06/0013/0003).
and degradation of the mis-folded Muc2 from the secretory cells by up-
regulation of proteins involved in the ERAD pathway.

POS-MON-151 POS-TUE-152
KRüPPEL-LIKE FACTOR 3 (KLF3) AND THE UV- THE SECRETIN PROTEIN SUPER-FAMILY: NEW
RESPONSE PATHWAY STRUCTURES FOR BACTERIAL OUTER MEMBRANE
PROTEINS
Dewi V.A., Pearson R.C.M. and Crossley M.
School of Biotechnology and Biomolecular Sciences, University of Dunstan R., Alcock F., Webb C. and Lithgow T.
New South Wales, NSW, 2052. Department of Biochemistry & Molecular Biology, Monash University,
Clayton, 3800 Australia.
Following DNA damage, cells enter either cell cycle arrest or apoptosis
depending on the severity of the damage. Upon extensive DNA damage It was considered for some time that all integral membrane proteins in
by ultra violet (UV) light, a master regulator of cell cycle and apoptosis, bacterial outer membranes had an architecture referred to as a β-barrel,
p53, is phosphorylated by the kinase, Homeodomain-interacting protein and recently it has become clear that these proteins are assembled by
kinase 2 (Hipk2). This phosphorylation of p53 stabilises the protein the BAM complex. Very recently a new architecture of bacterial outer
and is recognised as a hallmark of apoptosis. The transcriptional co- membrane proteins was discovered: large, multimeric complexes
repressor C-terminal binding protein 2 (Ctbp2) is also phosphorylated by containing amphipathic helical transmembrane domains rather than
Hipk2 upon DNA damage. In contrast to p53, phosphorylation of Ctbp2 the conventional β-barrel structures. These “secretins” form pores for
marks it for degradation. Krüppel-like factor 3 (Klf3) is a transcriptional the secretion of proteins of the type II and type III secretion systems,
repressor known to recruit Ctbp2 to silence target genes. Using in vitro formation of type IV pili and the assembly of filamentous bacteriophage.
kinase assays we have shown that Hipk2 can also phosphorylate Klf3. They also form the outer membrane collar of bacterial flagella. The
This led us to hypothesise that Klf3 plays a role in the UV-response precise mechanism and kinetics of secretin assembly and insertion
pathway. Using NIH-3T3 as a model cell line, we conducted time course into the OM is unclear. Using a Hidden Markov Model (HMM) strategy,
experiments to study the effect of UV irradiation on the expression of based on sequences of known secretins, novel candidate secretins
Klf3 and its target genes. We observed downregulation of both Klf3 and have been identified from the enteropathogenic Escherichia coli strain
its co-repressor Ctbp2 following irradiation. We are currently exploring E2348/68. We are tailoring an assay in which the kinetics of assembly of
the hypothesis that changes in expression of Klf3 target genes play an these secretins can be determined and aim to determine the machinery
important role in the response to DNA damage. responsible for secretin assembly in bacterial outer membranes.

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POS-MON-153 POS-TUE-154
POLLEN-SPECIFIC DEPOLL GENE EFFECTS THE PROLIFERATIVE AND SURVIVAL PATHWAYS IN
EXPRESSION OF LTP PROTEINS IN ARABIDOPSIS OESOPHAGEAL CANCER
THALIANA
Esau L.E. and Hendricks D.
Duplakova N.1, 2 and Honys D.1, 2 University of Cape Town, Cape Town, Western Province, South Africa.
1
Laboratory of Pollen Biology, Institute of Experimental Botany ASCR,
Rozvojová 263, 165 02 Prague 6, Czech Republic. 2Department of Oesophageal cancer is the 7th most common cancer worldwide and
Plant Experimental Biology, Faculty of Science, Charles University, available treatment does not significantly enhance patient survival. The
Viničná 5, 128 44 Prague 2, Czech Republic. epidermal growth factor receptor EGFR is commonly over-expressed
in oesophageal cancer and its over-expression correlates with tumour
Plant cell wall is highly organized and complex structure consisting of aggression and survival. Reports exists that the insulin-like growth factor
carbohydrates, proteins and aromatic compounds surrounding and receptor 1 IGF-1R plays a role in survival and that interactions exist
separating cells from outer environment. It has been assigned many between EGFR and IGF-1R. The objective of this study is to determine
different functions like regulation of cell volume, protoplast protection, the role of EGFR and IGF-1R in proliferation and survival of oesophageal
cell shape determination and many others including its key role in cancer. EGFR and IGF-1R function in cultured oesophageal cancer
morphogenesis. The pollen wall represents its special type as it is cell biology was determined by using western blot analysis, inhibitor to
composed by two distinct layers - exine and intine. Moreover, pollen wall EGFR or IGF-1R shRNA. Receptors were activated with specific ligand.
is covered with unique and specialized lipid pollen coat with functions EGFR kinase domain phosphorylation activated IGF-1R as receptors
in pollen dispersal and pollen stigma recognition. The list of cell wall were shown to immunoprecicpitate with each other. Targeting EGFR
proteins is very extensive and one of these, lipid-transfer proteins resulted in decreased proliferation and an IC50 of +/-10uM however
(LTPs), are present in pollen wall separable fraction. LTP proteins are increased Akt activity was observed. IGF-1R knockdown resulted in
basic, soluble, 9-kDa proteins representing up to 4% of total soluble increased pEGFR and pERK1/2 with diminished Akt activity which lead
proteins in higher plants. They were found to be secreted and located to a 3 fold increased sensitivity to EGFR inhibitor compared to EGFR
extracellularly, in the cell wall, although earlier hypotheses considered inhibition alone. EGFR and IGF-1R show potential as drug targets in
their function in intracellular lipid dynamics. Several roles for LTPs Oesophageal cancer as combined targeting of these two receptors
were suggested in plant growth and development, defense reactions show increased response than targeting EGF receptor alone.
against phytopathogens, symbiosis and the adaptation of plants to
various environmental conditions. Here we show that T-DNA insertion
in pollen-specific gene depoll encoding 21 kDa (185 aa) C2 protein
caused increased expression of numerous LTPs in male gametophyte
accompanied by defects in pollen cell wall formation or cell death.
Acknowledgment: Authors gratefully acknowledge the financial support
from the Czech Science Foundation (grant 522/09/0858) and Ministry
of Education, Youth and Sports of the Czech Republic (grants LC06004
and OC10054).

POS-MON-155 POS-TUE-156
TARGETS OF RNA-BINDING PROTEIN MUSASHI-1 IN THE GOLGI-LOCALISED CALCIUM ATPASE IN BREAST
MOUSE GERM CELL DEVELOPMENT CANCER
Fraser B.A.1, 2 , Sobinoff A.2, Pye V.J.2, Roman S.D.1, 2, Hime G.R.1, 3, Grice D.M.1, Vetter I.2, Faddy H.M.1, Kenny P.A.3, Monteith G.R.1 and
Siddall N.A.1, 3, Koopman P.1, 4 and McLaughlin E.A.1, 2 Roberts-Thomson S.J.1
1
ARC Centre of Excellence in Biotechnology & Development. 1
School of Pharmacy, The University of Queensland, Brisbane, QLD,
2
Reproductive Science Group School of Environmental & Life Science, 4072, Australia. 2Institute for Molecular Biosciences, The University
University of Newcastle, Callaghan NSW 2308. 3Dpt of Anatomy & of Queensland, Brisbane, QLD, 4072, Australia. 3Department of
Cell Biology, University of Melbourne, Parkville VIC 3010. 4Institute for Developmental and Molecular Biology, Albert Einstein College of
Molecular Biosciences, University of Queensland, St Lucia QLD 4072. Medicine, Bronx, New York, USA.
RNA-binding proteins, such as Musashi-1 (Msi-1), can contribute to Golgi-localised calcium ATPases maintain the calcium concentration
posttranscriptional control and are believed to have an important role of the Golgi apparatus and as such are associated with cell signalling,
in the maintenance of germline stem cells and germ cell differentiation. apoptotic signalling and post-translational modification of proteins.
We have previously confirmed that Msi-1 is predominately expressed We assessed the mRNA expression of one of these Golgi-localized
in gonocytes and mitotic spermatogonia. Protein-RNA pulldown and calcium ATPases in 295 breast tumour samples classified into five
microarray and bioinformatic analysis has provided us with a number transcriptional subtypes each associated with a different prognosis and
of possible targets of Msi-1 in the testes. Further to this, we aimed: i) response to therapeutic treatment. The Golgi-localised calcium ATPase
to generate mice that over-express Msi-1 in differentiating male germ was enriched in particular breast cancer subtypes. To characterize
cells and examine the resultant phenotype; (ii) to use RNA interference the role of this calcium ATPase in breast cancer, its expression was
to knockdown the expression of Msi-1 in cultured spermatogonial cells; silenced in MDA-MB-231 MCF7 and T-47D breast cancer cell lines
and (iii) to use a Musashi-1 specific antibody to immunoprecipitate target using Dharmacon OnTarget plus Smartpool siRNA. We assessed
RNA from a spermatogonial cell lysate. Initial phenotypic analysis of the the consequences of ATPase silencing on cell viability, response to
transgenic mice indicated that, whilst these mice produced spermatozoa, induction of endoplasmic reticulum stress and affects on G-protein
they have reduced fertility or are infertile. They have anomalous sperm coupled receptor cytosolic calcium signaling stimulated by trypsin,
formation and the sperm have a decreased ability to bind to mature thrombin and ATP using a high throughput FLIPRTETRA imaging system
oocytes. Increasing transgene expression resulted in male sterility and the potential regulation of Golgi-resident enzymes. Our results
due to spermatogenic arrest and a total absence of mature sperm. suggest that alterations in the expression of a Golgi-localised calcium
Quantitative gene expression of the putative targets demonstrated ATPase may be a feature of specific breast cancer subtypes, and that
significantly increased transcript levels with concomitant increase in inhibition of the expression of these calcium pumps differentially affects
Msi-1 transgene expression. The relative expression of the target genes pathways important in breast cancer progression.
was down-regulated in cultured spermatogonia following shRNA Msi-1
knockdown. Immunoprecipitated mRNA from spermatogonial lysate was
significantly enriched with Msi-2 mRNA. In conclusion, our preliminary
results indicate that Msi-1 is required for normal spermatogenesis and
that it may target a number of mRNAs, particularly Musashi-2, to achieve
posttranscriptional control of mouse spermatogonial differentiation.

OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 139


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POS-MON-157 POS-TUE-158
CONTROL OF CELL GROWTH AND SURVIVAL VIA OXIDATION AND INFLAMMATION INCREASE WITH
14-3-3 (ZETA) AND TYPE 1 INSULIN-LIKE GROWTH AGEING IN IMMUNE CELLS FROM MEN AND WOMEN
FACTOR RECEPTOR
Mate I.1, Arranz L.1, Garcia-San Frutos M.V.1, Vida C.1, Hernanz A.2 and
Hadzir S.1, Ramshaw H.2, Krake R.2, Delaine C.1, Guthridge M.2, De La Fuente M.1
Wallace J.C.1, Lopez A.F.2 and Forbes B.E.1
1
Department of Physiology. Faculty of Biology. Complutense University
1
School of Molecular and Biomedical Sciences, University of Adelaide, of Madrid. 2Biochemistry Department. La Paz Hospital. Madrid.
Adelaide SA,5005. 2Division of Human Immunology, Centre for Cancer (Spain).
Biology, Adelaide, SA, 5000.
A direct relationship exists between ageing and increasing oxidation
We have developed the first isoform specific 14-3-3ζ-/- mouse. 14-3-3ζ- and inflammation. The immune cell functions change with ageing
/-
mice exhibit post-natal growth deficiency (P7 onwards) with 20-30% and this immunosenescence seems to be a consequence of this
reduction in size compared to wild type littermates. 14-3-3 proteins are age-related oxidative and inflammatory stress. We have proposed a
intracellular phosphoserine/phosphothreonine binding proteins involved relevant role of the immune cells on oxi-inflamm-ageing. The aim of
in modulation of crucial biological processes such as proliferation, signal the present work was to determine the age-related changes in several
transduction, metabolism, cell cycle and apoptosis. Importantly, 14-3-3ζ oxidative and inflammatory parameters as well as in some oxidative
binds the type 1 insulin-like growth factor receptor (IGF-1R) and several stress-related functions in leukocytes from men and women. A total
proteins involved in IGF-1R downstream signalling pathways including of 334 healthy human volunteers were studied (171 women and 163
IRS-1 and 2, p85, Akt and Raf. These interactions modulate IGF-1R men), and divided into three age groups: adult (30-49 years of age),
survival, proliferation and transformation activities. Initial observations adult-mature (50-59 years) and mature (60-79 years). Blood samples
suggest a perturbation of the GH/IGF-I axis in 14-3-3ζ-/- mice. Preliminary were extracted and the following parameters analyzed in neutrophils
analyses have revealed low pituitary growth hormone (GH) levels and and lymphocytes: adherence to tissues, spontaneous mobility (SM),
low serum IGF-I (~ 2.7 fold), IGF binding protein-3 (IGFBP-3) and acid chemotaxis, phagocytosis, proliferation of lymphocytes in response to
labile subunit (ALS) levels. 14-3-3ζ-/- mice growth deficiency is evident mitogens, superoxide anion (SA) levels, TNF-α secretion in response to
before weaning (P7 onwards) whereas GH deficiency models show LPS, as well as plasma C-reactive protein (CRP). The results showed
growth deficiency only at puberty and IGF-I knockout mice are pre and statistically significant increases of CRP, SA, TNF-α, adherence and SM
postnatally growth deficient The phenotype suggests it is different to in mature groups with respect to adults, whereas the other functions
most mouse models of IGF-I deficiency, although it is reminiscent of the were decreased. These changes were more significant in women than
hypothalamic IGF-1R knockout phenotype. Interestingly, we have shown in men. Thus, oxi-inflamm-ageing seems to increase in postmenopausal
a perturbation in the IGF-1R signalling via Akt and Erk1/2 in embryonic women as a possible consequence of the loss of the protective role of
fibroblasts derived from the 14-3-3ζ-/- mice. Therefore, our evidence so oestrogens. Financial support: MICINN (BFU2008-04336); Research-
far suggests that the growth deficiency is driven by abnormalities in GH/ Group-UCM (910379ENEROINN); RETICEF (RD06/0013/0003).
IGF-I axis and perturbation in 14-3-3ζ action involving IGF-1R signalling
(via Akt and Erk). The mechanism underlying the GH deficiency is still
not clear.

POS-MON-159 POS-TUE-160
ASSEMBLY OF MITOCHONDRIAL PORINS: MARSUPIAL INTERFERON INDUCIBLE
ADAPTATION TO AN ENDOSYMBIOTIC LIFESTYLE TRANSMEMBRANE PROTEINS
Hewitt V.L., Gabriel K., Traven A. and Lithgow T.J. Hickford D.E., Shaw G. and Renfree M.B.
Department of Biochemistry and Molecular Biology, Monash Zoology Department, The University of Melbourne.
University, Melbourne.
The Interferon Inducible Transmembrane (IFITM) family is a group of
The outer membrane of mitochondria is the interface between the cell surface proteins with roles in diverse cellular processes, including
organelle and its host. The SAM (sorting and assembly machinery) is promoting homotypic cell adhesion, mediating cell differentiation
embedded in this membrane where it helps insert and assemble vital and acting as a transducer of anti-proliferative signals. In humans,
membrane proteins and protein complexes. The core components of IFITM genes are up-regulated in some cancers and IFITM2 acts as
the SAM complex are conserved across eukaryotes but it appears to be a pro-apoptotic factor. In the mouse and human the IFITM genes are
able to co-opt additional subunits for some tasks. While previous studies clustered; five Ifitm genes within a 68 kb locus on chromosome 7 in
have identified some of these subunits there is still very little known the mouse and four genes within a 26.5 kb locus on chromosome 11 in
about how the core of the sorting and assembly process really works humans. Sequence comparison suggests that gene duplications at the
even for the most abundant proteins, such as porin (VDAC). Though mouse and human IFITM locus have occurred independently in both
extensively studied in Saccharomyces cerevisiae, understanding the species. Despite the broad range of processes the IFITM genes have
details of the assembly and insertion of porin has been hampered by been implicated in, nothing is known about marsupial IFITMs. We have
the instability of the porin complex. Preliminary studies in Candida identified five IFITM homologs in the tammar wallaby Macropus eugenii,
albicans show porin assembles more quickly and into a more robust four of which are clustered in a 74.5 kb locus. There are four homologs
complex than in Saccharomyces. Import of radiolabeled proteins into in the opossum Monodelphis domestica, three of which are clustered on
purified mitochondria is used to elucidate the role of the SAM complex chromosome 3. Amino acid identity between the various IFITM proteins
in this process. This more stable model system enables a more is 27-76% in the tammar and 35-64% in the opossum. Sequence
thorough comparison of the core SAM subunit, Sam50 and its bacterial analysis suggests that similar to the mouse and human, IFITM genes
homologue BamA (Omp85). Understanding the origins, interactions and have been duplicated independently in marsupials. Using RT-PCR, we
roles of these components can help reveal how free-living bacteria were analysed the expression of IFITM genes in adult organs, embryos and
assimilated into the cell to become mitochondria. fetuses of the tammar wallaby. Expression of the tammar IFITM genes
differs to that in the mouse, suggesting that the roles of the IFITM genes
may not be conserved between marsupials and eutherians.

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POS-MON-161 POS-TUE-162
DIFFERENTIAL EXPRESSION OF HEDGEHOG CYTOTOXICITY AND OPTICAL-PHYSICAL STUDIES OF
SIGNALING COMPONENTS AND SNAIL/E-CADHERIN 6-ALKYNYL-COUMARIN DERIVATIVES
IN HUMAN BRAIN TUMORS
Hisao C. and Hong F.
Hong Y.R.1, 2 and Chou C.H.1, 2 Department of Chemistry, National Chung Hsing University, Taiwan.
1
Department of Biochemistry, Faculty of Medicine, College of Medicine,
Kaohsiung Medical University, Kaohsiung, Taiwan. 2Department of Many coumarin derivatives are biologically important molecules that
Biological Sciences, National Sun Yat-Sen University, Kaohsiung, possess antifungal, antibacterial, antimicrobial insecticidal, anti HIV and
Taiwan. antibiotic activites. The coumarin moiety as chromophore of fluorescent
ion indicator is a useful physical property for the bio-imaging technique
The Hedgehog (Hh) transcription factor Gli induces transformation in biological systems. It was expected that modification on coumarine
of epithelial cells via induction of Snail, a repressor of E-cadherin. with conjugated multiple bonds might bring about the desired change
Epithelial-mesenchymal transition is also a determinants of the in either red-shift of wavelength or intensity of the emission. It is our
progression of tumorigenesis, following downregulation of E-cadherin. intention to apply these compounds to biological systems by combining
However, the role of Hh signaling components and Snail/E-cadherin in both the cytotoxicity and fluorescence property. Several attempts in
brain tumors is not yet fully understood. We analyzed the expression of the modification of 6-Br-coumarine with substituted alkynes by the
Hh signaling components and Snail/E-cadherin in 69 brain tumors by Sonogashira reaction were pursued. Indeed, it led to the formation of
reverse transcription–polymerase chain reaction (RT-PCR). The data several coumarine derivatives with expected red-shift of wavelength
showed that overexpression of Smo (35/69), Ptch (50/69), Gli1 (56/69), in fluorescence. It was also found that optical property was affected
Gli2 (29/69) and N-myc (39/69) might contribute to brain tumorigenesis. greatly by slight change the substitutents on the phenyl moiety which
Our results also indicated that Snail and E-cadherin showed opposing is linked to coumarin mainframe through a triple bond. Moreover, we
expression in malignant tumors (high grade astrocytoma and metastasis). applied these coumarine derivatives to biological systems, especially,
Snail and E-cadherin showed less correlation in benign brain tumors. We on the study of cytotoxicity towards HeLa cells. These newly-made
further investigated mutations of Gli2 and Snail by RT-PCR and direct compounds indeed showed higher cytotoxicity towards cells compared
sequencing. No mutation was observed on Gli2 but several sporadic with the unmodified one.
mutations on Snail were found, including S96G, S111L, S111L/S119Y
and one nonsense mutation at codon 158 (Y158*). An in vitro E-cadherin
promoter assay showed that S96G, S111L, S111L/S119Y Snail mutants
were decreased 15%, 25% and 50%, respectively, whereas Y158* was
increased 40% compared to wild type. Furthermore, our data showed
that wild type Snail and S96G, S111L, S111L/S119Y translocated into the
nucleus, while the Y158* mutant failed to translocate into the nucleus.
Taken together, our results demonstrate that Hh signaling components,
the expression and mutations of Snail, and the expression of E-cadherin
may play an important role in human brain tumorigenesis.

POS-MON-163 POS-TUE-164
IDENTIFICATION OF PROTEIN-PROTEIN THE HIJACKING OF MITOCHONDRIAL IMPORT
INTERACTIONS REGULATING SODIUM-PROTON MECHANISMS BY PORB FROM NEISSERIA
ANTIPORTER ACTIVITY MENINGITIDIS
Huynh D.1, 2 and Gendall T.1 Jiang J.-H.1, 2 , Davies J.3, Strugnell R.A.2, Lithgow T.1 and Gabriel K.1
1
Department of Botany, La Trobe University, Bundoora, Victoria 3086, 1
Department of Biochemistry and Molecular Biology, Monash
Australia. 2Cuulong Delta Rice Research Institute, Cantho, Vietnam. University, Victoria, Australia. 2Department of Microbiology and
Immunology, the University of Melbourne, Victoria, Australia.
In plants, the concentration and ratio of Na+ and K+ is crucial for 3
Department of Microbiology, Monash University, Victoria, Australia.
homeostasis, particularly in stressful conditions, such as salinity, cold
and drought. Ion homeostasis in plants is regulated by a large number PorB is a β-barrel protein from the pathogen Neisseria meningitidis
of ion exchangers that control the movement of ions across the plasma that is targeted to host cell mitochondria and modulates apoptosis
and intracellular membranes. The class of sodium-proton exchangers during bacterial invasion. The sorting and assembly machinery (SAM)
designated as NHEs in animals or NHXs in plants and yeast, is highly is required for eukaryotic β-barrel protein import into mitochondria.
conserved throughout eukaryotes. NHX5 and NHX6 are intracellular- To investigate the mechanisms behind PorB import, S35 -labeled PorB
localized proteins that are widely conserved in diverse plants. The was in vitro translated and incubated with mitochondria isolated from
C-terminal tail of NHX5 and NHX6 is also well conserved in many plant mouse tissue or import machinery deficient yeast strains. Folded
species suggesting that it may mediate a protein-protein interaction and PorB was distinguished from unfolded protein using semi-native gel
act as a part of pH sensing mechanism. To investigate the functional electrophoresis. The rate of PorB folding in mammalian mitochondria
significance of this well conserved C-terminal tail in NHX5 and NHX6, is decreased if the mitochondrial outer membrane is ruptured by hypo-
a yeast 2-hybrid screen has been performed to identify interacting osmotic shock. This treatment releases soluble inter-membrane space
proteins. The predicted C-terminal tail and the highly conserved regions (IMS) proteins such as the small Tim(s) and hence this suggests a role for
of both NHX5 and NHX6 have been used as baits to screen existing IMS proteins in PorB folding. Mitochondria isolated from Sam50 mutant
2-hybrid libraries. Positive colonies will be identified, and the interaction which are deficient in import of native mitochondrial β-barrel proteins
further will be analyzed using by deletion analysis of both the C-terminal also fold PorB at a slower rate. Taken together, the import of PorB into
region of NHX5 and NXH6, and the interacting protein(s). These results mitochondria is SAM pathway dependent. The exact localization of
will be validated by in vitro pull down assays using recombinant proteins. PorB in mitochondria and the roles of other SAM components will also
To determine if NHX5 and NHX6 function as homo- or hetero-dimers, be assessed in the future.
interactions between NHX5 and NHX6 have also been investigated by
yeast two-hybrid system, bimolecular fluorescence complementation
and co-immuno-precipitation.

OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 141


POSTERS MONDAY & TUESDAY

POS-MON-165 POS-TUE-166
EXPRESSION AND REGULATION OF THIOREDOXIN IN PHYSIOLOGICAL SIGNIFICANCE OF D-AMINO
CANCER CELLS DURING HYPOXIA ACID DEGRADATIVE ENZYMES IN NEMATODE
CAENORHABDITIS ELEGANS
Karlenius T.C.1, 2 , Shah F.L.1, 2, Clarke F.M.1, 2 and Tonissen K.F.1, 2
1
School of Biomolecular and Physical Sciences, Griffith University, Katane M.1, Saitoh Y.1, Seida Y.1, Kawata T.1, Maeda K.1, Sekine M.1,
Nathan QLD 4111, Australia. 2Eskitis Institute for Cell and Molecular Furuchi T.1, Kobuna H.2, Sakamoto T.1, Inoue T.3, Arai H. 3, Nakagawa Y.
Therapies, Griffith University. 1
and Homma H. 1
1
Department of Pharmaceutical Life Sciences, Kitasato University,
Redox homeostasis is crucial for cell survival. Too much oxygen in the Tokyo, Japan. 2School of Medicine, Tokyo Women’s Medical University,
cell leads to oxidative stress through the production of ROS, which Tokyo, Japan. 3Graduate School of Pharmaceutical Sciences, The
reacts with the cells macromolecules causing cell damage and finally University of Tokyo, Tokyo, Japan.
cell death. The cells defend themselves against oxidative stress through
the production of antioxidants, which either neutralise ROS or reverse Among free D-amino acids existing in living organisms, D-serine
ROS-induced damage. In contrast, low oxygen levels in the cell lead (D-Ser) and D-aspartate (D-Asp) are the most actively studied. D-Ser
to hypoxia. Under hypoxic conditions a signalling pathway involving a has been proposed as a neuromodulator that regulates L-glutamate-
key regulator termed hypoxia-inducible factor (HIF) is switched on. HIF mediated activation of the N-methyl-D-Asp (NMDA) receptor by acting
drives the induction of many genes controlling multiple cell functions as a co-agonist. On the other hand, D-Asp has been proposed to
such as angiogenesis, metabolism and apoptosis/survival. Thus, the play important roles in regulating developmental processes, hormone
level of oxygen in a cell dictates the molecular response of cells through secretion and steroidogenesis. D-Amino acid oxidase (DAO) and D-Asp
modulation of gene expression. Furthermore, both oxidative stress and oxidase (DDO) are known as stereospecific degradative enzymes that
hypoxia are common features of solid tumours. Both oxidative stress and catalyze the oxidative deamination of D-amino acids. DAO and DDO
hypoxia lead to changes in the cellular redox balance within cancer cells. reportedly regulate endogenous D-Ser and D-Asp levels, respectively,
High levels of antioxidants and redox control systems, especially the as well as mediate the elimination of accumulated exogenous D-amino
Thioredoxin system, are often observed in cancer cells and are believed acids in various organs. Previously, we demonstrated that nematode
to play a major role in cancer progression. We are currently investigating Caenorhabditis elegans has at least one active DAO gene and
the expression and regulation of Thioredoxin in breast cancer cell lines three active DDO genes, and that these enzymes exhibit different
cultured in hypoxic conditions and after re-oxygenation. and distinctive enzymatic properties. In this study, to elucidate the
physiological roles of the C. elegans DAO and DDOs, we examined the
localization of these enzymes within the whole body of C. elegans using
green fluorescent protein-based gene expression analysis, revealing
that the spatiotemporal distributions of these enzymes differ from one
another. We also examined several phenotypes of four C. elegans
mutants in which each gene for these enzymes is partially deleted and
inactivated. We will report the phenotypes of these C. elegans mutants
in comparison with those of wild-type C. elegans, as well as alterations
in D-amino acid levels within the body.

POS-MON-167 POS-TUE-168
CIAP ANTAGONISTS AND IFNγ ACTIVATE CASPASE INVESTIGATION OF HOW RASACT COOPERATES
AND RIPK1 DEPENDANT DEATH PATHWAYS IN WITH RHOGEF2 OVER-EXPRESSION AND SCRIBBLE
CANCER CELLS MUTANTS IN TUMOURIGENESIS
Khan N.1, Miasari M.1, Chau D.1, Wong W.W.-L.1, Mckinlay M.2, Khoo P., Brumby A.M., Humbert P.O. and Richardson H.E.
Chunduru S.K.2, Benetatos C.A.2, Condon S.M.2, Vaux D.L.1 and Silke Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne,
J.1 Victoria.
1
La Trobe University, Melbourne, VIC 3086, Australia. 2TetraLogic
Pharmaceuticals 343 Phoenixville Pike Malvern, PA 19355. scribble (scrib) is a tumour suppressor gene which regulates cell polarity
in Drosophila epithelial cells. Expression of activated Ras (RasACT) in
Synthetic IAP antagonists have been shown to kill tumour cells as scrib - clones in the Drosophila eye disc results in the development of
single agents or sensitise them to existing anti-cancer treatments, tumours, characterised by decreased cell death, loss of cell polarity,
validating Inhibitor of apoptosis (IAPs) proteins as targets for anti- hyper-proliferation and reduced differentiation. RhoGEF2, a gene
cancer therapeutics. We have previously shown that a peptide-mimetic involved in cell shape changes, also cooperates with RasACT in eye
IAP antagonist compound has similar effects on cells as the TNF family disc clones to result in tumourigenesis. Jun kinase (jnk) activity is both
cytokine, TWEAK. Because some cancer lines can be sensitised to up-regulated and required for tumourigenesis in RhoGEF2 and RasACT
TWEAK cytotoxicity by co-treatment with IFNγ, we hypothesised that tumours and scrib - + RasACT tumours. We compare the development
IFNγ might synergise with IAP antagonists to kill tumor cells. Consistent of tumours arising from the co-expression of RhoGEF2 and RasACT in
with our hypothesis, tumor cells that are sensitive to TWEAK/IFNγ eye disc with scrib - + RasACT tumours and investigate the pathways
were killed when treated with IFNγ and IAP antagonist while primary downstream of RhoGEF2 in its cooperation with RasACT in clones.
untransformed lines were unaffected. JAK/STAT signaling was We show that RhoGEF2 + RasACT clonal tissue over-grows but not to
required for cell death because synergistic killing could be blocked the same extent as in scrib - + RasACT tumours and is not as invasive.
by SOCS1 overexpression, but the TNFR1 pathway was not. Another Preliminary data indicate that when Rho 1, Rho kinase or Zipper levels
distinguishing feature of this IAP antagonist/IFNγ death was that it could are reduced in RhoGEF2 + RafGOF (a downstream effector of Ras) clones
not be blocked by caspase inhibition alone even though cells displayed using UAS-RNAi transgenes, morphological defects are suppressed and
classic apoptotic features. Surprisingly, caspase inhibition together with differentiation is restored. These results suggest that Rho1, Rho kinase
a RIPK1 inhibitor (Nec1) significantly attenuated synergistic killing by and Zipper are required for tumourigenesis arising from RhoGEF2 and
Compound A and IFNγ, as did RIPK1 knockdown. Our results suggest RafGOF co-expression in clones.
that IAP antagonists can activate both apoptotic and non-apoptotic cell
death which may extend their clinical use.

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POS-MON-169 POS-TUE-170
PHYSIOLOGICAL FUNCTION OF A NOVEL NF-κB- REACTIVE OXYGEN SPECIES GENERATED THROUGH
REGULATING MOLECULE, NUCLING, IN IMMUNE NADPH OXIDASE MEDIATE UV-INDUCED GSK-3BETA
SYSTEM ACTIVATION
Kim S.M., Sakai T., Tran N.H. and Fukui K. Kim H.S., Kwon Y.H., Kim J.Y. and Sohn J.
Division of Enzyme Pathophysiology, The Institute for Enzyme Department of Biochemistry & Molecular Biology, Korea University
research, The University of Tokushima. College of Medicine, Seoul 136-705, Korea.

Nucling is a novel stress-induced protein isolated as a marker for Previously, we have reported that UV induced ATR-dependent GSK-3β
cardiac development. Recent our studies have revealed that Nucling is a activation and subsequent proteasomal degradation of p21 protein in an
regulator of nuclear factor-kappa B (NF-κB) operated by its cytoplasmic ubiquitination-independent manner. Here, we show that reactive oxygen
retention through the physical interaction with Nucling. Notably, Nucling species (ROS) are generated by UV irradiation and GSK-3β activation.
is considered to be important for the regulation of NF-κB signals in liver. ROS generation was detected in the cytosol and mitochondria at 5 min
Nucling-KO mice showed several liver dysfunctions including hepatitis after UV exposure. NADPH oxidase contributed to the ROS generation
and cancer with some defects of NF-κB signal in liver. In addition, a since chemical inhibitors of NADPH oxidase, DPI and AEBSF, abrogated
it. NOX4 mRNA was expressed in MCF-7 cells, and transfection of the
number of kupffer cells, residential macrophages in liver, and hepatic cells with NOX4 siRNA prevented UV-induced ROS generation. DPI
dendritic cells apoptotically decreased in the Nucling-KO mice. Thus we and apocynin also inhibited UV-induced GSK-3β activation indicating
focused on the function of Nucling in immune system. Further analyses that ROS generated by NADPH oxidase mediated GSK-3β activation.
revealed that macrophages of the Nucling-KO mice were decreased in Further supporting redox-sensitive activation of GSK-3β, treatment of
lung. On the other hand, Nucling-KO mice were resistant to endotoxin cells with hydrogen peroxide activated GSK-3β. ROS scavengers and
(Lipopolysaccharide) similarly to TNFα-KO mice. It is correspond to our NOX4 siRNA abolished p21 protein degradation after UV-irradiation,
previous report showing the defect of NF-κB signal in Nucling-KO mice. confirming the previous finding that p21 phosphorylation by GSK-3β
Our recent studies indicated that the spleen of Nucling-KO mice showed was required for p21 degradation. To investigate whether changes in
abnormally activated germinal centers without immunologic stimulation. the [Ca2+]i mediated NADPH oxidase activation after UV irradiation,
Besides, we observed dissimilarity of B cell subset distribution between the effect of Ca2+ chelators on ROS production was assessed. Indeed,
Nucling-KO mice and wild-type mice. Therefore, we speculate that there pretreatment with EGTA or BAPTA-AM blocked ROS generation
are some differences between Nucling-KO mice and wild-type mice indicating Ca2+-dependent activation of NADPH oxidase. In MCF-7
concerning the functions of macrophages in the aspect of its quantity or cells, increase in [Ca2+]i was detected < 5 min after UV irradiation and
activity. Nucling is considered to play an important role in the immune continued up to 2 h. Since GSK-3β activation by UV was shown to be
system with the regulation of NF-κB signal pathway. dependent on ATR, it was next studied whether an increase in [Ca2+]i
and ROS generation were upstream to ATR activation. Phosphorylation
of ATM/ATR substrates after UV irradiation was observed 15 min after UV
irradiation. Pretreatment of cells with BAPTA-AM, Tiron or NAC prevented
their phosphorylation indicating that ATR activation was dependent on
intracellular Ca2+ and ROS. Taken together, UV mobilizes intracellular
Ca2+ and thereby activates NADPH oxidase. ROS generated through
NADPH oxidase then activate ATM/ATR and that, in turn, induces GSK-
3β activation and p21 degradation.

POS-MON-171 POS-TUE-172
A ROLE FOR SODIUM/PROTON EXCHANGERS AND GSK3β INTERACTS WITH AND PHOSPHORYLATES
INTRACELLULAR HYDROGEN ION CONCENTRATION BCL2L12 IN BRAIN CANCER CELLS
IN REGULATING VITAMIN C-DRIVEN ELECTRON
TRANSPORT ACROSS THE PLASMA MEMBRANE Lin C.C., Chen W.J, Lin R.C., Chou C.H., Wu C. and Hong Y.R.
Department of Biochemistry, Faculty of Medicine, College of Medicine,
Lane D.J.R. , Robinson S.R. , Czerwinska H. and Lawen A.
1 2 2 1
Kaohsiung Medical University, Kaohsiung, Taiwan.
1
Department of Biochemistry and Molecular Biology, School of Biomedical
Sciences, Monash University. 2School of Biomedical Sciences and A novel GBM oncoprotein, Bcl2-Like 12 (Bcl2L12, a BH2 containing
Psychology & Psychiatry, Monash University. protein), was recently identified which is significantly expressed in the
Ascorbate is the major electron donor to a transplasma membrane majority of primary GBM tumor specimens and distantly related to
electron transport (tPMET) system that was originally identified in human canonical Bcl-2 proteins. By using large scale yeast 2-hybrid screening,
erythrocytes [1]. This plasma membrane redox system appears to transfer Bcl2L12 was found as a GSK3β binding partner in testis cDNA library.
electrons from intracellular ascorbate to extracellular oxidants (e.g., non- Our data showed that Bcl2L12 middle fragment (118-240 residues)
transferrin-bound iron). Though this phenomenon has been observed in which locates outside of Bcl2L12 C-terminal BH2 motif is responsible
nucleated cells, its mechanism and regulation are not well understood. for the binding to GSK3β and also confirmed by far-western blotting
Here we have examined both facets of this phenomenon in K562 cells and experiment. In vitro kinase assay showed that GSK3β phosphorylates
primary astrocyte cultures. Using ferricyanide as the analytical oxidant Bcl2L12 at S156 but not Bcl2L12A (Bcl2L12 splicing transcript variant),
we demonstrate that tPMET is enhanced by dehydroascorbate uptake LiCl (GSK3 inhibitors) and phosphatase were used to confirm that
via facilitative glucose-transporters, and subsequent accumulation of GSK3β indeed phosphorylates Bcl2L12 at S156. Interestingly, the S156
intracellular ascorbate. Additionally, we demonstrate that this stimulation phosphorylation site on Bcl2L12 does not fit the GSK3β consensus
is not due to ascorbate that is released from the cells, but is dependent sequence (S/T-X-X-X-S/Tp or S/T-P). To explore the significance of
only on a restricted intracellular pool of the vitamin. Substrate-saturation the interaction between GSK3β with Bcl2L12 and Bcl2L12 S156A
kinetics suggest an enzyme-catalysed reaction across the plasma for assessing the morphological changes and caspases 2, 3 and 7of
membrane by an as-yet-unidentified reductase that relies on extensive apoptosis, we transfected Bcl2L12 and Bcl2L12 S156A into the HeLa
recycling of intracellular ascorbate. Inhibition of ascorbate-stimulated and Glioma cell lines (U87MG and GBM 8401).The data showed that
tPMET by the Na+/H+-exchange inhibitors amiloride and 5-(N-ethyl-N- Bcl2L12 induced apoptosis in Hela cell, whereas in GBM and U87MG
isopropyl)amiloride, which is diminished by bicarbonate, suggests that may function as anti-apoptosis. Ongoing studies will combine TMZ (an
tPMET activity may be regulated by intracellular pH. In support of this alkylaing agent against recurrent glioma) to examine how Bcl2L12 gets
hypothesis, tPMET in astrocytes was significantly inhibited by ammonium involved and which may function as a chemotherapy biomarker in brain
chloride-pulse-induced intracellular acidification, while it was significantly tumorgenesis.
stimulated by bicarbonate-induced intracellular alkalinisation. These
results suggest that ascorbate-dependent tPMET is enzyme-catalysed and
is modulated by NHE activity and intracellular pH [2]. Though the identity of
the putative enzyme(s) responsible for electron transfer across the plasma
membrane has yet to be identified, it has been suggested that members of
the ubiquitous cytochrome b561 family (e.g., Dcytb) are likely to be involved.
[1] Lane and Lawen (2009) Free Radic. Biol. Med. 47, 485-495. [2] Lane et
al. (2010) Biochem. J. 428, in press.

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POS-MON-173 POS-TUE-174
PLASTIDAL STARCH PHOSPHORYLASE IN SWEET P21-ACTIVATED KINASE 1 IS IMPORTANT FOR
POTATO ROOTS IS PROTEOLYTIDY REGULATED BY SURVIVAL OF COLORECTAL CANCER CELLS
THE 20S PROTEASOME
Lin Y.C., Chou I.M., Chen A.N., Cheng C.H., Chang S.C. and Juang R.H. Liu H., Huynh N., Baldwin G. and He H.
Department of Biochemical Science and Technology, Institute of Department of Surgery, Austin Health, The University of Melbourne,
Microbiology and Biochemistry, National Taiwan University, Taipei 106, Heidelberg, VIC, Australia 3084.
Taiwan.
Background and Aim: P21-activated kinase 1 (PAK1) functions
Starch phosphorylase (SP, EC 2.4.1.1) catalyzes the reversible as a key node in various signalling pathways leading to cell survival,
phosphorolysis of starch and produces glucose 1-phosphate (Glc-1-P) migration and growth. PAK1 is overexpressed and activated in several
as one of its products. Plants express two isoforms of SP, which are human tumors including colorectal cancer (CRC), which is the second
classified as low-affinity (L-form SP, L-SP or Pho1) and high-affinity most common cause of cancer death. PAK1 is also required for vascular
(H-form SP, H-SP or Pho2) types according to their binding affinities endothelial growth factor (VEGF) expression and consequently for
to starch. Although the role of L-SP in starch metabolism is unclear, angiogenesis, which in turn promotes tumor growth and metastasis.
several studies have found that the gene expression and catalytic VEGF is a downstream target gene of hypoxia-inducible factor 1α (HIF-
activity of L-SP correlate with starch content in plants, indicating that 1α) which is involved in cellular adaptation to hypoxia and important for
L-SP might be involved in starch biosynthesis. In addition, L-SP in cell survival. The aim of this study was to investigate the importance of
Arabidopsis leaves might play a role in the tolerance of abiotic stress PAK1 in CRC cell survival. Methods: PAK1 knock-down (KD) clones of
(Zeeman et al., 2004). Previous studies with sweet potato roots have the human CRC cell line DLD1 were obtained by stable transfection with
shown that the activity of L-SP may be regulated by proteolysis of the shRNA targeting PAK1. Hypoxia was mimicked by treatment with CoCl2
central 78-amino acid peptide (L78). Removal of L78 increased the and cell survival was measured by [3H]-Thymidine incorporation. Protein
catalytic activity of L-SP in a phosphorolytic direction (Chen et al., expression was determined by Western Blot and VEGF production was
2002). During the purification of L-SP from sweet potato roots, Chang measured by ELISA. Results: After treatment with 150μM CoCl2, cell
(1999) observed an unknown high molecular weight complex (HX), survival was significantly reduced by 40% in PAK1 KD cells (P<0.05). The
whose mobility is significantly slower than the typical L-SP on non- expression of HIF-1α and the production of VEGF were both significant
denaturing polyacrylamide gel electrophoresis, also performed L-SP lower in PAK1 KD cells compared to control cells. Conclusion: This
activity. Following this observation, we utilize mass spectrometry, study demonstrates that PAK1 is required for cell survival and VEGF
coimmunoprecipitation, agarose-gel based double diffusion, two- production of CRC cells. PAK1 may regulate these cellular processes
dimensional gel electrophoresis, and confocal microscopy as the through a HIF-1α-dependent pathway.
tools and demonstrate that HX was composed of L-SP and the 20S
proteasome. Furthermore, we found that HX was reduced immediately
after 45°C heat treatment, and then a stepwise degradation of L-SP
was observed in a time-dependent mode which was strongly inhibited
by MG132, suggesting that the 20S proteasome was involved in L-SP
turnover. In addition, kinetic studies indicated that the proteolytic
modification of L-SP might increase the binding affinity of L-SP against
soluble starch and subsequently enhance its phosphorolytic activity.
This work demonstrates the role of 20S proteasome as a regulator of
L-SP activity which might be controlled by environmental heat stress.

POS-MON-175 POS-TUE-176
PSPC1 IS AN IMPORTIN ALPHA REGULATION OF EMBRYONIC GLOBINS BY THE
NUCLEOCYTOPLASMIC TRANSPORT CARGO KRüPPEL-LIKE FACTORS
Major A.T.1, 3, Hogarth C.A.4, Miyamoto Y.2, 3, Young J.C.2, 3, Jans D.A.2, Mak K.S.1, Funnell A.P.W.1, Pelka G.J.2, Radziewic T.2, Power M.2, Tam
3
and Loveland K.L.1, 2, 3 P.P.2, Pearson R.C.M.2 and Crossley M.1
1
Department of Anatomy and Developmental Biology, Monash 1
School of Biotechnology and Biomolecular Sciences, University of
University, Australia. 2Department of Biochemistry and Molecular New South Wales, NSW 2052. 2Embryology Research Unit, Children’s
Biology, Monash University, Australia. 3The ARC Centre of Excellence Medical Research Institute, Westmead, NSW, Australia.
in Biotechnology and Development, Australia. 4School of Molecular
Biosciences, Washington State University, USA. Basic Krüppel-like Factor (Bklf/Klf3) is one of 17 members of the Krüppel-
like family of transcription factors that regulate biological processes
Importin proteins facilitate classical nuclear import by binding nuclear such as cell proliferation, differentiation and apoptosis. Bklf is highly
localisation sequences (NLSs) on cargo proteins and translocating expressed in erythroid tissues, consistent with its direct activation by
them into the nucleus. Through a Y2H screen of an E12.5 mouse Erythorid Krüppel-like Factor (Eklf), an erythroid-specific transcriptional
testis cDNA library, we identified paraspeckle protein 1 (PSPC1) as activator. Bklf knockout mice exhibit a mild deficiency in the erythroid
an importin α2 cargo. The long isoform of PSPC1 is 523aa, contains population Chromatin immunoprecipitation experiments have shown that
two putative NLSs and is highly expressed in the testis. PSPC1 is Bklf directly binds to the Klf8 gene which is up-regulated in the Bklf-null
a marker for paraspeckles, a distinct subnuclear domain formed erythroid tissues. Klf8, like Bklf, functions primarily as a transcriptional
around the non-coding RNA transcript, NEAT1, and present only in repressor, recognising similar DNA sequences and repressing genes by
differentiated cells. Paraspeckles are rich in RNA transcripts and two recruiting the same co-repressor, Ctbp. Given the similarities between
other core paraspeckle proteins, PSF and NONO. Recent research Bklf and Klf8, we hypothesize that Klf8 might functionally compensate
suggests that paraspeckles form a structural base by which A to I edited for the absence of Bklf and that this may account for the mild phenotype
RNA transcripts may be retained within the nucleus. We addressed of the Bklf knockout mouse. We have intercrossed Bklf and Klf8 mutant
the hypothesis that trafficking of PSPC1 into the nucleus and its mice and purified erythroid cells from compound mutants of various
subsequent localisation into paraspeckles is mediated specifically by genotypes. Affymetrix arrays have been performed to study the
importin α2. Binding between PSPC1 and both importin α2 and α6 differences in gene expression resulting from the disruption of Bklf or
was confirmed using recombinant proteins in an ELISA based binding Klf8 or both. Eliminating Klf8 activity in erythroid cells should reveal not
assay. The extent of PSPC1 nuclear import was quantitated following only the full biological function of Bklf, but also distinguish target genes
transient transfection of HeLa cells with constructs that over-express that are shared between or specific to Bklf and Klf8.
GFP-tagged full length and truncated (dominant negative) constructs
encoding several importins. Data are collected following co-transfection
of DsRed2-tagged PSPC1 or assessed following endogenous PSPC1
detection via immunostaining. Dominant negative importin α2 appears
able to reduce the number of detectable paraspeckles, indicating the
importance of regulated importin synthesis to cell function.

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POS-MON-177 POS-TUE-178
HEPATOCYTE GROWTH FACTOR (HGF) ACUTELY ACTIVATION OF INSULIN-LIKE GROWTH
REGULATES THE EPITHELIAL JUNCTIONAL FACTOR BINDING PROTEIN-3 (IGFBP-3) BY DNA-
CYTOSKELETON AND ZONULA ADHERENS BY DEPENDENT PROTEIN KINASE (DNA-PK) OR
CONTROLLING JUNCTIONAL MYOSIN VI ATAXIA TELANGIECTASIA MUTATED (ATM) IS NOT
NECESSARY FOR ITS PROAPOPTOTIC FUNCTIONS
Mangold S.
Institute for Molecular Bioscience, 306 Carmody Rd., Brisbane 4072, Marzec K.A., Martin J.L. and Baxter R.C.
QLD. Kolling Institute of Medical Research, University of Sydney, Royal
North Shore Hospital, St Leonards, New South Wales 2065, Australia.
HGF plays an important role in various morphogenetic events during
embryonic development, as well as being implicated in tumor invasion Chemotherapeutic drugs such as doxorubicin, which induce apoptosis
and metastasis. Many studies have investigated the long-term effect of by causing DNA double strand breaks (DSB), are opposed by the
HGF treatment on epithelial cells. HGF binds with high affinity to the phosphoinositide-3-kinase-related protein kinases, DNA-PK and ATM,
receptor tyrosine kinase c-Met. Activation of c-Met leads to recruitment which promote DSB repair. Our laboratory has previously described the
of adaptor proteins and in turn to activation of a multitude of down stream phosphorylation by DNA-PK of IGFBP-3, which was reported by others
signalling molecules, including PI3-Kinase, Src and Ras/MAPK. The to be required for the pro-apoptotic function of IGFBP-3. The aim of
resulting signalling cascades initiate a broad range of cellular responses this study was to investigate the dichotomy where DNA-PK inhibition
such as proliferation, migration, invasion and even complex events like enhances apoptosis by preventing DSB repair, yet opposes apoptosis
branching morphogenesis in 3D matrices. All of these effects occur by inhibiting IGFBP-3 phosphorylation. ATM phosphorylates similar
after long-term treatment (several hours to days) with HGF. Our lab is sites to DNA-PK and may also have a role in activating IGFBP-3. We
interested in the formation and function of E-cadherin based cell-cell assessed the response of phenotypically normal breast cells (MCF-10A)
adhesions and their significance both under physiological conditions and breast cancer cells (Hs578T, MDA-MB-231 and MDA-MB-468) to
and in disease. To allow morphogenetic changes or processes like cell doxorubicin, alone or with the ATM inhibitor KU55933 or the DNA-PK
migration, the zonula adherens and the cytoskeleton must be able to inhibitor NU7441 (KuDOS Pharmaceuticals), monitoring caspase-3
respond quickly and in an orchestrated fashion to the initiating HGF signal. activity in cell lysates as a marker of apoptosis. Apoptosis induced over
In this study we investigate the acute effects of early HGF treatment on 24-72 h by 0.3-1 μM doxorubicin was significantly enhanced in all cells
the epithelial junctional cytoskeleton and the zonula adherens in CaCo2 treated with 1 μM NU7441 or 10 μM KU55933. To investigate its role
cells. We report a dramatic re-organization of the apical actin ring within in this response, endogenous IGFBP-3 was downregulated in Hs578T
the first 15min of HGF treatment, which is accompanied by a loss of cells ≥80% with small interfering RNA. Doxorubicin-induced apoptosis
Myosin VI from the adherens junctions. Myosin VI is an unconventional was significantly reduced in IGFBP-3-silenced cells compared to control
actin binding motor protein that has previously been shown to play a role cells, confirmed by decreased cleavage of the caspase-3 substrate,
in the stabilization of adherens junctions and the regulation of F-actin poly(ADP-ribose) polymerase. These data suggest that endogenous
organization. We identify Myosin VI as a novel down stream target of IGFBP-3 potentiates doxorubicin-induced apoptosis, and this activity is
HGF signalling and elucidate that Calcium signalling plays a crucial role not prevented by DNA-PK or ATM inhibition.
in the HGF induced regulation of junctional Myosin VI.

POS-MON-179 POS-TUE-180
GENETIC TRANSFORMATION IN COLLETOTRICHUM PROTEOMIC STUDIES ON A HIGH AMYLOLYTIC
TRUNCATUM ASSOCIATED WITH ANTHRACNOSE NIGERIAN MAIZE CULTIVAR
DISEASE OF CHILI PEPPER
Awoyinka O.A.1, 2 , Adebawo O.O. 1, 2 , Daini O.A.2 and Dipanker C.3
Auyong A.S.M., Ford R. and Taylor P.W.J.
1
Babcock University, Ilisan-Remo, Nigeria. 2Olabisi Onabanjo
BioMarka/Plant Health Centre, Department of Agriculture and Food University, Ago-Iwoye, Nigeria. 3Indian Institute of Science, Bangalore
Systems, 3010 Parkville, The University of Melbourne, Australia. India.

Colletotrichum truncatum is the causal agent of chili pepper This study sought a high amylolytic Nigerian maize cultivar. It also
anthracnose causing severe economic loss through reduced yield and established the amino acid sequence that forms the primary structure of
marketability of infected fruit. To this extent an efficient and reliable the β amylase found in the maize cultivar. Purification steps comprising
transformation system of the pathogen is required to prove the function of fractional precipitation by ammonium sulphate, gel filtration and
of isolated pathogenicity genes through gene silencing or mutagenesis. anion exchange chromatography was used respectively to purify β
Agrobacterium tumefaciens-mediated transformation (ATMT) system amylase from the malt of TZEE*TZEE-W*DEMARSCUS*TZEE-W
was developed for C. truncatum. A. tumefaciens carrying a hygromycin before determination of the primary structure of the β-amylase with the
phosphotransferase gene (hph) and a green fluorescent protein (GFP) aid of Matrix assisted laser desorption ionization time- of- flight (MALDI-
gene was used to transform the conidiospores of two C. truncatum ToF) mass spectrometry. Results obtained showed that An apparent
pathotypes (F8-3B and BRIP26974). Optimum transformation efficiency 60 KDa monomeric protein was detected on a single dimensional 10%
was obtained when equal volume of A. tumefaciens strain AGL1 carrying sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-
either pJF1 or pPK2 binary vector was transformed with 1000000 PAGE). Well complete primary structure deduced showed a signature
C. truncatum conidiospores/ml and co-cultivated at 24°C for three of a highly conserved ubiquitous not yet reported β-amylase comprising
days. Southern blot analysis and TAIL-PCR indicated that most of the of 505 amino acid sequence. However, mass spectrometry analysis
transformants contained randomly inserted, single copies of the T-DNA. further carried out on concomitant proteins eluted with the β-amylase
Infection and colonization of chili pepper fruit at the mature red stage during partial purification gives a hypothetical insight into the metabolic
with F8-3B-GFP and BRIP26974-GFP confirmed the virulence of these activities of the high amylolytic maize cultivar during malting. This
transformed pathotypes. Analysis by fluorescent microscopy showed could be explored in quest of improving the diastatic power of the
that colonization of parenchyma cells of fruit pericarp tissue occurred by Nigerian maize cultivar. Keywords: β-amylase, Malting, Maize, Mass
subcuticular intramural infection. In the susceptible Capsicum annuum spectrometry and Proteomics.
genotype, fungal hyphae was detected between the cell walls of
parenchyma cells up to 1 cm in advance of the visible lesion, indicating
that C. truncatum entered a short endophytic stage in its disease cycle
before becoming necrotrophic.

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POS-MON-181 POS-TUE-182
NON-DESTRUCTIVE HIGH-THROUGHPUT MOLECULAR ANALYSIS OF PHOSPHITE INDUCED
PHENOTYPING IN THE PLANT ACCELERATOR TO RESPONSES IN ARABIDOPSIS
STUDY PLANTS UNDER SALT AND WATER STRESS
CONDITIONS Berkowitz O.1, Jost R.2, Hardy G.S.E.J.1 and O’Brien P.A.1
1
Murdoch University, School of Biological Sciences and Biotechnology.
Berger B.1, 2, 3 and Tester M.1, 2, 3
2
The University of Western Australia, School of Plant Biology.
1
The Plant Accelerator. 2Australian Center for Plant Functional
Genomics. 3University of Adelaide, Waite Campus, Urrbrae SA, 5064. Phosphite (H2PO3-) is a phosphate analog widely used to protect
plants from oomycete pathogens such as Phytophthora and Phytium.
Salinity and drought have a major impact on agricultural productivity, Phytopthora species are prominent pathogens in agriculture, e.g.
likely to increase with climate change. To secure global food production, Phytopthora infestans being the causing agent of potato blight (Irish
crop plants with increased yields under these stress conditions are potato femine). Phytopthora cinnamomi has devastating effects
urgently needed. Whereas gene and marker discovery have become (“dieback disease”) on native ecosystems with over 2000 plant
faster than ever, it is now the phenotyping process that is increasingly species at risk in Western Australia alone. Phosphite is the only known
limiting the generation of more tolerant crop plants (Finkel, 2009; Furbank, protectant of plants and exhibits a complex mode of action. At elevated
2009; Tester and Langridge, 2010). Both salinity and drought trigger concentrations it directly inhibits the pathogen’s growth by interference
dynamic physiological responses that require continuous phenotypic with its phosphate-dependent metabolism which is paralleled in plants
measurements to be able to dissect overall tolerance mechanisms into grown on high phosphite concentrations. At the same time it also inhibits
individual traits. The Plant AcceleratorTM combines automated plant the plant’s phosphate starvation response, e.g. the up-regulation of
handling and high-throughput imaging to alleviate the phenotyping high-affinity phosphate transporters, and thus has constrictive effects
bottleneck. Water stress regimes are controlled and recorded with on plant growth under low phosphate supply. In addition to these direct
automated watering and weighing stations. At the same time, various effects phosphite also induces some of the plant’s defence responses,
phenotypic traits can be assessed non-destructively by digital imaging e.g. treatment of plants leads to increased expression of defence
in different wavebands. Colour images can be used to estimate biomass genes. However, the underlying mechanism of this indirect effect is not
production and to measure growth rates and the degree of senescence. understood. We have started to characterise the impact of phosphite on
Temperature differences between control and stressed plants, commonly plant defence responses by analyses of gene expression and metabolic
used as a surrogate for stomatal conductance, are determined with pathways. Transgenic plants have been generated that express a
infrared cameras. Furthermore, changes in leaf water content over time microbial phosphite dehydrogenase which converts phosphite into
are monitored using near infrared imaging. The Plant AcceleratorTM, phosphate. These plants are a valuable tool to dissect direct from
therefore, enables researchers to monitor important aspects of the indirect phosphite effects.
physiological changes occurring under salt and water stress conditions.
The high-throughput capacity provides the opportunity to screen large
populations, making possible forward genetics approaches to the
study of many relevant aspects of plant responses to salt and drought
stress. Finkel, E. (2009) Science 325, 380-381. Furbank, R.T. (2009)
Functional Plant Biology, 36, v-vi. Tester, M. and Langridge, P. (2010)
Science 327, 818-822.

POS-MON-183 POS-TUE-184
GENE EXPRESSION OF PONCIRUS TRIFOLIATA, AUXINS: CRUCIAL TO GRAPE BERRY RIPENING
CITRUS SUNKI AND THEIR HYBRIDS UNDER
INFECTION OF PHYTOPHTHORA PARASITICA USING Bottcher C.1, Harvey K.1, Forde C.2, Boss P.K.1 and Davies C.1
REAL-TIME QUANTITATIVE PCR
1
CSIRO Plant Industry, PO Box 350, Glen Osmond SA 5064, Australia.
2
Nestle S.A., 1800 Vevey, Switzerland.
Boava L.P., Cristofani-Yaly M., Mafra V.S., Kubo K.S., Stuart R.M. and
Machado M.A. Fruit ripening is a complex process which seems to be largely regulated
Centro APTA Citros Sylvio Moreira, CP4, 13490-970, Cordeiropolis- by plant hormones. In contrast to climacteric fruit, such as bananas and
SP, Brazil. tomatoes, the ripening of non-climacteric fruit, e.g. strawberries and
grapes, seems to be less dependent on ethylene and might be controlled
by several other hormones. We are investigating the role of hormones
Phytophthora nicotianae Breda de Haan (Phytophthora parasitica during grape berry development and are testing their ability to manipulate
Dastur) is an important oomycete causing damage in Citrus nurseries ripening. While the application of some hormones, e.g. absicic acid and
and orchards and around the world. To integrate breeding and genomics brassinosteroids, can advance the onset of berry ripening (veraison)
programs to provide resistance against P. parasitica, the identification others, e.g. auxins, delay it. The work presented here focuses on the
of genes involved in disease response is required. In this study, we ripening-delaying effects of auxins that have been reported for a number
proposed that genes differentially expressed between resistant and of climacteric and non-climacteric fruit. Of particular interest is the ability
susceptible hybrids of P. trifoliata and C. sunki (resistant and susceptible, of synthetic auxins like 1-naphthaleneacetic acid (NAA) to retard grape
respectively) may provide key candidates to identify transcripts involved berry ripening. This ability to delay ripening, and therefore harvest,
in disease resistance. We investigated gene expression in pools of four has potential advantages for the wine industry. NAA applied to berries
resistant and four susceptible hybrids in comparison to their parents 48 approximately two weeks prior to veraison delayed ripening by about
hours after P. parasitica inoculation using real-time quantitative PCR. ten days as measured by reduced sugar levels and skin colouration.
Our analysis searched up regulated genes (fold ≥ 2 and p-value ≤ 0.05) A decrease in levels of indole-3-acetic acid (IAA), the most abundant
in the resistant genotypes relative to the susceptible, found in previous auxin in plants, occurs during the development of a number of different
microarray study. These genes were selected due to the biological fruit. This decrease also occurs in grapes and the potential for IAA to act
interest based on their function according to CitEST database, among as an endogenous inhibitor of grape berry ripening is a current research
them, genes that encode enzymes participating in defense-related focus. In this context the role of IAA-conjugation in the initiation of berry
metabolic pathways such as the biosynthesis of phenylpropanoids and ripening is examined.
antimicrobial compounds such as phytoalexins, flavonoid biosynthesis
and resistance genes such as CC-NBS-LRR and TIR-NBS-LRR.
Our results showed that all analyzed transcripts were up regulated in
P. trifoliata relative to C. sunki and in the resistant pool relative to C.
sunki and to susceptible pool. This suggests that a number of defense
strategies are activated following the recognition event encoded by
Phytophthora resistance genes. The genes that we have identified as
up regulated across the resistant genotypes will be valuable for ongoing
work in eQTL mapping. Financial support: FAPESP-INCT/CNPq.

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POSTERS MONDAY & TUESDAY

POS-MON-185 POS-TUE-186
IN VIVO ROLE FOR FLAVONOIDS IN AUXIN SPECTRAL EXTENSION OF PHOTOSYNTHESIS
TRANSPORT AND GRAVITY RESPONSES
Chen M.
Buer C.S. and Djordjevic M.A. University of Sydney.
ARC Centre of Excellence for Integrative Legume Research; Research
School of Biology; College of Medicine, Biology, and Environment; The Chlorophyll a is typically the major photosynthetic pigment in all oxygenic
Australian National University; Canberra ACT; Australia. photosynthetic organisms (plants, algae and Cyanobacteria) with Chl b
and c playing only an accessory light-harvesting function. The newly
Flavonoids are bioactive plant secondary metabolites with a myriad discovered cyanobacterium Acaryochloris marina contains Chl d as its
of important functions (Buer et al., 2007, 2009, 2010). The well- major pigment. Chl d in Acaryochloris is involved in both the accessory
characterised Arabidopsis thaliana transparent testa (tt) mutants are light-harvesting processes and as the primary donor pair in Photosystem
compromised in various flavonoid pathway structural enzymes and I and Photosystem II. This is the first known example where a chlorophyll
regulatory genes (Buer et al., 2007, 2008). Several lesions result in other than Chl a is involved in the primary chemical reaction in a reaction
differential root flavonoid accumulation when compared to wild-type center as is the case in all other oxygenic photosynthetic organisms.
roots (Buer et al., 2010). This differential accumulation allowed testing The unique optical properties of chlorophyll d fill in the absorption gap
whether certain flavonoids controlled auxin transport and subsequent between chlorophylls (Chl a/b) and bacteriochlorophylls which has
root gravity responses. The greatest changes in gravity response, shattered the old scheme that Chl a is the only major chlorophyll of
root elongation, and auxin transport were in the mutants accumulating photosynthesis in oxygenic organisms, and has broadened the potential
quercetin (tt3, tt8, tt10, and ttg1). Increased quercetin accumulation to extend the range of photosynthesis into a new spectral region. Chl
caused rapid gravitropic curvature, faster root elongation rates, and d is the only Chl known so far that can replace all roles of Chl a in
higher auxin transport inhibition compared to the wild type. Those oxygenic photosynthesis. Here, I wish to report the discovery of the
mutants with no or low quercetin accumulation (tt4, tt5, and tt6) were biochemical steps, at gene and protein levels, leading to the formation
inhibited in gravity responses and had increased auxin transport rates of the unique pigment chlorophyll d, which will lead us to the possibility
confirming earlier experiments with tt4 (Buer and Muday, 2004). These for genetic engineering of these important chlorophylls. A potential
in vivo assays extend earlier in vitro assays (Jacobs and Rubery, 1988) new biotechnology application has been developed. We are seeking
showing that flavonoids inhibit auxin transport with quercetin showing the application in improving the crop spectral properties, by extending
the greatest effects and that flavonoids are important for normal auxin their light absorption properties into a new spectral region. Recently,
flux and plant development. Buer C, Muday G, Djordjevic M (2007) Plant we have discovered an unknown chlorophyll from stromatolites pigment
Physiology 145: 478-490. Buer C, Muday G, Djordjevic M (2008) Plant extractions. Using various spectral analyses, we confirmed that it is a
Signalling and Behavior 3: 415-417. Buer C, Djordjevic M (2009) Journal new type of chlorophyll molecule, which showed a even further red-shift
Experimental Botany 60: 751-763. Buer C, Imin N, Djordjevic M (2010) absorption Qy peak. This discovery challenges the limitation of spectral
Journal of Integrative Plant Biology 52: 98-111. Buer C and Muday G wavelength region for oxygenic photosynthesis, which may open new
(2004) Plant Cell 16: 1191-1205. Jacobs M and Rubery P (1988). Science bioenergy applications.
241: 346-349.

POS-MON-187 POS-TUE-188
RNAI-MEDIATED MANIPULATION OF NITROGEN CHARACTERISATION OF TWO NECROSIS AND
DEFENCES IN PLANTS USING PYRIDINE ALKALOID ETHYLENE INDUCING PROTEIN-LIKE (NLP) GENES
BIOSYNTHESIS IN NICOTIANA AS AN EXPERIMENTAL FROM SCLEROTINIA SCLEROTIORUM
SYSTEM
Dinsdale A.J.1, Van Den Elsen F.2, Barnett R.1 and Plummer K.1
Dalton H.L., DeBoer K.D., Neale A.D. and Hamill J.D.
1
La Trobe University, Bundoora, Victoria, Australia. 2Wageningen UR
School of Biological Sciences, Monash University, VIC, Australia. Plant Breeding, AJ Wageningen, The Netherlands.

Alkaloid synthesis in plants usually involves the diversion of amino The necrotrophic phytopathogen, Sclerotinia sclerotiorum, induces
acid precursors from primary into to secondary metabolism. A good plant cell death in order to colonise host plants and release nutrients.
example is the production of toxic pyridine alkaloids, which are a This pathogen is known to secrete various compounds, including oxalic
feature of all species in the genus Nicotiana and some other genera acid and lytic enzymes during infection. Infection is often facilitated in
in the Solanaceae family. Alkaloids are recognised as being important other necrotrophs (such as Fusarium oxysporum and Botrytis cinerea)
defensive compounds, the synthesis of which can increase in response by the secretion of small, phytotoxic effector molecules (necrosis and
to wounding in order to provide protection from predators in native ethylene inducing peptides, NEPs). We have cloned two genes from
habitats. Wound- or jasmonate-induced production of nicotine in Sclerotinia sclerotiorum with significant similarity to NEPs called NEP-
Nicotiana tabacum is correlated with elevated transcript and enhanced like1 (Nlp1) and Nlp2. Both NLPs appear to induce necrosis when
activity of a number of key alkaloid biosynthetic enzymes such as transiently expressed in Nicotiana tobaccum and N. benthamiana.
ornithine decarboxylase (ODC) and arginine decarboxylase (ADC). Multiple 35S fusion constructs, designed to investigate the effect of in
Our previous research has indicated that antisense-mediated down planta NLP protein expression (with or without signal peptides) have
regulation of ADC transcript levels has very little effect upon capacity of also been completed and transformations are currently underway. Both
transgenic N. tabacum to produce nicotine. In contrast, RNAi-mediated genes have also been expressed in Pichia pastoris resulting in purified
down regulation of ODC transcript levels in N. tabacum produced a NLP1 and NLP2 that will be used for further transient assays to assess
substantial decrease in nicotine levels which was accompanied by a the specific response induced in host plants when presented with one
marked increase in concentrations of anatabine. Treatment of ODC- or both proteins. Multiple Nlp1RNAi mutant lines have been generated
RNAi hairy root cultures and regenerated plants with methyl-jasmonate which display inhibited growth rates both in vitro and in planta; further
did not restore the plants capacity to produce normal amounts of nicotine characterisation of these will be presented. Expression localisation
and anatabine. These results suggest that ODC has an important role studies are also underway using Nlp promoter:GFP fusion constructs.
in providing the putrescine used in alkaloid biosynthesis in N. tabacum,
particularly in the wound response. Ongoing work is examining the
wider effects of these RNAi manipulations upon activity of related genes
and enzymes in primary and secondary metabolism and the capacity of
these transgenic Nicotiana tissues to redirect nitrogen from secondary
metabolism back into primary metabolism and growth – particularly in
response to abiotic and biotic stresses.

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POS-MON-189 POS-TUE-190
CHLAMYDOMONAS REINHARDTII - A MODEL Evaluation of growth, nodule development
ALGA FOR THE STUDY OF TRIACYLGLYCEROL and the presence of SAT1 homologs in the
BIOSYNTHESIS AND LIPID BODY BIOGENESIS Australian wild soybean relative Glycine
canescens
Djordjevic M.A., Chen H.-C., Hillier W., Hocart C. and James G.
Plant Science Division, Research School of Biology, The Australian Ebrahimie E., Mazurkiewicz D., Mohammadidehcheshmeh M.,
National University, Canberra, Australia. Chiasson D. and Kaiser B.
School of Agriculture Food and Wine, The University of Adelaide,
C. reinhardtii is an emerging model algal system to study triacylglycerol Australia.
biosynthesis and storage in lipid bodies (Wang et al., Eukaryot. Cell,
2009; 8: 1856-1868; Moellering et al., Eukaryot. Cell, 2010; 9: 97-106; SAT1 is a basic helix-loop-helix transcription factor located on the
Yanto et al., Metabolic Engineering, doi:10.1016/j.ymben 2010.02.002; peribacteroid membrane in soybean nodules (Kaiser et al 1998). Loss of
Dean et al., Bioresource Technology, 2010; 12: 4499-4507). Normally, SAT1 activity in soybean limits nodule development and nitrogen fixation
C. reinhardtii, when cultivated under nutrient limited conditions, stores producing a typical nod plus, fix minus nodule phenotype (Loughlin et
carbon predominantly as starch with only low levels as triacylglycerols. al unpublished results). We are interested in the role of this protein in
Mutations in ADP-glucose pyrophosphorylase inhibit starch synthesis symbiotic nitrogen fixation and the signaling cascades it participates in.
and concomitantly redirect carbon into lipid biosynthesis and storage of We have identified SAT1 homologs in all sequenced plants (legumes
triacylglycerols in lipid bodies when nitrogen deprived. We have found and non-legumes) often present in multiple gene families. As part of this
C. reinhardtii can store 40% of dry cell weight as lipid under nitrogen examination we have looked for the presence of SAT1 in other Glycine
starvation at low light (100 µmol m-2 s-1). Fourier transform infrared species. The perennial soybean, Glycine canescens F.J.Herm. is widely
(FTIR) spectroscopy was used to develop high throughput methods distributed throughout inland regions across Australia. G. canesscens is
for semi-quantitative measurements of cellular protein, carbohydrate a wild relative of G. max however it is often found growing in extreme
and lipid content. Targeted transcriptional profiling by quantitative real- environments where both water and nutrients are often limiting. Very
time PCR was performed to investigate the temporal relationships of little is known about the genetic and physiological properties of G.
key genes involved in the induction of lipid biosynthesis and those canesscens. In this study we examined the physiological properties of
associated with lipid body formation. Proteomic analysis on isolated G. canescens growth and nodule development and evaluated the level
lipid bodies identified a key lipid body associated protein. We conclude of conservation in nodule expressed SAT1 genes. G. canescens was
that C. reinhardtii is an alga that can be manipulated to produce high found to contain both determinate (young) and indeterminate nodules
levels of triacylglycerols making it a novel model to study triacylglycerol when inoculated with Bradyrhizobium japonicum (CC1601a). The
biosynthesis and lipid body biogenesis. nodules were capable of nitrogen fixation as plants grew adequately in
the absence of external nitrogen. We identified four SAT1 homologs in
G. canescens, which are related to those found in G. max. A summation
of our findings and a bioinformatic analysis of the SAT1 clones will
presented.

POS-MON-191 POS-TUE-192
THE IMPACT OF CHLOROPLAST NUMBER ON THE MODIFICATION OF FLOWER COLOUR IN
PHOTOSYNTHETIC PROPERTIES OF TOBACCO DIANTHUS CARYOPHYLLUS
Evans J.R.1, Tazoe Y.1, Pengelly J.J.L.1, Nugent G.D.2 and Von Fiorito S.D.1, Parish R.W.1, Brugliera F.2 and Gendall A.R.1
Caemmerer S.1 1
Department of Botany, La Trobe University, Bundoora, Victoria,
1
Research School of Biology, Australian National University. 2RMIT. Australia, 3086. 2Florigene PTY LTD, 1 Park Drive, Bundoora, Victoria,
Australia, 3086.
The mesophyll conductance to CO2 diffusion between intercellular
airspaces and chloroplast stroma depends on the surface area of Flower colour is an important evolutionary aspect for many plants,
chloroplasts exposed to intercellular airspace. We characterised attracting a variety of pollinators such as insects, birds and small
chloroplast division mutants in tobacco to investigate the effects of mammals. The interaction of pigments, co-pigments, metal ions, inter-
chloroplast number on mesophyll conductance to CO2 diffusion. and intra- molecular staking and vacuolar pH can contribute to an
We analysed four different lines: two lines which had only one to two array of colours in petals, which is valuable to the floricultural industry.
chloroplasts per cell, one line which had more chloroplasts compared Anthocyanin compounds, synthesized via the flavonoid biosynthetic
to control and control plants. Tobacco plants were grown under full pathway, are the most abundant group of pigments associated with
sunlight in a greenhouse. Gas exchange was measured concurrently flower colours, occurring in the majority of plant species. Recombinant
with carbon isotope discrimination to assess photosynthetic properties DNA technology has been utilised by the floricultural company
and the drawdown in CO2 concentration between intercellular airspaces Florigene, to create novel flower colours, with a particular emphasis on
and chloroplasts. Leaves were subsequently sampled for Rubisco the generation of blue petal colour. Florigene were the first company to
and chlorophyll content and quantitative anatomy. Mesophyll cells in release the purple coloured carnation (Dianthus caryophyllus) varieties
leaves from plants with 1-2 chloroplasts per cell were distorted. The known as the FLORIGENE Moon series TM. Various transcription factors
large chloroplast covered most of the cell wall area, but was not always and putative pH regulators genes will be identified in carnation using a
in close contact with the cell wall, buckling away in places. Despite degenerate primer approach. A rapid transient system has been applied
large differences in chloroplast numbers between different lines, CO2 to carnation floral tissues to generate transient down regulation of
assimilation rates, mesophyll conductance, Rubisco content and leaf known genes within the flavonoid biosynthetic pathway and putative pH
anatomy were similar. The relationship between Rubisco content and regulators. Constructs have been generated and the transient system
CO2 assimilation rate at a given CO2 concentration in the chloroplast tested; with GUS expression optimised. A selection of constructs will
will be assessed. be stably transformed into carnation to confirm if the technology is
functional in carnation. Once these genes have been identified amiRNA
or RNAi constructs will be generated and transient and/or stable down
regulation applied.

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POS-MON-193 POS-TUE-194
PROTEOMIC ANALYSIS OF THE RESPONSE OF THE AFFECTS OF LIGHT ON CYANOGENESIS IN
WHEAT CULTIVARS THAT DIFFER IN THEIR CAPACITY FORAGE SORGHUM
TO WITHSTAND DROUGHT
Fromhold S.M.1, O’Donnell N.H.1, Blomstedt C.K.1, Stuart P.N.2,
Ford K.L., Cassin A. and Bacic A. Gleadow R.M.1, Hamill J.D.1 and Neale A.D.1
Australian Centre for Plant Functional Genomics, School of Botany,
1
School of Biological Sciences, Monash University, VIC, AUS. 2Pacific
The University of Melbourne. Seeds, QLD, Australia

Using a series of multiplexed (iTRAQ) experiments we have studied Sorghum is a C4, drought resistant plant species, which is also
the changes in protein abundance of three Australian bread wheat cyanogenic in all tissues except the mature seeds. The cyanogenesis
cultivars (Triticum aestivum). The three cultivars differ in their ability pathway in sorghum produces the stable cyanogenic glucoside,
to maintain grain yield under drought conditions. Plants were grown in dhurrin. When the plant tissue is disrupted the dhurrin is hydrolysed
the glasshouse with cyclic drought treatment that mimics conditions in to release hydrogen cyanide (HCN). At low concentrations HCN can
Southern Australia’s wheat growing regions. Buffer soluble proteins were be metabolised safely by mammals but at higher concentrations it is
isolated from the leaves of the wheat plants and the iTRAQ system with toxic as it causes metabolic asphyxiation. The HCN potential (HCNp) of
multiple rounds of chromatography was used to follow the changes in sorghum is important in the cattle industry as forage sorghum is used
protein abundance, a pooled internal standard was used to allow cross as an alternate feed crop over summer. Environmental factors, such as
experiment comparison. The study contributed to a wheat database of drought, are known to increase the HCNp of sorghum but little is known
2,183 proteins, as well as identifying 110 leaf proteins that changed in regarding the affects of other conditions, including light levels. Analysis
abundance in response to drought. The number of significant changes in of the putative promoter regions of the three biosynthetic genes in the
the cultivars at the different time points reflected their differing response cyanogenesis pathway; CYP79A1, CYP71E1 and UGT85B1, showed
to drought. In general we observed an increase in proteins involved that in all three sequences there are potential light and circadian
in oxidative stress metabolism and a down regulation of proteins response elements. It has been postulated that sorghum may also go
involved in photosynthesis and the Calvin cycle. Drought responsive through a circadian or diurnal rhythm in relation to its cyanogenesis
proteins, including dehydrins and LEA proteins were also significantly pathway. An experiment has been conducted to compare different light
upregulated. treatments on forage sorghum that were hydroponically grown and
placed in cabinets with controlled temperature and nutrients. The plants
subjected to three light treatments; total light, total darkness and 14/10
light/dark cycle, and plants were harvested every four hours for 60
hours. This experiment was designed to determine if there is a circadian
or diurnal effect on the cyanogenesis pathway in forage sorghum and
the results will be discussed.

POS-MON-195 POS-TUE-196
A MODEL DESICCATION-TOLERANT GRASS, SCLEROTIAL FORMATION IN SCLEROTINIA
SPOROBOLUS STAPFIANUS GANDOGER
Greenhill A.1, Gendall A.1, Benny U.2, Rollins J.2, Porter I.3 and
Gaff D.F. , Blomstedt C.K. , Neale A.D. , Hamill J.D. , Ghasempour
1 1 1 1 Plummer K.1
H.R.2 and Sutaryono Y.A.3
1
La Trobe University, Victoria, Australia. 2University of Florida, Florida,
1
School of Biological Sciences, Monash University, Clayton 3800, USA. 3Victorian Department of Primary Industries, Victoria, Australia.
Australia. 2Department of Biology, Razi University, Taghbostan,
Kermanshah, Iran 6. 3Lembaga Penelitian Universitas Mataram, Sclerotinia diseases cause over $100M loss to vegetable crops annually
Jl.Pendidikan No. 37, Mataram, 83125 NTB, Indonesia. in Australia, with significant losses worldwide. Control of these diseases
is severely impeded by the pathogen’s ability to produce sclerotia;
Of the ~40 grasses that can recover from dehydration to air-dryness, highly melanized structures crucial to the pathogen’s propagation,
Sporobolus stapfianus is a most versatile tool for research into reproduction and survival. T-DNA mutants have been created via
desiccation tolerance in vegetative grass tissue. This African tropical- Agrobacterium-mediated transformation. In this process a short DNA
subtropical perennial grass is easily propagated by subdivision or from sequence (T-DNA) is inserted randomly into the fungal genome. As the
seed. A number of comparisons of desiccation-tolerant and desiccation- sequence of this T-DNA tag is known, the gene or region into which
sensitive leaves can be made, eg. leaves become desiccation-tolerant it has been inserted can be identified. Approximately 1200 T- DNA
while they dry attached to intact plants in the light, whereas leaves mutants were created and screened for altered sclerotium phenotypes,
detached before drying remain desiccation-sensitive, - this permits with approximately 40 of these presenting a sclerotia-minus, or aberrant
comparison of leaves with identical genotypes. Many desiccation- sclerotia phenotype. T-DNA insertion points have been identified in a
sensitive Sporobolus species and 6 desiccation-tolerant ones are number of these mutants and work is underway to characterise the
available for comparison with S. stapfianus. S. stapfianus is salt tolerant interrupted genes. In addition to this genetic study the role of melanin
and its roots adapt to withstand waterlogging if they grow into water. Of in sclerotial formation is also being examined. Vectors to knock out or
7 desiccation-tolerant grass species tested in preliminary field trials in silence melanin biosynthesis genes have been constructed. Additionally,
the Northern Territory, S. stapfianus, a C4 grass, produced the greatest chemical melanin inhibitors have been tested against Sclerotinia in vitro.
dry matter increment per unit basal area. S. stapfianus has favorable This work is supported by funding from Horticulture Australia Limited
digestibility, and satisfactory protein and phosphate levels. S. stapfianus and an Australian Postgraduate Award.
exemplifies an advanced stage of an evolutionary trend in desiccation
tolerant plants toward increased importance of the dehydration phase
for induction of desiccation tolerance accompanied by synthesis of
protectants and extensive proteomic changes. In the resurrection
monocot Borya contracta and dicot Craterostigma plantagineum, the
induction of desiccation tolerance during drying is controlled by the
phytohormone abscisic acid (ABA), the induction in S. stapfianus is ABA-
independent. S. stapfianus offers a valuable resource for future research
on desiccation tolerance in a most important family economically.

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POSTERS MONDAY & TUESDAY

POS-MON-197 POS-TUE-198
EXPRESSING RED RUBISCOS IN GREEN PLANTS MANIPULATION OF TOXIC NITROGEN SECONDARY
METABOLISM IN PLANTS AND OPPORTUNITIES IN
Gunn L.H. and Whitney S.M. PLANT BIOTECHNOLOGY
Plant Sciences Division, Research School of Biology, The Australian
National University, Canberra A.C.T. 0200, Australia. Hamill J.D., Blomstedt C.K., Dalton H.L., DeBoer K.D., DeGuzman G.,
Gleadow R.M., Ling H.Y., O’Donnell N.H., Neale A.D. and Walmsley A.M.
The CO2-fixing enzyme Ribulose-1,5-bisphosphate carboxylase/ School of Biological Sciences, Monash University, Melbourne, Victoria
oxygenase (Rubisco) has a pervasive influence on photosynthetic 3800.
carbon assimilation in plants. In higher plants Rubisco is comprised
of eight catalytic large subunits (L, rbcL gene, plastome encoded) Many plants produce high levels of toxic nitrogen-containing specialized
and eight small subunits, essential for maximal activity (S, rbcS gene, metabolites, such as alkaloids and cyanogenic glycosides, which
nuclear encoded). Despite millions of years of evolution, Rubisco is an generally are regarded as offering environmental protection against
inefficient enzyme as O2 competes with CO2 as a substrate producing predators. Using Nicotiana (dicot - alkaloids) and Sorghum (monocot
a product (2-phosphoglycolate) whose dissipation via photorespiration - cyanogenic glycosides) as experimental species, we are studying the
consumes energy. In nature, Rubisco shows considerable catalytic interplay between primary and secondary metabolism and the extent to
diversity with some variants in red algae having properties that surpass which movement of nitrogen can be experimentally redirected in vivo.
those in higher plants. Replacement of higher plant Rubisco with that of A screening program to identify mutations in key genes necessary for
some red-algae (in theory) has the potential to improve photosynthetic cyanide production in Sorghum is well underway. We have also produced
C-assimilation, however such endeavours have been thwarted by the transgenic lines of Nicotiana with markedly altered patterns of pyridine
finding that the folding and assembly properties of Galdieria sulphuraria alkaloids, both qualitatively and quantitatively. These studies have
Rubisco (a red algae) could not be met by tobacco plastids. Here relevance to biotechnology, in relation to the redirection of metabolites to
we expand on this work to introduce L- and S-subunits from the red produce plants with altered growth potential. They also have relevance
algae Griffithsia monilis and a “red-like” Rubisco from the bacterium to other ongoing research interests involving the production of plant-
Rhodobacter sphaeroides into inverted repeat regions of a plastome made vaccines in plants without concomitant negative connotations
transforming tobacco-master line (cmtrL1). To evaluate whether the that may be associated with the co-production of toxic nitrogen-based
putative molecular chaperone CbbX (coded by the cbbX gene located 3’ secondary metabolites.
to the rbcL-S genes in red algae) influences assembly of G.sulphuraria
Rubisco in tobacco, the rbcL-S-X operon have also been introduced into
cm
trL1. The current status of this research will be presented.

POS-MON-199 POS-TUE-200
IDENTIFICATION OF FLAGELLAR MASTIGONEME MOLECULAR ANALYSIS OF THE EARLY STAGES OF
PROTEINS FROM PHYTOPHTHORA SOYBEAN NODULATION
Blackman L.M.1, Arikawa M.2, 3, Yamada S.2, Suzaki T.2 and Hardham A.R.1 Hayashi S.1, Reid D.1, Lorenc M.2, Stiller J.2, Edwards D.2, Ferguson B.J.1
1
Plant Sciences Division, Research School of Biology, Australian and Gresshoff P.M.1
National University, Canberra, ACT 2601, Australia. 2Department 1
ARC Centre of Excellence for Integrative Legume Research, The
of Biology, Graduate School of Sciences, Kobe Univesity, Nada-ku, University of Queensland, St. Lucia, QLD 4072, Australia. 2Australian
Kobe 657-8501, Japan. 3Department of Cardiovascular Control, Kochi Centre for Plant Functional Genomics, School of Land, Crop and
Medical School, Nankoku, Kochi 783-8505, Japan. Food Sciences, The University of Queensland, Brisbane, Qld 4072,
Australia.
Motile, flagellated zoospores of Phytophthora and Pythium species play
a key role in pathogen dissemination and the initiation of infection of host Nitrogen is typically a limiting factor for plant growth. Legumes can form
plants. The diseases these pathogens cause are highly destructive and ‘nitrogen-fixing root nodules’ de novo through a symbiotic association
result in extensive losses in agriculture and natural ecosystems worldwide. with soil-living bacteria called rhizobia. This provides them with nitrogen,
Tripartite tubular hairs called mastigonemes on the anterior flagellum of thus enabling them to grow in nitrogen-poor environments. Nodule
Phytophthora and Pythium and other protists in the Stramenopile taxon formation is initiated via a chemical exchange between the plant and
are responsible for reversing the thrust of flagellar beat and for cell motility. the bacteria. This is followed by a series of signalling cascades in the
Immunoprecipitation experiments using antibodies directed towards root, which induces cell proliferations leading to the formation of nodule
mastigonemes on the flagella of zoospores of Phytophthora nicotianae primordia. In recent decades, mutagenesis programs using several
have facilitated the cloning of a gene encoding a mastigoneme shaft legume species have identified numerous components required for
protein in this pathogen. Expression of the gene, designated PnMas2, nodule development. These studies also indicated that root epidermal
is up-regulated during asexual sporulation, a period during which many and cortical/pericycle cells must tightly coordinate their development
zoospore components are synthesized. Analysis of the sequence of the during the early stages of nodulation in order to establish functional
PnMas2 protein has revealed that, like other Stramenopile mastigoneme nodules. To further identify genes that are activated during the early
proteins, PnMas2 has an N-terminal secretion signal and contains four stages of nodulation, we conducted a root transcriptome analysis using
cysteine-rich epidermal growth factor (EGF)-like domains. Evidence high-throughput deep-sequencing technology (Illumina’s GAIIx) in
from non-denaturing gels indicates that PnMas2 forms large oligomeric soybean (Glycine max). Soybean root tissue was harvested from the
complexes, most likely through disulphide bridging. Bioinformatic zone of nodulation to specifically focus on nodulation-expressed genes.
analysis has revealed that Phytophthora species typically have three The tissue was harvested at various times following rhizobia inoculation.
or four putative mastigoneme proteins containing the four EGF-like Over 6,000 differentially expressed genes were identified, including
domains. These proteins are similar in sequence to mastigoneme known, and many previously unknown, nodulation-dependent genes.
proteins in other Stramenopile protists including the algae Ochromonas In addition, we identified a number of novel molecular pathways (i.e.,
danica, Aureococcus anophagefferens and Scytosiphon lomentaria and hormone biosynthesis and signalling) that are differentially regulated
the diatoms Thalassiosira pseudonana and T. weissflogii. during nodulation. Findings from these studies will be presented.

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POSTERS MONDAY & TUESDAY

POS-MON-201 POS-TUE-202
SILENCING OF THE PHOSPHOLIPASE C GENE SEQUENCE DYNAMICS OF AN ARABIDOPSIS
IN PROTOPLASTS FROM COFFEA ARABICA L. THALIANA GENOMIC LOCUS UNDER HIGHLIGHT (HL)
SUSPENSION CELLS STRESS
Poot-Poot W.A., De Los Santos-Briones C., Munoz- Sanchez J.A. and Ichim M.C.1, Milcamps A.2, Papazova N.3, Depicker A.4, De Loose M.3
Hernandez-Sotomayor S.M.T. and Van Den Eede G.2
Centro de investigacion Cientifica de Yucatan. 1
Stejarul Research Centre for Biological Sciences. 2Joint Research
Centre. 3Institute for Agricultural and Fisheries Research. 4Flanders
Phospholipase C (PLC) is an enzyme involved in signal transduction Interuniversity Institute for Biotechnology.
pathways. PLC catalizes the hydrolysis of phosphatidylinositol
4,5 bisphosphate generating two second messengers: inositol The integration of the T-DNA into the plant genome is reported to
1,4,5-triphosphate (IP3) and diacylglycerol (DAG) which is immediately be often accompanied by simple or complex rearrangements at the
phosphorylated to phosphatidic acid (PA) by diacylglycerol kinase integration genomic locus. It is suspected that transgenic events,
(DGK) in plant cells. Likewise, PLC has been observed that is involved in instability can arise upon plant exposure to abiotic stresses. The
the response to different types of stress such as drought, salinity, cold, stability of the transgene and the sequences flanking the T-DNA is
pathogens and metals such as Al3+. RNA interference (RNAi) technology an important aspect for the approval of GMPs. The structural stability
is a widely used tool to analyze gene function in different organisms. In of transgenes and their integration locuses is a concern for risk
order to obtain a protocol for silencing PLC from protoplasts of C. arabica assessment, food labeling, traceability and post-release monitoring. We
suspension-cell, cells were treated with a mixture of hydrolytic enzymes. have performed sequence analysis of a genomic locus, corresponding
To determine the concentration of polyethylene glycol (PEG) required for to a T-DNA insertion point from an Arabidopsis thaliana transgenic line.
the transformation, we made a curve to different concentrations of PEG The analyses of the target locus will allow defining if and what kinds
and viable protoplasts were quantified and the optimal concentration of of rearrangements are specifically induced under highlight (HL) stress
PEG to use for transformation was 20%. Protoplasts were exposure to 20 (PPFD of 600 μmole/m2/s), known to be a mutagenic oxidative stress due
% (PEG) and the plasmid for RNAi which contains the catalytic fragment to the generation of ROS molecules. In order to be able to detect even
of PLC from C. arabica plus the red fluorescent protein. Protoplasts the smallest mutations possible (SNPs) we have tested and optimized
were observed with fluorescence microscopy at 12, 24, 48, 72 hours. the Single Strand Conformational Polymorphism analysis coupled with
Our results showed greater transformation with of 20% PEG and the Capillary Electrophoresis (SSCP-CE). The results indicate that in wt
suitable time to analyze the RNAi-PLC of 72 hours. These results allow plants, both those grown in standard conditions and those exposed to
us to obtain information in a short time on the role of the PLC to different highlight stress, the sequence considered does not undergo any major
stimuli. Acknowledgments: Scholarship to WPP CONACYT (157993) or minor re-arrangement. Moreover, as method for mutation detection,
and a CONACYT grant (98352) to SMTHS. the SSCP-CE has proved to have the potential to be used in the field
of GMO research for stability analyses and post-release monitoring
purposes.

POS-MON-203 POS-TUE-204
THE ARABIDOPSIS PHYTOSULFOKINE RECEPTOR A COMPARATIVE PROTEOMICS APPROACH FOR
CONTAINS A FUNCTIONAL GUANYLYL CYCLASE IDENTIFICATION OF EFFECTORS IN VENTURIA
DOMAIN ENABLING CGMP-DEPENDENT INAEQUALIS
DOWNSTREAM SIGNALLING IN VIVO
Jones D.1, 2, Mesarich C.3, 4, Hill G.3, 4, Shiller J.2, Bowen J.3, Templeton M.3
Kwezi L.1, 2, Ruzvidzo O.2, Wheeler J.I.1, Govender K.2, Iacuonne S.1, and Plummer K.1, 2
Thompson P.E.1, Gehring C.2, 3 and Irving H.R.1
1
Cooperative Research Center for National Plant Biosecurity. 2La
1
Monash Institute of Pharmaceutical Sciences, Monash University, Trobe University. 3Plant and Food Research, New Zealand. 4The
381 Royal Parade, Parkville VIC 3052, Australia. 2University of the University of Auckland, New Zealand.
Western Cape, Bellville 7535, South Africa. 3Computational Bioscience
Research Centre, 4700 King Abdullah University of Science and Venturia inaequalis and V. pirina are the causative agents of apple and
Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia. pear scab (respectively). This project aims to identify and characterise
effectors in these species. Effectors are pathogen proteins involved
Phytosulfokines (PSKs) are sulphated pentapeptides that stimulate cell in infection. Effectors can also be recognised as foreign by plant
growth and differentiation in plants. The PSK receptor (PSKR1) is a receptors, which then initiate a signal transduction cascade that results
leucine rich repeat receptor like kinase but its role in mediating PSK in plant resistance. Breaking of resistance can occur if either the plant
signals is undefined at a biochemical level. We identified a putative receptor gene or the effector gene is lost, or if the receptor is unable
guanylate cyclase (GC) catalytic centre in PSKR1 that is encapsulated to recognise the effector due to a mutation. Due to this evolutionary
within the kinase domain. This is a different architecture to known animal pressure, effectors often vary across species and races. Effectors
receptor GCs where a linker domain separates the GC catalytic centre are therefore of interest for their direct role in infection, for their role in
from the kinase homology domain. Hence we tested the hypothesis that activating plant resistance, and for use in developing molecular tests
the GC worked in conjunction with the kinase to signal downstream that can differentiate strains of V. inaequalis and/or species of Venturia.
events in PSK signalling. We expressed the recombinant complete kinase We are developing a PCR-based test using a putative effector, a fungal
domain of AtPSKR1 and show that it has serine/threonine kinase activity hydrophobin; this test would be of use in surveillance, particularly in
using the Ser/Thr peptide 1 as a substrate with an approximate Km of 7.5 Western Australia, which is currently free of V. inaequalis but requires
μM and Vmax of 1800 nmol min-1 mg-1 protein. This same recombinant ongoing surveillance to verify area freedom. Using 2-D DIGE, we intend
protein also has GC activity in vitro that is significantly enhanced when to compare proteins from Venturia sp. grown on cellophane/PDA culture
calcium is present. Overexpression of the full length AtPSKR1 receptor against proteins on PDA culture, since V. inaequalis grows infection
in freshly isolated protoplasts from Arabidopsis thaliana leaves results in structures (stroma) on cellophane but not PDA. Therefore, proteins
the endogenous basal cGMP levels being raised over 20-fold indicating present only in cellophane culture may be involved in infection structures.
that the receptor has GC activity in vivo. In addition, we demonstrate Isolated proteins can be identified either by de novo sequencing, or by
that PSK α itself induces small and rapid increases in cGMP levels Peptide Mass Fingerprinting. To this end, the genomes of V. inaequalis
and that the relatively inactive non-sulphated peptide backbone n-PSK and V. pirina are being sequenced using pyrosequencing.
does not. Together these results indicate that the AtPSKR1 contains
dual functioning GC and kinase catalytic activity and represents a novel
enzymatic class of kinases with overlapping catalytic domains.

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POS-MON-205 POS-TUE-206
DOES PHOSPHITE MIMIC PHOSPHATE IN MODIFICATION OF THE INTERCELLULAR CONTACTS
REGULATING THE PHOSPHATE-STARVATION IN PLANT SUSPENSION CELLS THROUGH
RESPONSE IN PLANTS? ADAPTATION TO ENVIRONMENTAL STRESS
Jost R.1, Pharmawati M.2, Berkowitz O.3, Lambers H.1 and Finnegan P.M.1 Kasprowicz A.K.1, Michalak M.J.1, Baluska F.2 and Wojtaszek P.1
1
School of Plant Biology, The University of Western Australia, Crawley 1
Department of Molecular and Cellular Biology, Faculty of Biology,
WA 6009, Australia. 2Biology Department, Faculty of Mathematics and Adam Mickiewicz University, Umultowska 89, 61-614 Poznan. 2Institute
Natural Sciences, Udayana University Campus Bukit, Jimbaran, Bali. of Cellular and Molecular Botany, University of Bonn, Kirschallee 1,
3
School of Biological Sciences and Biotechnology, Murdoch University, 53115 Bonn, Germany.
Murdoch WA 6150, Australia.
The cell wall-plasma membrane-cytoskeleton continuum is responsible
Phosphate is one of the most important macronutrients for plant for mechanical integration of plant cells. Long-term water deprivation
growth and elaborate mechanisms have evolved to sense and maintain leading to osmotic stress activates adaptory mechanisms which enable
phosphorus homeostasis. Phosphite, a reduced form of phosphate, is cell to function more or less normally. During stepwise adaptation of
considered to be metabolically inert when taken up by plants, and is in tobacco BY-2 suspension cells to growth in the presence of various
wide use as an effective fungicide against a wide range of devastating osmotically active agents, cell lines diverged into independent,
plant diseases caused by oomycetes, particularly Phytophthora osmoticum type-specific lines. The changes in growth patterns were
species, e.g. P. infestans, the causative agent of the potato famine in accompanied by the alterations in the composition of cell walls. It
18th century Europe, and P. cinnamomi, the dieback pathogen that was determined that cell walls of cells grown in the presence of ionic
currently threatens the long-term survival of many Australian native agents were homogenous, while longitudinal walls and cross-walls in
ecosystems. The effects on plant productivity upon phosphite treatment cells adapted to nonionic agents were significantly different. In plants,
in the field vary and depend on the presence of phosphite-oxidizing cross-walls within cell files of axial organs exhibit specific properties
microorganisms in the soil, as well as the internal phosphorus status that allow them to act as domains of contact and intense intercellular
of the plant. Phosphite has been demonstrated to suppress phosphate communication e.g. via endocytosis, and the sites of anchorage of
starvation responses in plants with low phosphorus status, but to date cytoskeleton. Similar properties were also observed in cross-walls
there is very little understanding of how phosphite interferes with the connecting cells grown in the presence of nonionic osmoticum. The
plant phosphorus signalling networks and how/if this is linked to the presented results were revealed with immunolocalization, live confocal
long-term resistance to P. cinnamomi observed in plants in the field microscopy and proteomics approaches. This research was founded
after aerial spraying of the host canopy. Our project aims to characterize by the Polish Ministry of Science and Higher Education grants: PBZ-
the molecular effects of phosphite on the phosphate-sensing machinery KBN-110/P04/2004 and 2944/B/P01/2008/34 to PW and grant of Dean
within an Arabidopsis thaliana model. of Biology Faculty for PhD students and scholarship from European
Social Fund Programme for best PhD students from Wielkopolska to
AK.

POS-MON-207 POS-TUE-208
EVOLUTION OF A CIS-ELEMENT THAT REGULATES NOVEL TRANSIENT GENE-REPLACEMENT SYSTEM
PETAL LOSS EXPRESSION IN THE PERIANTH OF THE TO INVESTIGATE RNA PROCESSING ACTIVITIES OF
ARABIDOPSIS FLOWER DICER-LIKE ENZYMES IN PLANTS
Kilinc A. and Smyth D.R. Kim K.W.1, Eamens A.L.1, 2, Fusaro A.1, 2 and Waterhouse P.M.1, 2
School of Biological Sciences, Monash University, Melbourne, Vic 1
University of Sydney NSW 2006. 2CSIRO Enquiries Locked Bag 10
3800. Clayton South VIC 3169 Australia.

An outstanding issue in biology is the evolution of complex traits, and Since their discovery, RNA silencing pathways have been the focus of
how changes in regulatory networks drive this process. Flower structure intense research in both plants and animals. The use of HELA cells and
in the Brassicaceae is remarkably conserved. The outermost whorl Drosophila embryo lysates have allowed animal RNA silencing pathways
consists of four sepals, and the second whorl contains four petals to be studied rapidly in vitro, whereas in plants, RNA silencing pathways
arising between them, giving a distinctive tetrameric appearance. The have been studied mostly in vivo, within different stable mutants of
PETAL LOSS (PTL) gene is a transcription factor of the trihelix family Arabidopsis thaliana. A.thaliana, has been used as the model plant for
with 29 members in Arabidopsis, a member of the Brassicaceae. The ptl studying plant RNA silencing pathways because of its short generation
mutant phenotype is characterised by a loss of petals and aberrant sepal time, complete annotated genome and its capacity to be transformed by
development. PTL is expressed between developing sepal primordia a non-tissue culture, floral dip method with stable transgenes. A rapid
(termed the Inter Sepal Zone, ISZ) where it functions to suppress sepal alternative to stable transformation is the use of transient Agrobacterium-
enlargement and to promote petal initiation in nearby internal spaces. mediated transformation (agro-infiltration). However, this technique
We have identified a specific 16 bp regulatory enhancer (RE) within the is only compatible with an Australian native, Nicotiana benthamiana,
sole intron of PTL which is required to activate its expression in the ISZ. whoes genome remains to be fully sequenced. In an attempt to combine
This element is highly conserved within orthologous genes in tetrameric the benefits of both systems, we developed a novel transient gene-
species of the related Brassicaceae, Cleomaceae and Capparaceae replacement strategy to investigate the RNA processing activities of
families tested, but surprisingly, was not detected in more distantly Arabidopsis Dicer-like proteins in N. benthamiana. This involved the
related pentameric families within the order Brassicales. The ISZ RE transient knock-down of endogenous N. benthamiana DCL1 protein
shows close homology to a sequence within the LTRs of some gypsy- expression using an artificial miRNA (amiRNA) that is specific to the
like retrotransposons, although the latter sequences are 2 bp longer. endogenous NbDcl1 gene, followed by the transient over-expression
Deletion of these 2 nucleotides is sufficient to activate ISZ expression. of functional exogenous Arabidopsis DCL1 protein via agro-infiltration.
Evolution of the distinct tetrameric perianth pattern of Brassicaceae may This resulted in the successful complementation of DCL1-dependent
have arisen through insertion of a retroelement into the intron of the processing of miRNA-precursors, and therefore demonstrated that the
PTL ancestral gene, followed by simple mutational change in the LTR concept of transient gene-replacement was achievable for studying
sequence to create an active ISZ RE and thus recruit PTL function to specific components of the miRNA silencing pathway.
this region.

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POS-MON-209 POS-TUE-210
STRUCTURAL BASIS OF DISEASE RESISTANCE IN IDENTIFICATION OF PLANT HOST PROTEINS
FLAX AGAINST FLAX RUST TARGETED BY FLAX RUST EFFECTOR PROTEINS
Ve T.1, Williams S.1, 2, Valkov E.1, Stamp A.1, Sornaraj P.2, De Courcy- Koeck M.1, 2 , Dodds P.N.2, Jones D.A.1, Ellis J.G.2 and Hardham A.R.1
Ireland E.2, Ellis J.G.3, Dodds P.N.3, Anderson P.A.2 and Kobe B.1 1
Australian National University, CMBE, RSB, PS. 2CSIRO Plant
1
School of Chemistry and Molecular Biosciences, University of Industry, Black Mountain, Canberra.
Queensland, Brisbane 4072, Australia. 2School of Biological Sciences,
Flinders University, G.P.O. Box 2100, Adelaide 5001, Australia. Secreted rust fungal proteins found inside the plant cell during infection
3
CSIRO Plant Industry, Canberra, Australia. are thought to manipulate host metabolism and defence responses.
Flax (Linum usitatissimum) and its rust pathogen (Melampsora lini) is
Plant diseases significantly affect economically important crops. Plant a useful model system for this infectious disease, and several secreted
immunity is triggered by the recognition of a pathogen effector protein effectors have been identified from flax rust, including AvrL567. The
by a plant resistance (R) protein, leading to the activation of plant aim of this study is to identify proteins that interact with AvrL567, which
defences and a localized cell death response. The effectors usually may represent such host targets. Using a Yeast-2-Hybrid screen with
have roles in virulence and are structurally diverse, while R proteins cDNA from infected flax leaves, three interactors of AvrL567 could
generally fall into a few conserved families. The effector-R recognition be identified, namely LuCKX1, C2MP and SIA2. Interaction between
event is poorly understood at molecular and structural levels. We have AvrL567 and these three targets was confirmed by a Bimolecular
used the fungal pathogen flax rust interaction with flax as a model Fluorescence Complementation assay in planta. LuCKX1 (Linum
system to characterize this process. The flax R proteins consist of a core usitatissimum cytokinin oxidase 1) is a cytosolic plant gene closely related
nucleotide-binding, N-terminal Toll-interleukin-like (TIR), and C-terminal to AtCKX7, one of seven genes in Arabidobsis thaliana responsible
leucine-rich repeat (LRR) domain. Previously, we have shown the direct for irreversible degradation of cytokinin. These plant hormones are
interaction of the effector proteins AvrL567 and AvrM with R proteins L6 involved in cell development such as lateral root formation and their
and M, respectively (1,2). We also determined the crystal structure of cellular concentration changes upon pathogen infection. Homologues
AvrL567 (3). Here, we report the first crystal structure of a TIR domain of the plant gene C2MP (C2-domain containing membrane-targeting
from a plant R protein (L6) at 2.3 Å resolution. The structure reveals protein) have been reported to be involved in pathogen responses in
important differences from the structures of mammalian TIR domains, rice. A.thaliana lines expressing these rice genes were more resistant
and highlights three separate functionally important protein surfaces, to Pseudomonas syringae infection compared to the wild type plants.
involved in dimerisation, interaction with a downstream signalling The third interactor, SIA2 (Secreted Interactor of AvrL567a), is another
partner, and regulatory intramolecular interactions, respectively. We also secreted fungal protein that may be a co-factor for AvrL567. SIA2 also
determined the crystal structure of flax rust effector protein AvrM, which shows binding to two other rust effector proteins (AvrM and AvrP),
has no significant sequence similarity with proteins of known structure. suggesting it may have an important general role in protein translocation
The 2.7 Å resolution structure reveals a novel L-shaped helical fold, and infection. Additionally, homologous of SIA2 are associated with
with two chains forming a dimer with an unusual non-globular shape. pathogenicity in powdery mildew and rice blast fungi.
Our results bring us a step closer to understanding the molecular basis
for the disease resistance process and the ability to engineer novel
resistance specificities. 1. Dodds et al (2006) Proc Natl Acad Sci USA
103: 8888. 2. Catanzariti et al (2010) Mol Plant Microbe Interact 23: 49.
3. Wang et al (2007) Plant Cell 19: 2898.

POS-MON-211 POS-TUE-212
DE NOVO ASSEMBLY OF THE NICOTIANA ALATA CHARACTERISATION OF LEGUME ANGIOGENIC
POLLEN TRANSCRIPTOME USING NEXT-GENERATION ACTIVE METABOLITES
SEQUENCING
Kordbacheh F.1, Hocart C.1, Du Fall L.1, Oakes M.1, Bezos A.2, Parish C.1, 2
Koh P.1, Cassin A.M.2, Edwards D.3, Bacic A.1, 2 and Newbigin E.1 and Djordjevic M.1
1
Plant Cell Biology Research Centre, School of Botany, University of
1
ARC Centre of Excellence for Integrative Legume Research,
Melbourne, VIC 3010, Australia. 2Australian Centre for Plant Functional Plant Science Division, Research School of Biology, GPO Box
Genomics, School of Botany, University of Melbourne, VIC 3010, 475, Australian National University, Canberra ACT 2601, Australia.
Australia. 3Australian Centre for Plant Functional Genomics and School
2
Department of Immunology and Genetics, The John Curtin School of
of Land Crop and Food Sciences, University of Queensland, Brisbane, Medical Research, The Australian National University, PO Box 334,
Australia. Canberra, ACT, 2601 Australia.

We report the sequencing of the Nicotiana alata pollen grain transcriptome Legumes have many beneficial health effects in humans and animals
using a Next-Generation or deep-sequencing approach followed by de due to their nutritional properties and they are a known source of
novo sequence assembly. Next-Generation sequencing is increasingly bioactive metabolites that affect plant, animal and bacterial cells (Buer
being used to profile transcriptomes although to date this approach has et al, Journal of Integrative Plant Biology, 2010, 52: 98-111; Dixon and
mainly been used with model organisms where a sequenced genome is Sumner, Plant Physiology 2003; 131: 878-885; Banunas and Kinghorn
used to align the transcriptome sequence reads. De novo transcriptome 2005 Life Sciences 78: 431-441). We have isolated, identified and partially
assembly offers a way of using this powerful technology in non-model characterised metabolites from legumes that modulate angiogenesis.
organisms such as N. alata, for which there are few genomic resources. In Angiogenesis is the formation of new blood capillaries, veins and
de novo assembly, long RNA transcripts are first converted into a library arteries from pre-existing blood vessels to supply blood and nutrients
of short cDNA fragments that are then sequenced in a high-throughput and remove metabolic wastes from the tissues. Size filtration and high
manner to obtain small lengths of sequence information from one end of performance liquid chromatography (HPLC) were used to purify and
each cDNA fragment. Computer programs then reconstruct full-length isolate the bioactive metabolites from the crude extract. The bioactive
and partial transcripts from this vast amount of sequence information. components were identified using activity-guided assays. An in vitro
Here we describe the workflow used to de novo assemble the N. alata angiogenesis bioassay based on in vivo blood vessel formation (Parish
pollen transcriptome, some of the features of this transcriptome, and et al, Cancer Research 1999; 59: 3433-3441) was used to discover at
RT-PCR validation of selected cDNAs. least three different molecules that possess pro-angiogenic activity.
Mass spectrometry strategies including gas and liquid chromatography
(GC/MS, LC/MS) were used to determine the molecular masses of the
compounds, establish elemental formulas and determine structural
details of the bioactive compounds. These compounds may find utility
in cardiovascular disease and wound healing.

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POS-MON-213 POS-TUE-214
GENOME-WIDE TRANSCRIPTOME ANALYSIS FORMATION OF SECOND WHORL ORGANS IN THE
OF CITRUS HYBRIDS PLANTS RESISTANT OR ARABIDOPSIS FLOWER INVOLVES AUXIN INFLUX
SUSCEPTIBLE TO CITRUS LEPROSIS C (CILV-C) AND IS SENSITIVE TO DISTORTION OF THE FLOWER
MERISTEM SHAPE
Kubo K.S.1, 2 , Cristofani-Yaly M.1, Ribeiro-Alves M.3, Boava L.P.1,
Costa F.M.1, 5, Kishi L.1, Mafra V.S.1, 2, Freitas-Astua J.1, Bastianel M.1 Lampugnani E.R., Kilinc A. and Smyth D.R.
and Machado M.A.1 School of Biological Sciences, Monash University, Melbourne, Vic
1
Centro de Citricultura Sylvio Moreira/IAC, CP4, 13490-970, 3800.
Cordeiropolis-SP, Brazil. 2Institute of Biology, CP 6109, State
University of Campinas – UNICAMP, 13083-970, Campinas, SP, We are interested in how organ primordia are initiated within flowers, and
Brazil. 3Centro de Desenvolvimento Tecnologico em Saude (CDTS), in identifying the genetic factors and pathways involved in this process.
Fundacao Oswaldo Cruz (Fiocruz), 21040-900. Rio de Janeiro, RJ, Flowers of mutants of the PETAL LOSS (PTL) gene of Arabidopsis show
Brazil. 4Embrapa Cassava and Tropical Fruits, 44380-000, Cruz das a progressive loss in the ability to generate petals, and the sepals are
Almas, BA, Brazil. 5Department of Agronomy, Universidade Federal wider, closer together and sometimes fused. PTL encodes a trihelix
Rural de Pernambuco, 52171-900, Recife, PE, Brazil. transcription factor that is expressed between sepal primordia and in
sepal margins during early stages of flower development. Measurements
The molecular response of citrus plants against Citrus leprosis C of the numbers of cells that express PTL in this region indicate that there
(CiLV-C) is still unknown. To identify differentially expressed genes in are more when PTL function is lost. Thus PTL may have a role in the
response to CiLV-C, pools of seven highly tolerant and seven highly suppression of growth between sepals and in sepal margins. Disruption
susceptible hybrids from Murcott tangor (Citrus sinensis x C. reticulata) of petal initiation in ptl mutants may be the consequence of disruption
and Pera sweet orange (C. sinensis) were evaluated by microarray of a signal required for nearby petal initiation. It seems likely that auxin
analysis. In order to define susceptible and tolerant hybrids, they were provides such a signal because disruption of auxin influx in aux1 ptl
infested by viruliferous mites in 2002 and the symptoms were evaluated double mutants further compromises petal initiation. This is supported
during six years. Symptomless leaves of each hybrid were collected by observations on further disruptions to petal number in mutant
for RNA extraction. The experiment was designed in ten experimental combinations of ptl, and aux1, with other genes, including cuc, and
blocks, the leaves were collected from four blocks, each being a eep1, which further disrupt flower meristem shape when their function is
biological repetition and each repetition containing seven individuals. lost. Changes in flower meristem shape apparently cause disruptions to
The microarray chip contained 32,121, 18,873 and 12,873 unigenes auxin signalling, and the initiation of second whorl primordia is especially
from Pera sweet orange, Ponkan mandarin (C. reticulata) and Poncirus sensitive to variation in its strength.
trifoliata, respectively. These unigenes were obtained from the Citrus
ESTs database (CitEST) of Centro de Citricultura Sylvio Moreira/IAC.
The microarrays were performed by Roche Nimblegen and Bayesian
moderated T-test yielded 466 diferentially expressed genes (p.val ≤ 0.05
and fold-change ≥ 2). From these, the majority was related to metabolism,
cell rescue, defense and virulence, and protein fate. Financial support
FAPESP 2007/00117-4/INCT/Cnpq.

POS-MON-215 POS-TUE-216
TAPETUM AND POLLEN DEVELOPMENT REGULATED INVESTIGATING CELLULAR SIGNALLING PATHWAYS
BY ATMYB103 AND ITS APPLICATION IN HYBRID SEED IN CANCER CELL LINES TREATED WITH A NOVEL
PRODUCTION COMPUTATIONALLY DESIGNED MYXOMA VIRUS T5
PEPTIDE ANALOGUE
Li S.F., Phan H., Iacuone S., Avdic A., Xu Y., Pham H. and Parish R.W.
Botany Department, School of Life Sciences, La Trobe University, Almansour N.M.1, Pirogova E.2 and Istivan T.1
Melbourne, Vic. 3086, Australia. 1
School of Applied Sciences, Health Innovations Research Institute,
RMIT University, Bundoora West, Australia. 2School of Electrical and
The Arabidopsis AtMYB103 gene codes for a R2R3 MYB domain protein Computer Engineering, Health Innovations Research Institute, RMIT
whose expression is restricted to the tapetum of developing anthers and University, Melbourne, Australia.
to trichomes. We have previously shown that blocking the function of the
AtMYB103 gene using either an insertion mutant or an AtMYB103EAR M-T5 is a myxoma virus ankyrin-repeat host range protein that is shown
chimeric repressor construct results in complete male sterility and failure to to be required for virus replication in rabbit lymphocytes and the majority
set seed. These plants exhibit similar abnormalities in tapetum and pollen of human tumor cells. We investigated the cytotoxic effects of RRM-
development, with the tapetum and pollen degenerating prematurely. We MVT5, a short peptide analogue for this protein, on melanoma cell lines
are now studying the molecular network regulated by AtMYB103 protein as a future cancer therapeutic candidate. This novel computationally
and developing male sterility systems for hybrid seed production in crop designed bioactive peptide has significant apoptotic/ necrotic effects
plants. AtMYB103 regulates the expression of genes that are involved in on cancer cell lines and a negligible toxic effect on normal cells. Akt
tapetum and pollen development including the release of microspores activation is essential for normal cell processes such as programmed
from tetrads. These putative target genes of AtMYB103 protein are being cell death, proliferation and angiogenesis. Yet, the regulation of Akt
characterized and they will provide clues to the AtMYB103 molecular activation is impaired in cancer cells. It was previously shown that viral
network. To develop a reversible male sterility system and an inducible M-T5 binds with phospho-Akt to regulate Akt signalling in some human
male sterility system for hybrid seed production, several AtMYB103 cancer cell lines. In this study we investigated the effect of RRM-MVT5
homologues were cloned from crop plants. They are highly conserved in treatment on Akt activation in skin cancer cell lines to detect the possible
their deduced amino acid sequences and their promoters are only active cellular pathway that is affected by this peptide. The cellular expressions
in developing anthers. These homologues are the functional equivalents of total Akt and phspho-Akt in cell cultures of the malignant melanoma
of AtMYB103 in regulating tapetum and pollen development as they were cell line, MM96L, the human squamous carcinoma cell line COLO-16
able to restore male fertility of a male sterile atmyb103 mutant. The male and B16-F0 murine melanoma were detected using western blotting.
sterility systems using chimeric MYB103EAR repressors for hybrid seed The levels of phospho-Akt expression were assessed in cell cultures
production are discussed. treated with RRM-MVT5 peptide analogue, in the presence or absence
of the PI3 kinase inhibitor (LY294002) Our preliminary results indicated
that the cytotoxic effects of RRM-MVT5 are not targeted towards this
pathway as the expression of both total and phospho-Akt in cancer cells
were not affected by this peptide analogue.

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POS-MON-217 POS-TUE-218
ANALYSIS OF SUBUNIT INTERACTIONS WITHIN THE A MODULAR BAM COMPLEX IN THE OUTER
NUCLEOSOME REMODELLING AND DEACETYLASE MEMBRANE OF THE α-PROTEOBACTERIUM
(NURD) COMPLEX CAULOBACTER CRESCENTUS
Alqarni S., Thong S., Josep F. and Mackay J. Anwari K.1, Poggio S.2, Jacobs-Wagner C.2 and Lithgow T.J.1
School of Molecular Bioscience, University of Sydney, NSW, Australia. 1
Department of Biochemistry and Molecular Biology, Monash
University, Melbourne, Australia. 2Department of Molecular, Cellular
Our lab is interested in the structure and function of the Nucleosome and Developmental Biology and Microbial Pathogenesis Section, Yale
Remodelling and Deacetylase (NuRD) complex, which is conserved University, New Haven, Connecticut, United States of America.
across a wide range of eukaryotes and expressed broadly in mammalian
tissues. This ~2-MDa complex appears to comprise at least ten The BAM (β-barrel assembly machinery) complex is a multi-subunit
polypeptides, including Mi-2/CHD-family proteins, HDAC1/2, RbAp46, protein complex present in the outer membrane of all Gram-negative
RbAp48, MTA-family proteins (MTA1, MTA2 or MTA3), MBD2 or MBD3, bacteria. It comprises of a core β-barrel protein, BamA and associated
p66α and p66β. Despite its importance, almost nothing is known about lipoproteins that collectively participate in the folding and insertion
the structure, assembly or biochemical mechanism of action of this of β-barrel proteins. In this study, we demonstrate that the BAM
complex. RbAp46/RbAp48 and MTA1/MTA2 are core members of the complex possesses modular characteristics and contains BamA and
complex and have been shown previously to interact with each other. three outer membrane lipoproteins (BamB, BamD and BamE) in the
To identify the residues of MTA proteins that are critical for mediating α-proteobacterium, Caulobacter crescentus. In addition to the three
RbAp46/48 interactions, we generated a series of constructs encoding known lipoproteins, we identify an essential subunit of the BAM complex
truncations and mutants of MTA1 and MTA2. These constructs were referred to as peptidoglycan-associated lipoprotein (Pal). We propose
used to generate in vitro translated 35S-labeled proteins. Using pulldown that Pal is a protein that anchors the BAM complex to the peptidoglycan
experiments, we tested the interactions between RbAp46/RbAp48 and layer and promotes proximity to the inner membrane Sec machinery for
different truncations of MTA1 and MTA2. Our data indicate that RbAp46/ efficient outer membrane protein assembly.
RbAp48 proteins interact with MTA1 constructs in the same manner and
we have identified a short motif in MTA1 that appears to be necessary for
the interaction with RbAp48. We have used a range of RbAp48 mutants
to delineate the MTA1-binding surface of RbAp48. Together, these data
provide the first detailed information on the inter-subunit interactions that
facilitate the assembly of the NuRD complex, and also provide insight
into the manner by which NuRD interacts with promoter-bound proteins
to regulate gene expression.

POS-MON-219 POS-TUE-220
STRUCTURE OF DIHYDRODIPICOLINATE SYNTHASE MECHANISTIC PERSPECTIVE FOR POLYMERIZATION
FROM BACTERIA AND PLANTS: INSIGHTS INTO OF ADH AND ROLE OF α-CYCLODEXTRIN AS AN
REGULATION AND INHIBITION OF A VALID DRUG ARTIFICIAL CHAPERONE FOR SOLUBILIZATION OF
TARGET LARGER AMORPHOUS NANOAGGREGATES
Atkinson S.C., Dogovski C., Dobson R.C.J. and Perugini M.A. Barzegar A.1, 2 and Moosavi-Movahedi A.A.2
Department of Biochemistry and Molecular Biology, Bio21 Molecular 1
Research Institute for Fundamental Sciences (RIFS), University
Science and Biotechnology Institute, The University of Melbourne, of Tabriz, Tabriz, Iran. 2Institute of Biochemistry and Biophysics,
Victoria 3010, Australia. University of Tehran, Tehran, Iran.

Agrobacterium tumefaciens is a Gram-negative bacterium and the A crucial problem of protein-based agents in biotechnology and
causative agent of Crown Gall disease, which is characterised by the biopharmaceuticals is their long-term stability or so-called shelf-life.
formation of tumours at sites of environmental damage in plants. The The shelf-life of biotechnological potent enzymes is limited by thermal
disease is common to stone fruit, including grape vines, causing millions stress because of self-assembly of proteins into nanoaggregates such
of dollars of damage each year to the wine industry worldwide. Despite as nanoensembles or nanofilaments. Since biotechnological application
its widespread occurrence, there is currently no treatment for Crown Gall of mesophilic alcohol dehydrogenases (ADH) in the pharmaceutical
disease. Accordingly, there is an urgent need to discover, characterise industry is limited because of easily polymerization during thermal
and validate new antimicrobial targets in this organism. One such target process. We have evaluated the mechanism of ADH aggregation
is dihydrodipicolinate synthase (DHDPS), which is an essential bacterial with a view to establish of thermal stabilization in order to improve the
enzyme that catalyses the condensation of aspartate semialdehyde shelf-life of the ADH enzymes. The fluorescence, circular dichroism,
and pyruvate in the diaminopimelate (DAP) pathway, ultimately yielding
the amino acid lysine. Previous studies in Gram negative bacteria and UV-Vis spectrophotometry, dynamic light scattering (DLS) technique,
plants demonstrate that the downstream product of the DAP pathway, enzymatic activity assay, molecular dynamic and molecular docking
lysine, can feedback inhibit DHDPS allosterically This project aims to methods have been used for the mentioned aim. Based on the findings
characterise the structure, regulation and inhibition of DHDPS from we have proposed a new model of self-assembly for ADH enzymes that
both Agrobacterium tumefaciens (AgT) and the common grape vine, construction of nuclei and growing to formless nanoaggregates without
Vitis Vinifera (Vv). Both AgT-DHDPS and Vv-DHDPS have been cloned, enzymes denaturation and unfolding. Enzymes quaternary structural
expressed and purified to homogeneity. The recombinant enzymes are changes delocalization of subunits lead to enzymes polymerization
enzymatically active, folded and form tetramers in solution. We have without unfolding. Alpha cyclodextrin (α-CyD) caused two types thermal
solved the crystal structure of AgT-DHDPS in the apo, pyruvate-bound stabilization of ADH enzymes including kinetic and thermodynamic
and pyruvate & lysine bound forms and have used the apo structure to stabilities by masking delocalized regions of ADH chains. Delocalization
conduct an in silico screen using a virtual library comprised of more than of ADH subunits, which causes the exposure of hydrophobic domains,
4 million drug-like compounds. The screen has yielded 100 potential takes part in the enzyme polymerization and has proven to be beneficial
inhibitors, targeting either the active or allosteric sites. 10 of these for aggregation inhibition and solubility enhancement within the host
compounds show greater than 10% inhibition in vitro at a concentration α-CyD-nanocavity. The thermostabilization of ADH against aggregation
of 25 μM. Inhibition studies using the Vitis vinifera form of the enzyme as proposed for its biotechnological applications.
a control reveal that several of these hits show specificity towards AgT-
DHDPS and do not inhibit the plant enzyme in vitro. We are currently in
the process of further validating these hits both in vitro and in vivo.

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POS-MON-221 POS-TUE-222
ANALYSIS OF THE CHROMATIN BINDING PROPERTIES UNRAVELLING THE ENZYMES OF THE TARTARIC ACID
OF CHD4 BIOSYNTHETIC PATHWAY IN VITIS VINIFERA
Bendak K., Mansfield R.E., Burdach J., Crossley M. and Mackay J. Burbidge C.A.1, Soole K.L.1, Hayes M.A.2 and Ford C.M.2
School of Molecular Bioscience, University of Sydney, NSW 2006, 1
School of Biological Science, Flinders University, GPO Box 2100, SA
Australia. 5001, Australia. 2School of Agriculture, Food, and Wine, University of
Adelaide, Adelaide, SA 5005, Australia.
Chromosome helicase DNA–binding protein 4 (CHD4) is a major
component of the nucleosome remodeling and deacetylase (NuRD) Tartaric (TA) and malic acid (MA) account for 90% of total acid in the
complex. The NuRD complex contains at least 10 subunits and developing grape berry. While the majority of MA is metabolised before
plays an important role in transcription regulation and development harvest, TA levels remain constant throughout development. Little work
throughout a wide range of eukaryotic species. Mammalian CHD4 has been conducted into the synthesis of TA due to its rarity in higher
contains two plant homeodomain (PHD) fingers. Recent results from plants. Previous work has identified ascorbic acid (Asc, Vitamin C) as
our laboratory show that these fingers are capable of binding in vitro the principle precursor of this acid, with additional evidence for a minor
to the amino-terminus of histone H3. This interaction is enhanced by synthetic route via D-gluconic acid. To date only one enzyme, L-idonate
acetylation or methylation of H3Lys9, whereas H3Lys4 methylation or dehydrogenase has been identified as having an active role in TA
H3Ala1 acetylation inhibit the interaction. To examine the functional production. Bacteria including Erwinia spp and Escherichia coli possess
significance of this interaction, we have sought to determine whether enzymes capable of catalysing reactions identical to those involved in
CHD4 can bind to chromatin in vivo. Mutations have been introduced synthesising TA in V. vinifera. Comparative analysis of the E. coli and
into the PHD domains of full-length CHD4 that compromise binding to V. vinifera genomes has identified candidate genes that may encode
H3 and chromatin immunoprecipitation (ChIP) experiments have been previously unidentified enzymes of the TA biosynthetic pathways.
used to assess the ability of the mutant CHD4 to colocalize in cells Candidates were selected based on criteria including the presence of
with H3 bearing trimethylated Lys9. Additionally immunoprecipitation cofactor binding sites and conserved bases. Cloning and expression
experiments have been carried out, to establish recruitment of other of enzymes pertaining to two separate steps of the biosynthetic
NuRD complex components through CHD4. pathway has resulted in high amounts of soluble protein. Analysis of
these enzymes indicates activity towards their respective substrates.
Determination of TA levels, enzyme activity and gene expression over
berry development were used to confirm an enzymatic role for these
enzymes within the TA biosynthetic pathway.

POS-MON-223 POS-TUE-224
AP180 BINDING TO CLATHRIN IN ENDOCYTOSIS: A EXPRESSION AND PURIFICATION OF HUMAN G72 AND
NEW INSIGHT DAO, TWO SCHIZOPHRENIA-RELATED PROTEINS
Chan L.S., Robinson P. and Graham M. Chen S.Y.1, 2 , Chen Y.J.3, Wang C.M.1, Lane H.Y.4 and Chang H.T.1, 4
Children’s Medical Research Institute, The University of Sydney. 1
Graduate Institute of Molecular Systems Biomedicine, China Medical
University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan, R.O.C.
The assembly protein AP180 is involved in membrane nucleation, which 2
Master Program of Life Sciences, National Chung Hsin University,
is the first step of synaptic vesicle endocytosis (SVE). AP180 controls No. 250, Kuo Kuang Road, Taichung 402, Taiwan R.O.C. 3Department
the uniformity of synaptic vesicle size and shape. It has two known of Public Health, China Medical University, No. 91, Hsueh-Shih
binding partners – the adaptor protein AP-2, which recruits accessory Road, Taichung 404, Taiwan, R.O.C. 4Graduate Institute of Clinical
endocytic proteins to the budding membrane, and clathrin heavy chain, Medical Science, China Medical University, No. 91, Hsueh-Shih Road,
which is the main coat component of the budding vesicle. Similar to other Taichung 404, Taiwan, R.O.C.
major SVE proteins, AP180 is dephosphorylated when the synapse
is stimulated (depolarization). Our aim was to investigate the binding Schizophrenia, a mental disease, is caused by multigene dysfunction
of clathrin to different domains of AP180 and to determine if clathrin or neurontransmitter dysregulation such as N-methyl D-aspartate
binding is phopsho-regulated. We made domain-specific constructs for (NMDA) receptor agonist decreases and dopamine deficits. Previous
AP180, and found a new clathrin binding site in AP180 that challenges studies showed that patients with schizophrenia might have abnormal
previous assumptions about clathrin binding. We have also completed a regulations of D-amino acid oxidase (DAO) and G72. DAO is responsible
phosphorylation map of AP180 and found that phosphorylation regulates for D-amino acid oxidation to pyruvates and G72 is defined as a DAO
clathrin binding to AP180. The location of the new clathrin binding site activator. However, the regulatory mechanisms of G72 and DAO are still
and the regulatory phosphorylation sites suggests a staged interaction unclear. Thus, how G72 regulates DAO is the main theme of this study. In
between clathrin and AP180. This has profound implications on the order to characterize the interaction between G72 and DAO, we produed
mechanism of clathrin recruitment during stimulus-dependent SVE. and purified recombinant DAO-6H, G72-6H, MBP-DAO and GST-G72
from E. coli BL21(DE3)pLysS cells. The yield was obtained by growing
the cells overnight at 25 °C under induction with 0.5 mM IPTG during the
exponential phase of growth. We have succeeded to express DAO-6H,
G72-6H, and MBP-DAO. However, in which only MBP-DAO was purified
from soluble fraction. Initial testing showed that the enzymatic activity
of MBP-DAO is not high. It may be due to the MBP tag obstructing the
DAO activie sites. Thus the MBP tag will be removed to increase DAO
activities. Furthermore, for production of soluble G72-6H, BL21(DE3)
containing pG-KJE6 chaperone plasmids will be used. DAO is a key
enzyme for oxidizing D-amino acids in brain and its enzymatic activities
are thought highly correlated with schizophrenia. We expect to verify
DAO abnormally regulated by G72 in vitro and in vivo.

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POS-MON-225 POS-TUE-226
A POTASSIUM SWITCH OF ATP-INDUCED GROEL STRUCTURE-ACTIVITY ANALYSIS OF CAROCIN S2
CONFORMATIONAL CHANGES
Chuang D.Y. and Wu J.L.
Chen J.1, Makabe K.1, 2 and Kuwajima K.1, 2 Department of Chemistry, National Chung-Hsing University.
1
Okazaki Institute for Integrative Bioscience and Institute for Molecular
Science. 2Department of Functional Molecular Science, the Graduate Carocin S2, which contains CaroS2K and CaroS2I, is the low-molecular-
University for Advanced Studies (Sokendai). weight bacteriocin produced by Pecctobacterium carotovorium 3F-3.
CaroS2K is an 85kDa protein that consists of a membrane-tranlocation
Bacterial chaperonin GroEL utilizes the co-chaperone GroES to assist domain, a receptor-binding domain, and a catalytic domain-in their
folding of cellular proteins in an ATP-dependent manner. An ATP-induced primary sequences. These phytopathogenic cells also produce
allosteric communication through GroEL double rings is prerequisite inhibitor protein CaroS2I (Mw=10kDa) located downstream of the
for the overall chaperonin cycle. It has been shown by Horovitz et.al. caroS2K gene in the carocin operon. Fruthermore, in order to minimize
(1995) that ATP-induced allosteric transitions of GroEL consist of two potential activity of carocin within the producing cell, the association of
phases with one at relatively low ATP concentrations (≤ 100 μM) and the immunity protein with its cognate CaroS2K is rapid, specific, and
the second at higher concentrations of ATP with a midpoint in the range high affinity. Our results demonstrate clearly that the minimal inhibitory
of 16 to 160 μM. Here we studied the effect of essential metal cofactor, concentration of CaroS2K is closed to 4μg/ml with indicator strain SP33.
potassium ion, on the ATP-driven allosteric transitions of GroEL by Here, we use several bioinformatics tools to predict the structure and
using a tryptophan mutated GroEL. Within the test range of potassium functional properties of CaroS2K. The analysis accompany with site-
concentration, two distinct patterns of ATP-induced allosteric transitions directed mutagenesis reveal that the catalytic pocket centred around
were observed. We showed that at 50 mM potassium concentration, His696, the general base of the cyclization step of the reaction. The
a remarkable increasing second-phase of ATP-induced allosteric nucleophilic attack participates in the abstraction of a proton from the
transition was observed which is contrary to the decreasing phase at 2’-hydroxy group of the substrate ribose and in the protonation of the 5’-
10 mM potassium concentration. References [1] Yifrach O, Horovitz A. oxy group of a leaving substrate. The negatively charged pentacovalent
Nested cooperativity in the ATPase activity of the oligomeric chaperonin transition state can than be stabilized by nearby positively charged
GroEL. Biochemistry. 1995, 34(16):5303-8. [2] Inobe T, Kuwajima residues (Lys692 and Lys695). Other mutant bacteriocin affecting the
K. Phi value analysis of an allosteric transition of GroEL based on a main residues forming the central groove (Phe760, Ser762, Trp764) or
single-pathway model. J. Mol. Biol. 2004, 339(1): 199-205. [3] Poso D, its loop (Tyr734) for stacking on the RNA base are also not active.
Clarke AR, Burston SG. A kinetic analysis of the nucleotide-induced
allosteric transitions in a single-ring mutant of GroEL. J. Mol. Biol. 2004,
338(5):969-77.

POS-MON-227 POS-TUE-228
STRUCTURE AND FUNCTION OF THE TIMA PROTEIN BIOSYNTHESIS OF CIRCULAR PROTEINS IN PLANTS
FROM CAULOBACTER CRESCENTUS
Conlan B.1, Gillon A.D.1, Barbeta B.L. 1, Colgrave M.3, Craik D.J.2, and
Civciristov S. , Clements A. , Purcell A. , Williamson N. and Lithgow T.
1, 2 1 2 2 1 Anderson M.A.1
1
Department of Biochemistry, Monash University, Clayton. 2Bio21
1
Department of Biochemistry, La Trobe University, Melbourne VIC
Institute, The University of Melbourne, Parkville. 3086. 2Institute for Molecular Bioscience, University of Queensland,
Brisbane QLD 4072. 3CSIRO Livestock Industries, Brisbane, QLD
Mitochondria have evolved from a bacterial endosymbiont which was 4067.
probably related to present day α-proteobacteria. They possess complex
molecular machines that are found in both mitochondrial membranes. Plant cyclotides are a large family of naturally occurring circular proteins
An example is the TIM23 protein translocase of the inner mitochondrial that are produced from linear precursors and are thought to be processed
membrane that is responsible for the final step of translocation of the by asparaginyl endopeptidases to produce the circular peptide. The
proteins destined to the mitochondrial matrix. The key components of production of the circular peptide through excision of the cyclotide
this complex are the Tim23 translocation channel, the mitochondrial domains and ligation of the free N- and C-termini occurs through a little
motor Hsp70 and the Tim44 protein which is required for docking to studied mechanism. Within the plant Oldenlandia affinis, the prototypic
the channel. C.crescentus is an α-proteobacterium which has a protein cyclotide producing plant, the linear precursor Oak1 is cleaved at two
which we named TimA and it’s a protein homologue of the yeast protein sites releasing the kalata B1 domain that is then cyclised resulting in
Tim44. The aim of my study is to identify if the TimA protein interacts the mature cyclotide. Previous work in our lab has shown that O. affinis
with any other proteins as part of an inner membrane protein complex produces only the cyclic form of the kalata B1 peptide from the Oak1
in C.crescentus and to investigate its function. We found that TimA is a precursor, whilst transgenic expression of the same precursor in plants
part of a complex in the inner membrane of Caulobacter by BN-PAGE. that do not normally produce cyclotides results in the formation of both
Immunoprecipitation with the TimA antibody followed by LC-MS/MS linear and circular forms of the peptide. We have studied the importance
revealed that TimA has two partner proteins: FtsH and HflC. These two of the residues surrounding the processing site of the cyclotide domain
proteins are part of a complex in E.coli together with a third protein- by site-directed mutagenesis, which revealed a number of residues and
HflK. The function of this complex in E.coli is in inner membrane quality motifs essential for cyclisation. The substitution of a highly conserved
control of misfolded proteins or jammed translocation machines such as asparagine residue at the C-terminal processing site of the cyclotide
SecY and YidC. HflK/C were shown to negatively regulate the activity domain with an alanine completely inhibited cyclization. Similarly, when
of FtsH protease in E.coli. Having in mind that TimA proteins are only the conserved C-terminal tri-peptide motif was truncated no cyclic
found in α-proteobacteria, we are investigating the function of TimA in product was detected. The mutagenesis of a leucine in the C-terminal
Caulobacter as a novel component of the FtsH complex as well as its tri-peptide motif produced a unique processing product with implications
involvement in inner and outer membrane remodeling. for a novel mechanism of cyclisation. The observed processing may be
the result of enzyme activity never before documented in planta.

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POS-MON-229 POS-TUE230
THE USE OF CELL PERMEABLE PEPTIDES TO INHIBIT MECHANISMS OF TRANSTHYRETIN AMYLOID
SPRY DOMAIN-CONTAINING SOCS BOX PROTEIN 2 FORMATION
(SPSB2) REGULATION OF INDUCIBLE NITRIC OXIDE
SYNTHASE (INOS) D’Souza D.G.1, Altland K.3, Richardson S.J.1, 2 and Pattenden L.K.1, 2
1
School of Medical Sciences, RMIT University, Bundoora, Australia.
D’Cruz A., Lewis R.S., Kuang Z., Norton R.S., Babon J.J. and
2
Health Innovations Research institute, RMIT University, Bundoora,
Nicholson S.E. Australia. 3Department of Human Genetics, University of Giessen,
The Walter and Eliza Hall Institute of Medical Research. Giessen, Germany.

The production of nitric oxide (NO) and related reactive nitrogen Transthyretin (TTR) is a β-sheet rich homo-tetrameric protein that
species is important for the killing of intracellular pathogens. iNOS/ transports retinol-binding protein and thyroid hormones. For unknown
NOS2 (inducible nitric oxide synthase) is rapidly expressed in response reasons, TTR has the potential to dissociate to dimers, then monomers.
to infection and catalyses the high-output production of NO. We Through protein misfolding, the monomers can form insoluble fibrils
have demonstrated that SPSB2 is a negative regulator of iNOS in (amyloids) that disrupt normal cellular functions and result in disease.
Poly(I:C) and LPS stimulated macrophages, targeting iNOS for poly- Preliminary data has shown human TTR forms insoluble amyloid fibrils
ubiquitination and proteasomal degradation. The SPSB2 SPRY domain under mild acidic conditions similar to those found in vivo, however
interacts with high affinity with a short linear sequence in the N-terminal wallaby TTR under the same conditions remains as stable dimers. Each
region of iNOS. We were interested in investigating whether intracellular human TTR monomer contains His31, Ser46, and His90, close to the
delivery of an iNOS peptide could disrupt the endogenous SPSB2/ dimer interface where they help stabilise the complex. Wallaby TTR has
iNOS interaction resulting in enhanced NO production. The highly different amino acid residues in these key positions; Lys31, Ala46, Tyr90.
cationic Arginine-8 (R8) and HIV-1 derived TAT sequences are known to Our work seeks to understand why human TTR dimers are unstable
facilitate the entry of covalently attached cargo molecules into cells via compared to wallaby TTR dimers. We hypothesise that protonation of
macropinocytosis. We utilized the R8 and TAT sequences to facilitate key Histidine residues in human TTR, absent in wallaby TTR, results
entry of iNOS peptide into macrophages. While the R8-iNOS and TAT- in destabilisation of the dimer, facilitating TTR amyloid formation. We
iNOS peptides bound the SPSB2 SPRY domain with high affinity in vitro, produced human, wallaby, and seven cross-species mutant TTRs.
much of the fluorescently labelled peptide was trapped in endosomes. SDS-PAGE and Western analysis proved the recombinant TTR subunits
We then utilized the influenza virus-derived HA2 sequence to enhance were the correct size. The proteins were purified via ion-exchange
endosomal escape of fluorescently labelled R8-iNOS and TAT-iNOS. chromatography. Stability assays were then performed under mild acidic
Despite observing cytoplasmic localization of the labelled peptides, we conditions (pH 7.4-6.4). Fibril formation experiments of human TTR,
saw no differences in the level of iNOS protein expressed in response wallaby TTR and their variants were performed at pH 7.4 (physiological),
to stimuli, highlighting the difficulties of using cell permeable peptides pH 6.5 (inflammation), pH 4.6 (standard non-physiological pH used to
to interfere with biological interactions. More experiments need to make amyloid fibrils). The application of evolutionary comparative
be conducted to determine if the iNOS peptides remain biologically biology to produce single amino acid variant TTRs provides a great
available upon delivery to the cytoplasm. opportunity to study structure/function relationships and understand
molecular mechanisms underlying disease states.

POS-MON-231 POS-TUE-232
ROLE OF THE BRCA1 BINDING PROTEIN BRAP2 IN STRUCTURAL STUDIES WITH CROTOXIN B FROM
THE TESTIS CROTALUS DURISSUS SUBSPECIES VENOM
Fatima S.1, 3, Davies R.G.1, Fulcher A.J.1, Wagstaff K.M.1, Whiley P.2, 3, Fernandes C.A.H.1, Salvador G.H.M.1, Colombi D.2, Soares A.M.3 and
Loveland K.L.2, 3 and Jans D.A.2, 3 Fontes M.R.M.1
1
Nuclear Signaling Laboratory,. 2Testis and Germ Cell Development 1
Departamento de Física e Biofísica, Univ Estadual Paulista, UNESP,
Laboratory, Department of Biochemistry and Molecular Biology. Botucatu-SP, Brazil. 2Departamento de Parasitologia, Univ Estadual
3
Australian Research Council Centre of Excellence for Biotechnology Paulista, UNESP, Botucatu-SP, Brazil. 3Departamento de Análises
and Development, Monash University, Clayton, Victoria, Australia. Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências
Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-
BRAP (BRCA1 binding protein) 2 is a phosphorylation-regulated negative USP, Ribeirão Preto-SP, Brazil.
regulator of nuclear import (NRNI) of specific proteins. It is highly
expressed in testis, including a testis specific isoform, consistent with Crotoxin B is a basic phospholipase A2 found in the venom of several
a specific role in testicular development. In situ hybridization analysis Crotalus durissus ssp. rattlesnakes and is one of the subunits that
indicates BRAP2 expression in spermatogonia and spermatocytes, and constitutes crotoxin, the mains component of the venom of these
to a lesser extent in round spermatids, essentially at the same stages snakes. This heterodimeric toxin is related to important envenomation
when molecules responsible for nuclear protein import (Importins - IMPs) effects such as neurological disorders, myotoxicity and renal failure.
are highly expressed. In cotransfection experiments, we determined Although crotoxin was first crystallized in 1938, the first structural data
that BRAP2 amino acid 442-592 is sufficient to inhibit nuclear import of only become available two years ago by our research group, with the
IMPα/β-recognised cargoes from testis. In an attempt to determine the crystallization of crotoxin B from Crotalus durissus terrificus venom.
interactome of BRAP2 in testis, we screened a yeast two-hybrid library However, this structure reveled an ambiguous result for the biological
from human testis using a C-terminal fragment of BRAP2 (343-592) as assembly, which could be dimeric or tetrameric. In this work, we present
bait. Among various clones identified, several appeared to interact with the crystal structure of crotoxin B from Crotalus durissus colillineatus
proteins binding to the actin cytoskeleton, or involved in mitotic spindle venom, an unambiguously dimeric complex formed by two crotoxin
function. Future work will involve documenting the apparent link between B isoforms (CB1 and CB2). A structural comparison between these
nucleus and cytoskeleton in mammalian testicular development and the crotoxin B structures reveals differences in salt bridges that are important
role therein of BRAP2 and its interactors. Keywords: BRAP2, Testis, to oligomerization and significant Cα deviation of 54-72 region. These
Negative regulator of Nuclear Import (NRNI). structural data combined with SAXS and CD experiments with inhibitors
and site-directed mutagenesis studies, currently in progress, may give
relevant insights into the understanding of its neurotoxic mechanism of
action.

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POS-MON-233 POS-TUE-234
CHARACTERIZATION OF ANTI-STAPHYLOCOCCAL EXPRESSION AND PURIFICATION OF RECOMBINANT
PROTEIN (P128) DERIVED FROM BACTERIOPHAGE BENZALDEHYDE DEHYDROGENASE AND BENZOATE
FROM IN PROCESS TO PURIFICATION USING MICRO- DIOXYGENASE FROM RHODOCOCCUS RUBER UKMP-5M
FLUIDIC BASED LC SYSTEM COUPLED TO AN
ADVANCED QTOF-MS Hamzah A., Rabu A. and Tvakoli A.
School of Biosciences and Biotechnology, Faculty Science and
Gudihal R.1, Sundaram P.M.2, Madhavi H.N.2, Asrani J.2 and Technology, University Kebangsaan Malaysia, Bangi 43600 Selangor,
Fouracre C.G.3 Malaysia.
1
Agilent Technologies India Pvt. Ltd, Bangalore, India. 2GangaGen
Biotechnologies Pvt. Ltd, Bangalore, India. 3Agilent Technologies, Rhodococcus ruber UKMP-5M strain utilizing aromatic compounds
Sydney, Australia. such as toluene and biphenyl was isolated from oil contaminated soils
in Malaysia. The bacterium was able to growth in 0.5 and 1 mM toluene.
P128 is recombinantly expressed in E.coli. P128 has potent bactericidal The enzyme activity for toluene degradation was determined with
activity against methycillin resistant Staphylococcus aureus (MRSA) Horseradish peroxidase (HRP) and Indole-indigo method. The catechol
and is currently under early development for use in humans. Liquid production was determined spectrophotometrically by monitoring
chromatography/Mass spectrometry (LC/MS) technology is a powerful the activity of catechol 1,2 dioxygenase (260 nm) and catechol 2,3
and sensitive technique for characterization and identification of proteins. dioxygenase (375 nm), respectively. The enzyme activity for catechol
Chemical modifications and degradation that can potentially occur during 1,2 dioxygenase was 0.0142 U/ml and for catechol 2,3 dioxygenase
manufacturing, formulation, and storage of proteins, necessitate reliable was 0.007 U/ml. These two enzymes, benzaldehyde dehydrogenase
and sensitive methods for the characterization of purity and structural (xylC) which oxidized benzaldehyde to benzoic acid and benzoate
integrity. If suboptimal characteristics of biologics are discovered after dioxygenase (benA) which involved in the catechol production
initiation of clinical trials can lead to serious and costly delays in time were identified from upper (hydrocarbon-carboxylic acid) and lower
to market. Here we present accurate molecular mass measurement, (carboxylic acid-tricarboxylic acid cycle) pathways. The selected genes
impurity profiling and peptide mapping for P128 which are some of were amplified by polymerase chain reaction and cloned into E. coli
the biophysical characterization for biopharmaceutical selection. expression system. The proteins were expressed at 37°C and 22°C with
Micro-fluidic based HPLC-Chip which integrates sample enrichment, 1mM and 0.5 mM IPTG (isopropyl β-D-thiogalactoside), respectively.
chromatographic separation and nano-electrospray formation for Benzaldehyde dehydrogenase (xylC) is a 24 KD protein with pI of 5.22
efficient and high sensitivity nanospray LC/MS was used for the study. and benzoate dioxygenase is 25 KD with pI of 10.38. The recombinant
Pure P128 as well as in process samples were analyzed using C8 proteins were purified by ion exchange chromatography on DEAE-
packed micro-fluidic HPLC Chip. Flow from the chip to the accurate- Sephacel, and SP sepharose for xylC and benA.
mass QTOF MS based system was 600nL/min and occurred through
a nanospray tip etched into the polyamide material of the chip. While
for peptide mapping, purified P128 protein was subjected to trypsin
digestion followed by peptide separation and mass determination on
C18 HPLC Chip at flow rate of 600nL/min coupled to QTOF-MS system.
Further the MS/MS experiments were performed so as to confirm the
peptide sequence.

POS-MON-235 POS-TUE-236
BEYOND PERMEABILISATION - THE ROLE OF PAS-DOMAIN INTERACTIONS OF DROSOPHILA AND
INTRACELLULAR TARGETS IN THE ACTION OF AN MOUSE PERIOD CLOCK PROTEINS
ANTIFUNGAL PEPTIDE
Hennig S.1, Strauss H.2, Arens J.1, Schulze S.3, Yildiz Ö3, Theiss C.1
Hayes B., Conlan B., Anderson M. and Van Der Weerden N. and Wolf E.1
La Trobe University, Department of Biochemistry, Plenty Road,
1
Max-Planck Institut für molekulare Physiologie, Dortmund, Germany.
Bundoora 3086.
2
Max-Planck Institut für Biophysik, Frankfurt, Germany. 3Nanolytics
Gesellschaft für Kolloidanalytik mbH, Potsdam, Germany.
Fungal disease is an ever increasing burden in the fields of medicine and
agriculture. Fungal pathogens are responsible for damage and loss of Most organisms exhibit a day-night activity cycle of approximately 24
many important agricultural crops as well as human illnesses that are often hours due to a circadian pacemaker (lat. circa: about; dies: a day) which
life-threatening, particularly in immunocompromised individuals. With is operated by autoregulatory translational and transcriptional feedback
the appearance of resistance to current treatments, novel approaches loops. The function of PERIOD proteins, as central components of
are required to treat and prevent fungal disease. Antimicrobial peptides the circadian clock, is controlled by synthesis, cellular localization,
have been the focus of intense research in recent years for their use as phosphorylation, degradation as well as specific interactions with other
antibacterial agents and this focus is now turning to include their use as clock components. Furthermore PERIOD is able to form homodimers
antifungal agents. Plants naturally express a vast array of antimicrobial via its tandemly organized PAS (PER-ARNT-SIM) domains. Here we
peptides as part of their innate immune system. One such protein, the solved crystal structures of PAS (PER-ARNT-SIM) domain fragments
plant defensin NaD1, from Nicotiana alata, has potent antifungal activity of Drosophila PERIOD (dPER) and mouse PERIOD2 (mPER2) to get
and is being trialed in the protection of transgenic crops. Its mechanism insights into the regulatory role in the circadian clock at an atomic level.
of action involves entry into the cytoplasm of fungal hyphae where it In the case of Drosophila PERIOD we were able to show the importance
potentially interacts with an intracellular target. We are using various of both the hydrophobic Trp482 and a C-terminal helix for homo dimer
fluorescence based techniques to elucidate how NaD1 traverses the stabilization. The mPER2 crystal structure represents the first three
cell wall and plasma membrane with the ultimate aim of identifying the dimensional structure of a mammalian clock protein. In contrast to
intracellular target. the Drosophila protein, the mouse homolog shows a homodimer
stabilized solely by interactions of the Trp419 (Trp482 in dPER) with
the PAS-B-β-sheet surface. By solving the PAS-domain structure of
both the Drosophila PERIOD and mammalian PERIOD2 and due to our
biochemical experiments, we were able to give the first insights into the
molecular mechanisms of their homo dimerization.

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POS-MON-237 POS-TUE-238
THE THREE-DIMENSIONAL STRUCTURE OF UNDERSTANDING HOW FLIPS INHIBIT DEATH
THE BIFUNCTIONAL FAD SYNTHETASE FROM RECEPTOR-MEDIATED APOPTOSIS
CORYNEBACTERIUM AMMONIAGENES REVEALS
OLIGOMERIC ASSEMBLIES Mirams R.E.1, Vajjhala P.R.1, Stojanovski S.1, Lambert L.K.2 and Hill J.M.1
1
School of Chemistry and Molecular Biosciences, The University of
Herguedas B.1, 2 , Martinez-Julvez M.1, Frago S.1, Medina M.1 and Queensland, Brisbane QLD 4072. 2Centre for Advanced Imaging, The
Hermoso J.A.2 University of Queensland, Brisbane QLD 4072.
1
Deparmento de Bioquimica, Biologia Molecular y Celular / Institute
for Biocomputation and Physics of Complex Systems (BIFI). Upon stimulation, death receptors such as Fas/CD95 recruit the adaptor
University of Zaragoza. Pedro Cerbuna 12, 50009-Zaragoza protein FADD and procaspase-8 into the death-inducing signalling
(Spain). 2Crystallography Deparment. Instituto de Quimica-Fisica complex (DISC). Assembly of the DISC promotes the dimerisation and
Rocasolano. Spanish National Research Council (CSIC). Serrano 119, activation of procaspase-8 via an induced proximity mechanism. This
28006-Madrid (Spain). process can be inhibited by a family of cellular and viral proteins known
as FLIPs. cFLIP exists as long (cFLIPL) and short (cFLIPS and cFLIPR)
FMN and FAD are essential cofactors in flavoproteins, a large group splice variants, all capable of protecting cells from apoptosis by blocking
of proteins involved in many vital processes including photosynthesis, procaspase-8 activation at the DISC. Several herpesviruses and
fatty acid metabolism, mitochondrial electron transfer chain, apoptosis poxviruses also express FLIPs to suppress apoptosis and promote their
and DNA repair. FMN and FAD are syntesized in vivo from Riboflavin survival in host cells. The hallmark of FLIPs is the presence of tandem
(RF, Vitamin B2) in two sequential steps, the first one is related with death effector domains (DEDs) that interact with the complementary DED
a Riboflavin Kinase (RFK) activity, and the second with an FMN of FADD and prodomain of caspase-8 to hinder caspase recruitment
adenylyltransferase (FMNAT) one. In prokaryotes, both activities and activation. However, the underlying mechanisms remain unclear.
reside in a bifunctional enzyme, FAD synthetase (FADS), whereas At present structural information on the assembly and regulation of the
in eukaryotes two independent enzymes catalyze each reaction. DISC is relatively limited and DED complexes have remained elusive.
Here we present the crystal structure of the bifunctional FADS from To further characterise the molecular basis of FLIP-mediated inhibition
Corynebacterium ammoniagenes (CaFADS) solved at 1.95Å. The of apoptosis, we have optimised the production of FADD for structural
structure of the protomer reveals two independent modules with the studies and successfully formed a stable FADD-FLIP complex. Here
catalytic sites placed 40Å away. In the protomer, the product of the we present a detailed structural and biochemical analysis of the FADD-
first reaction –FMN- must be released from the protein surface and FLIP complex. Our results offer new insights into the mechanism by
subsequently bind in the catalytic site of the second reaction, where which FLIPs subvert death receptor signalling.
it is a substrate. Crystal packing interactions also reveal an hexameric
assembly formed by the interaction of two trimers. In the oligomer, both
distance and orientation of the active sites from different protomers are
compatible with the direct transfer of FMN between modules during the
FAD synthesis. These results provide evidences, at molecular level, on
the mechanism of synthesis of FMN and FAD in prokaryotes.

POS-MON-239 POS-TUE-240
THE STRUCTURE OF BOO/DIVA REVEALS A BACTERIAL DIAMINOPIMELATE EPIMERASE FORMS
DIVERGENT BCL-2 PROTEIN AN ACTIVE DIMER
Hinds M.G.1, Day C.L.2 and Rautureau G.J.P.1 Hor L.1, 2, 3, Dobson R.C.J.2, 3, Dogovski C.2, 3, Hutton C.A.1, 3 and
1
Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Perugini M.A.2, 3
Australia. 2Department of Biochemistry, University of Otago, Dunedin 1
School of Chemistry, University of Melbourne, Parkville, VIC,
9054, New Zealand. Australia. 2Department of Biochemistry and Molecular Biology,
University of Melbourne, Parkville, VIC, Australia. 3Bio21 Molecular
Intrinsic cell death is mediated by interaction between pro-apoptotic Science and Biotechnology Institute, University of Melbourne,
and pro-survival proteins of the B-cell lymphoma-2 (Bcl-2) family of Parkville, VIC, Australia.
proteins. To initiate apoptosis the α-helix formed by the BH3 domain
of BH3-only proteins interacts with the hydrophobic groove of a pro- Given the rise in antibiotic resistance, it is has become necessary to identify
survival protein. Binding events leading to pro-survival neutralization novel antibacterial drug targets. One potential candidate is diaminopimelate
depend upon a combination of conserved and variable contacts and (DAP) epimerase, which is an enzyme in the lysine biosynthesis pathway
are typically of high affinity. We present the structure, binding properties of bacteria and plants. DAP epimerase catalyses the stereoinversion of
and biological implications of Bcl-2 protein Boo. Boo has a structure that LL-DAP to meso-DAP, which is a crucial component of bacterial cell walls.
is highly homologous to other multi-domain Bcl-2 proteins where seven This study has cloned, expressed and purified DAP epimerase from
amphipathic helices surround a central hydrophobic helix that forms the E. coli. The recombinant enzyme is folded, as determined by CD
base of a surface exposed hydrophobic groove. Although structurally spectroscopy, and is active in solution. Quaternary structure analysis
homologous to other Bcl-2 family proteins, Boo has significant sequence employing analytical ultracentrifugation indicates that DAP epimerase
differences in the groove, and it lacks affinity for the binding domain of is dimeric in solution. In addition, we have recently solved the crystal
pro-apoptotic proteins. In addition the canonical BH3 domain present structure of E. coli DAP epimerase (2.0 Å) in its open, active conformation.
in all pro-apoptotic proteins is absent. These observations suggest that The crystal structure crystallised as a dimer, which is consistent with
Boo may function in a distinct manner to modulate apoptotic signalling solution study results. We anticipate the crystal structure will aid in the
pathways. discovery of novel inhibitors of this enzyme.

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POS-MON-241 POS-TUE-242
SELF-INTERACTION OF THE SULPHATE VISUALIZING DIFFERENCE OF CONFORMATIONS OF
TRANSPORTER, SHST1 C-SRC IN LIVE CELLS USING TETRACYSTEINE TAGS
AND REASH REACTIVITY
Leves F.P.1, Tierney M.L.2, Price G.D.1 and Howitt S.M.1
1
Research School of Biology, Australian National University, ACT Irtegun S., Schweiggert J., Mulhern T. and Hatters D.
0200. 2John Curtin Sxhool of Medical Research, Australian National Bio21, The University of Melbourne.30 Flemington Road.The
University, ACT 0200. University of Melbourne. Victoria 3010 Australia.

Many transporter proteins form higher order structures, involving either c-Src is the prototypic member of the Src family kinases and its activity
self interaction or interaction with other proteins. The SulP (SLC26) is upregulated in a wide variety of human cancers. While the structural
family is a diverse anion transporter family found in all domains of life. basis of c-Src catalytic regulation is well understood, the temporal and
Genetic evidence suggests that members of this family may interact spatial features of SFK regulation in cells remain unclear. The activity of
with other members and therefore that these transporters may act all SFKs depends on whether their conformation is “open” or “closed”. In
as oligomers, rather than monomers. We investigated the ability of the open active conformation, the protein has an extended configuration;
the sulphate transporter, SHST1, to interact with itself using the split- while in the inactive closed conformation the protein is more globular
ubiquitin yeast two hybrid system. We identified an interaction between and compact. Because many cancers involve dysregulated Src
SHST1 molecules suggesting that in vivo, this transporter does form function, it remains critical to establish new approaches to monitor
oligomers. Transporters of the SulP family contain an intracellular, where, within cells, the active and inactive forms accrue and change
C-terminal domain, the STAS domain, which is homologous to anti- conformation in a dynamic fashion. Here we have developed novel
sigma factors These proteins have a role in protein/protein interaction reporters of c-Src conformation which enable us to distinguish between
making the STAS domain a good candidate for an interaction domain in the “open” active and “closed” inactive states of c-Src in live cells in
SHST1. Deletion experiments were used to test whether the interaction real time. The localization of c-Src has been determined using Green
between SHST1 molecules was due to the STAS domain. It appears Fluorescent Protein (GFP) fused to the C-terminus of the protein, while
that interaction is mediated at least partly by the STAS domain, but a the conformation of c-Src has been monitored by the presence of a
successful interaction requires that this domain be correctly oriented with genetically encoded tetracysteine (TC) tag, which binds the biarsenical
respect to the membrane. Mutagenesis of conserved residues within dye ReAsH in a conformation-sensitive manner. We have recently
the STAS domain has been used to investigate the role of particular validated that our c-Src reporters work independently of intrinsic kinase
residues on both sulphate transporter function and on self-interaction. activity, subcellular localization and cell morphological differences
Some of these mutations affect both transport function and interaction, attributed to Src activation.
confirming a role for the STAS domain in interaction between SHST1
molecules.

POS-MON-243 POS-TUE-244
DISSECTING THE MECHANISM OF BINDING OF DEVELOPMENT OF FLUORESCENT CHEMOSENSOR
ISOFORM-SELECTIVE PI 3-KINASE SMALL MOLECULE SUBSTRATES FOR HIGH THROUGHPUT SCREENING
INHIBITORS OF THERAPEUTIC INHIBITORS AND ASSAY OF
ONCOGENIC PROTEIN TYROSINE KINASE ACTIVITY
Jennings I.G.1, Zheng J.1, Imran S.I.1, Pinson J.1, Schmidt-Kittler O.2, IN CHRONIC MYELOGENOUS LEUKEMIA CELLS
Kinzler K.W.2, Vogelstein B.2 and Thompson P.E.1
1
Medicinal Chemistry and Drug Action, Monash Institute of Kamaruddin M.A.1, 2 , Hossain M.I.1, Jarasrassamee B.1, Thompson P.2,
Pharmaceutical Sciences, Parkville, Victoria, Australia. 2Johns Hopkins Scanlon D.1, Cheng H.C.1 and Graham B.2
University, Sidney Kimmel Cancer Centre, Baltimore, USA. 1
Department of Biochemistry and Molecular Biology, Bio21 Institute,
University of Melbourne. 2Department of Medicinal Chemistry and
A number of small molecule inhibitors of Class 1 phosphatidyl 3-kinases Drug Action, Monash Institute of Pharmaceutical Sciences, Monash
(PI3K) are now in Phase 1 clinical trials with potential to be used in the University.
treatment of a range of diseases. However there has been little progress
in the development of isoform-selective inhibitors against the highly The Src family of protein tyrosine kinases (SFKs) are the most
conserved class 1 PI3K isoforms, p110α, β, γ & δ. Selective inhibitors extensively studied protein tyrosine kinases involved in the formation
have the potential to reduce or eliminate unwanted side effects of these and progression of many types of cancers such as leukemia, colon and
treatments. We are interested in developing an α-specific inhibitor as this breast carcinoma. Recently, aberrant activation and over-expression
isoform as it has been shown to be mutated in some cancers resulting of the SFK members of Lyn and Hck kinases were found to induce
in increased enzyme activity. In an attempt to elucidate the structural development of drug resistant to chronic myelogenous leukemia (CML).
mechanism of selectivity we have examined the 3-dimensional crystal For this reason, development of small molecule compounds capable
structure of the PI3K α and γ isoforms and identified 2 regions of non- of inhibition of SFKs are urgently needed for the treatment of patients
conserved amino acids within the PI3K catalytic site which we postulate suffering from cancer. To facilitate the search and development of
will confer isoform selectivity towards small molecule PI3K inhibitors these therapeutic small molecular SFK inhibitors, we have designed
which bind at or near the ATP binding site. We have produced a range and synthesized fluorescent chemosensor peptides suitable for high
of in vitro mutants of these amino acids in p110α. These mutants have throughput assay for SFKs activity. In our design, we combine state
been co-expressed with the regulatory subunit, p85, in insect cells with of the art solid phase peptide technology and the flexibility of the click
the resulting complex purified. Results will be presented which correlate chemistry to develop a library of fluorescent chemosensor peptide
a change in inhibitor isoform selectivity with a mutation as a means to substrates for SFKs. Among them, some can be used to monitor SFK
identify amino acids critical for isoform selectivity. In addition several activity in CML cells with high selectivity and sensitivity. The availability
chemical classes of inhibitors have been assessed for their isoform of these chemosensor peptides permit high throughput screening of
selectivity against the four isoforms of PI3K. This has been followed by potential inhibitors of SFKs.
tuning of the α-selectivity by the addition of substituents to the original
scaffold.

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POS-MON-245 POS-TUE-246
A PROTEOMIC APPROACH TO INVESTIGATE CHARACTERISING A CASPASE-2 CLEAVAGE SITE
THE MECHANISMS OF SODIUM EXCLUSION IN CONSENSUS AND IDENTIFYING ITS SUBSTRATES
PHYSCOMITRELLA PATENS
Kitevska T.1, Roberts S.J.1, Scott F.L.2 and Hawkins C.J.1
Kelly L., Ford K. and Bacic A.
1
Biochemistry, La Trobe University, Bundoora, VIC, Australia.
Australian Centre For Plant Functional Genomics, School of Botany,
2
Burnham Institute for Medical Research, La Jolla, CA, United States.
The University of Melbourne, VIC, Australia.
Apoptosis is a vital pathway which maintains cellular homeostasis. This
Reduced productivity from agricultural land caused by increasing pathway activates cysteine-aspartate proteases known as caspases. To
levels of soil salinity is a growing problem. Plants have evolved a date, thirteen mammalian caspases have been identified and most have
variety of protective mechanisms to adapt to these conditions. These been well characterised. However, the role of some remains enigmatic,
mechanisms in general involve exclusion from the most sensitive including that of caspase 2. Substrates of caspases appropriately
tissue, compartmentalisation in the vacuole where elevated salinity illustrate their function. This study hypothesises that the identification
is less detrimental to cellular processes, and the production of of novel caspase 2 substrates may well explain the recently attributed
compatible osmolytes to compensate for the associated osmotic activity of caspase-2 in cell cycle control and tumour suppression.
effects. Physcomitrella patens has been shown to be tolerant to a The aim of this study is to determine the minimal substrate specificity
variety of abiotic stresses including salinity. This species is able to of caspase-2 and use that to screen for caspase-2 substrates using
survive in sodium chloride concentration up to 600mM, if exposure is a bioinformatics approach. The P5-P1’ minimal substrate specificity
gradual. Uniquely, among plants, Physcomitrella patens and the related of caspase 2 was determined using a unique transcriptional reporter
liverwort Marchantia Polymorpha possess Na+-ATPase (ENA) pumps system in the yeast, Saccharomyces cerevisiae. Based on this hexa-
capable of Na+ extrusion, in addition to the Na+/H+ antiporters utilised by peptide profile, a screen for proteins complying with this consensus
higher plants to transport sodium across the plasma membrane. Much was carried out using the program, Probability of Protease Specificity
remains unknown about this addition mechanism for sodium exclusion, (PoPS). The candidate substrates revealed by this screen are now being
absent from higher plants and how this process is regulated. Both the tested for caspase 2-mediated proteolysis in vitro. Substrates shown to
Na+/H+ antiporter and the ENA proteins have been identified by tryptic be susceptible to cleavage in vitro will then be further characterised.
digest and mass spectrometry (MS) in the membrane fraction from
Physcomitrella patens treated with 100 mM NaCl. The current work
describes the application of subcellular and posttranslational specific
enrichment strategies combined with analysis by MS to Physcomitrella
patens grown under various salt conditions to shed light on this recently
discovered mechanism for salinity tolerance.

POS-MON-247 POS-TUE-248
EXPLORING MOLECULAR DETERMINANTS OF MOLECULAR DESIGN AND TARGETED CELLULAR
PROTEIN-CARBOHYDRATE INTERACTIONS IN THE DELIVERY OF ENGINEERED HUMAN DEOXYCYTIDINE
CARBOHYDRATE BINDING MODULE ISOFORMS OF KINASE TO ENHANCE METABOLIC ACTIVATION OF
AMPK NUCLEOSIDE ANALOG PRODRUGS AND IMPROVE
ANTICANCER THERAPY
Koay A.1, Bieri M.1, Petrie E.J.1, Bailey M.F.1, Park K.2, Gooley P.R.1 and Konrad M.1, Ort S.1, Mcsorley T.1 and Lavie A.2
Stapleton D.1 1
Max-Planck-Institute for Biophysical Chemistry, D-37077 Goettingen,
1
Biochemistry and Molecular Biology, University of Melbourne, Germany. 2University of Illinois at Chicago, Dept. of Biochemistry and
Parkville, VIC, Australia. 2Biology, University of Incheon, Incheon, Molecular Biology, Chicago, IL 60607, USA.
Korea.
Nucleoside analogs (NA) are widely used in chemotherapy of cancer
The AMP-activated protein kinase (AMPK) is an evolutionarily conserved and viral infections. These compounds are commonly administered in
enzyme essential in sensing and regulating metabolic processes. their unphosphorylated form (prodrug). NAs cross the cell membrane via
Two mammalian β-subunit isoforms exist, each containing a central nucleoside transporters and are then converted to their 5’-triphosphorylated
carbohydrate-binding module (CBM). Using NMR and fluorescence states (NA-TP) by nucleoside and nucleotide kinases. The intracellular NA-
spectroscopy, we show that the ubiquitous β1-CBM and muscle specific TPs can efficiently terminate synthesis of nucleic acids in viral replication
β2-CBM possess different affinities and specificity toward glycogen and switch on the apoptotic cascade in cancer cells. Poor activation and
mimetics. We find that the β2-CBM binds carbohydrate up to ten-fold off-target cytotoxicity often limit the efficacy of these prodrugs. NAs, such
more tightly than the β1-CBM counterpart with an 80% sequence as AZT (3’-azido-3’-deoxythymidine) for the treatment of HIV infection, the
identity. Additionally, we observe that β2-CBM binds optimally to guanosine analogs acyclovir (ACV) and ganciclovir (GCV) used against
α->1,6 glucosyl maltoheptaose, a mimetic of single-glucose branched Herpes virus, or the anticancer compounds AraC (cytosine arabinoside)
portions in glycogen that will be exposed during glycogen degradation and gemcitabine (2’-deoxy-2’,2’-difluorocytidine), are phosphorylated by
by glycogen debranching enzyme. A three-fold increase in affinity for different kinases. The rate-limiting reaction in metabolic activation often is
glucosyl-maltoheptaose was observed in β1-CBM when a threonine the first, and in some cases the second phosphorylation step. Our aim is to
is inserted into position 102. This threonine is naturally present in β2- improve enzyme-NA systems that are of potential use in the treatment of
CBM (Thr101) but not in β1-CBM. Conversely, deletion of Thr101 in certain cancers. We engineered the human enzyme deoxycytidine kinase
β2-CBM saw a three-fold loss in affinity for glucosyl maltoheptaose. (dCK) such that it phosphorylates the prodrugs AraC and gemcitabine more
Thermodynamic and NMR studies reveal more favourable enthalpy efficiently than does the wildtype enzyme. Remarkably, this enzyme is highly
change and diminished chemical shift perturbations in β2-CBM over β1- active in phosphorylating non-physiological L-enantiomers of NAs that
CBM when titrated with ligand, suggesting an optimized carbohydrate- are less toxic in vivo and biologically more potent than the corresponding
binding site in β2-CBM. Using structural and biochemical approaches, D-forms. The modified dCK is delivered to tumour cells via Epidermal
we aim to define the molecular determinants governing specificity and Growth Factor Receptor (EGFR1 and 2)-specific single chain antibodies
affinity toward carbohydrates in the CBMs,ultimately contributing to our (scFv) or affibodies, and induces apoptosis upon administration of NA.
knowledge on the biological significance of isoform-specific roles in Thus, our work highlights the success of a novel Antibody-Directed Enzyme
AMPK. Prodrug Therapy (ADEPT) concept to significantly improve NA-dependent
cancer chemotherapy.- Ref.: Sabini, E. et al. (2003) Nat.Struct.Biol. 10, 513-
9. Sabini,E. et al. (2007) J.Med.Chem. 50, 3004-14. Sabini, E. et al. (2008) J.
Mol. Biol. 378, 607-21. Hazra, S. et al. (2009) Biochemistry 48, 1256-63.

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POS-MON-249 POS-TUE-250
CHANNELLING SCENT: NEW APPROACHES TO STRUCTURAL BASIS FOR THE INHIBITION OF
UNDERSTAND THE STRUCTURAL BASIS OF INSECT APOPTOSIS BY EPSTEIN-BARR VIRUS BHRF1
ODORANT RECEPTION
Kvansakul M.1, 2 , Wei A.1, Fletcher J.I.1, Willis S.N.1, Chen L.1,
Kralicek A.V.1, Law A.L.M.1, 2, Carraher C.1, 2, Christie D.L.2 and Colman P.M.1 and Huang D.C.S.1
Newcomb R.D.1, 2 1
La Trobe Institute of Molecular Sciences, La Trobe University,
1
The New Zealand Institute for Plant & Food Research Limited, 120 Kingsbury Drive, Bundoora, Victoria 3086, Australia. 2The Walter &
Mount Albert Rd, Auckland 1142, New Zealand. 2School of Biological Eliza Hall Institute of Medical Research, 1 G Royal Parade, Parkville,
Sciences, University of Auckland, Auckland 1010, New Zealand. Victoria 3050, Australia.

In animals the sense of smell is mediated by large families of membrane Viruses must evade host apoptotic defences to ensure their own
proteins known as odorant receptors (ORs). We are one of the first survival. Despite the complexity of mammalian cell death processes,
groups to show that insect ORs are not G protein-coupled receptors, viruses have evolved successful mechanisms for subverting the
as mammalian Ors are, but have a distinct membrane topology and apoptotic machinery, including homologs of the mammalian pro-survival
ability to function in the presence of G protein signalling inhibitors1. protein Bcl-2. The ubiquitous Epstein-Barr virus (EBV), a member
Instead they comprise a novel family of seven transmembrane ligand- of the γ-herpesviruses, infects the epithelium of the oropharynx and
gated ion channels2,3. As a major step towards understanding insect OR resting B cells. Acute infection manifests as infectious mononucleosis
structure/function we have overexpressed and reconstituted ORs into or glandular fever, whereas chronic EBV-associated transformation
artificial membranes. This offers a unique opportunity to investigate the is associated with Burkitt’s lymphoma, Hodgkin’s disease and
stoichiometry and structural nature of the interaction between the ligand- nasopharyngeal carcinoma. EBV BHRF1 is a sequence, structural
binding and ion channel subunits of the receptor complex. Additionally we and functional homologue of Bcl-2, however its mechanism of action
are using a comparative approach to understand where odor recognition remained unclear. Previous structural studies indicated that BHRF1
is encoded. We have identified orthologues of the Drosophila receptor, lacks an accessible BH3 binding groove, and shows only weak affinity
Or22a, that differ in their response to 2-heptanone using cell-based for BH3 ligands. We show that BHRF1 is a potent inhibitor of apoptosis,
assays4 (D. melanogaster EC50 = 1.8x10 -7 M, D. mauritiana EC50 = 5.4 and confers chemoresistance in mouse lymphoma models similar to
x10 -10 M) and determined EC50 values for a series of chimeric receptors mammalian Bcl-2. Next, we determined the crystal structures of BHRF1
to locate a region necessary for ligand binding. This work is contributing in complex with Bim and Bak BH3 peptides and show that in contrast to
to the design and development of real-time olfactory biosensors using previous predictions, BHRF1 interacts with these proteins in a manner
insect ORs and the design of compounds for pest control. 1) Smart et similar to its mammalian counterparts. Structure-based mutagenesis
al. (2008) Insect Biochem. and Molec. Biol. 38: 770-780, 2) Sato K et al. enabled us to address the molecular mechanisms underlying BHRF1
(2008) Nature 452: 1002-1006, 3) Wicher et al. (2008) Nature 452: 1007- activity. We demonstrate that BHRF1 can prevent Bak activation by
1011, 4) Kiely et al. (2007) J. Neurosci. Methods 159: 180-194. direct interaction, but prevents Bax activation indirectly by sequestering
the BH3-only proteins Bim, Puma and tBid. Unlike mammalian pro-
survival proteins, BHRF1 does not interact with the selective/sensitizer
BH3-only proteins. These studies indicate that BHRF1 might be targeted
by small molecule mimetics of BH3-only proteins.

POS-MON-251 POS-TUE-252
THE NATURAL HISTORY OF MITSUGUMIN-53 AND THE ANALYSIS OF HOMOTYPIC AND HETEROTYPIC
MEMBRANE REPAIR COMPLEX INTERACTIONS OF ARENAVIRUS Z MATRIX PROTEIN
Lemckert F.1, Waddell L.1, Tran J.1, Zheng F.1, Evesson F.1, Chen A.1, Loureiro M.E., Levingston MacLeod J.M. and Lopez N.
Clarke N.1, Ma J.2, North K.1 and Cooper S.1 Instituto de Ciencia y Teconologia Dr Cesar Milstein (CEVAN),
1
Institute for Neuroscience and Muscle Research, the Children’s CONICET. Saladilo 2468 (C1440FFX). Buenos Aires, Argentina.
Hospital at Westmead & the University of Sydney, Australia. 2Robert
Wood Johnson Medical School, the University of Medicine and Arenaviruses, such as Tacaribe virus (TacV) and its closely related
Dentistry of New Jersey, Piscataway, USA. pathogenic Junin virus (JunV), are enveloped viruses with a bipartite
negative-sense RNA genome which encodes the nucleocapsid
Defects in the muscle membrane repair pathway cause muscular protein (N), the precursor of the envelope glycoprotein complex
dystrophy. Dyferlin, responsible for limb-girdle muscular dystrophy 2B (GP), the polymerase (L) and a RING finger protein (Z). TacV-Z is a
and Miyoshi Myopathy, was the first muscle membrane repair protein multifunctional protein that has a key role on virus morphogenesis. In
identified. Caveolin-3 and annexinA1 have since been implicated in this addition, Z is able to self-associate, and exhibits an inhibitory effect
pathway; mutations in caveolin-3 cause human muscle disease. A new on viral RNA replication and transcription through its interaction with
protein, mitsugumin-53 (MG53), interacts with dysferlin and is involved the L polymerase (J.Virol. 77:10383-93, 2003). We have previously
in muscle membrane repair. MG53 accumulates at sites of membrane shown that the region comprised between the residues G36 and R85
damage in normal muscle fibres, and MG53 knockout mice display a of Z is sufficient to maintain Z-L interactions and Z inhibitory functions.
progressive muscular dystrophy and impaired resealing of isolated To learn more about the roles of individual amino acids in the different
muscle fibres. We sequenced MG53 in 50 patients with muscular interactions of Z, a panel of point mutants of TacV-Z and JunV-Z was
dystrophy of unknown genetic basis in which all other common causes of created by in vitro mutagenesis. The interaction between Z mutants
muscular dystrophy had been excluded. Although no primary mutations and L protein was analyzed by a coimmunoprecipitation assay and a
were identified, we still consider it a feasible disease gene candidate. minireplicon system was used to examine the effect of mutations on
Little is known about MG53’s role in skeletal muscle and how this viral RNA synthesis. The capacity of Z mutants to self-associate was
and other membrane repair proteins may be intrinsically modulated in also evaluated by coinmunoprecipitation and crosslinking essays. Our
patients with muscle disease. We characterised the expression of MG53 results show that single amino acid changes selectively interfere with
and other membrane repair proteins in control and diseased muscle Z-Z or Z-L interactions.
by Western blot and immunohistochemistry, to determine if levels and
localisation of these proteins (the membrane repair complex) change
with age or in response to disease, and if any correlation exists between
intrinsic modulation of membrane repair pathways and disease severity.
We show that dysferlin, MG53, caveolin-3 and annexinA1 are markedly
upregulated in muscular dystrophy, and by varying levels in different
forms of dystrophy. We assessed the degree of protein upregulation
through quantitative Western blot and examined the effects of primary
dysferlin, dystrophin and caveolin-3 protein deficiencies on modulation
of the other membrane repair components.

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POS-MON-253 POS-TUE-254
DIFFERENT DOMAINS OF THE DROSOPHILA CATHEPSIN B AS A TARGET FOR CONTROL OF
INSULATOR PROTEINS INTERACT WITH CP190 AND BLOOD-FEEDING PARASITES
E(Y)2 PROTEINS MEDIATING VARIOUS FUNCTIONS OF
DROSOPHILA INSULATORS Jilkova A.1, Horn M.1, Rezacova P.1, Kopacek P.2, Caffrey C.R.3 and
Mares M.1
Maksimenko O., Bonchuk A., Stakhov V. and Georgiev P.
1
Institute of Organic Chemistry and Biochemistry, Academy of
Institute of Gene Biology RAS, Moscow, Russian Federation. Sciences of the Czech Republic, 16610 Praha, Czech Republic.
2
Institute of Parasitology, Biology Centre of the Academy of Sciences
Insulators affect interactions between promoters and enhancers or of the Czech Republic, 37005 Ceske Budejovice, Czech Republic.
silencers and function as barriers for spreading of repressive chromatin.
3
Sandler Center for Drug Discovery, QB3, University of California San
A core of insulator’s protein part is highly specific DNA-binding protein Francisco, San Francisco, CA 94158, USA.
which binds DNA sequence of insulator. Such proteins responsible
for activity of the Drosophila insulators are dCTCF, Su(Hw) and Zw5 Hemoglobin digestion is an essential process for blood-feeding
proteins. It contain multi-zinc-finger DNA-binding domain connected parasites. In blood flukes and ticks, hemoglobin degradation is
with other poorly studied domains. Recently centrosomal protein CP190 orchestrated by a network of aspartic and cysteine peptidases operating
has been identified as functional component of Su(Hw) and dCTCF- at acidic pH. Our work focuses on cathepsin B, a cysteine peptidase
dependent insulators. CP190 co-precipitates insulator proteins but functioning as both an exo- and endopeptidase. In the hard tick, Ixodes
details of that interaction remain unknown. In this work we found that ricinus, a vector of Lyme disease and tick-borne encephalitis, we
CP190 is a major protein which precipitates with purified GST-tagged applied functional proteomic approaches to demonstrate that cathepsin
C-terminal part of dCTCF, middle part of Zw5 from S2 cells crude extract. B (IrCB) is the most abundant digestive enzyme critical for hydrolysis
Using GST pull-down assay we confirmed that CP190 and dCTCF, Zw5 of hemoglobin fragments. Accordingly, IrCB represents a candidate
directly interacts in vitro. We mapped region of CP190 sufficient for antigen for developing novel anti-tick vaccines. In the human blood fluke,
interaction with dCTCF, Su(Hw) and Zw5 insulator proteins in GST pull- Schistosoma mansoni, cathepsin B1 (SmCB1) is a therapeutic target for
down assay. Surprisingly such a region (308-470aa) overlaps with a the treatment of schistosomiasis. We have solved the crystal structure of
microtubule-interacting domain of CP190 and is independent of its zinc- SmCB1 and analyzed the inhibitory regulation of the enzyme active site.
finger domain. Then we have studied interactions with another insulator The identified structural relationships represent a potential tool for the
component - E(y)2/Sus1. Earlier we demonstrated the specific role of development of antischistosomal drugs. (This work was supported by
the E(y)2 protein in the barrier activity of Su(Hw) insulators. In this work grants P207/10/2183 (GACR) and IAA600960910 (GAASCR), research
we found that E(y)2 protein interacts with dCTCF in the yeast two-hybrid projects Z40550506 and Z60220518, and the Sandler Foundation.).
assay. Using GST pull-down assay we confirmed that the dCTCF and
Su(Hw) zinc fingers were essential for interaction with E(y)2. E(y)2 co-
precipitates insulator proteins dCTCF and Su(Hw) from S2 cells. Thus
we suggest that insulator complexes containing different DNA-anchor
proteins have general functional activities owing to the same protein
partners responsible for enhancer-blocking and boundary activities of
Drosophila insulators.

POS-MON-255 POS-TUE-256
THE N-TERMINAL EXTENSION OF CHARACTERIZATION OF ASSEMBLY FACTORS
HOLOCARBOXYLASE SYNTHETASE IS REQUIRED INVOLVED IN COMPLEX I BIOGENESIS AND DISEASE
FOR RECOGNITION OF PROTEIN SUBSTRATES
McKenzie M.1, Lazarou M.1, Thorburn D.R.2 and Ryan M.T.1
Mayende L., Swift R.D., Bailey L.M., Wallace J.C., Booker G.W. and
1
1Department of Biochemistry, La Trobe University, Melbourne,
Polyak S.W. AUSTRALIA. 22Murdoch Childrens Research Institute, Royal
Discipline of Biochemistry, University of Adelaide, Adelaide, 5005. Children’s Hospital, Melbourne, AUSTRALIA.

Protein biotinylation is an example of a protein:protein interaction Mitochondrial Complex I (NADH: ubiquinone oxidoreductase) is an
with exquisite specificity. Biotin protein ligase (BPL) catalyses the ~980 kDa multimeric enzyme composed of 45 subunits, with 7 subunits
activation of biotin-dependent enzymes, through the attachment of the encoded by mtDNA and the remainder by nuclear genes. The assembly
prosthetic group biotin. The catalytic region of all BPLs is contained of human Complex I is not well understood, complicated by its large size
in the conserved C-terminal region. Human BPL or holocarboxylase and its regulation by two genomes. In recent years a number of proteins
synthetase (HCS), contains a long N-terminal extension that is not have been described which aid the assembly of Complex I, however the
present in bacterial BPLs. The structure and function of the N-terminal exact functions of these ‘assembly factors’ remains unclear. We have
region is poorly understood. In order to delineate the role of this extension now defined the roles of various assembly factors which act at either
the domain structure of HCS was mapped using limited proteolysis. Two early, mid or late stages of Complex I assembly. C20orf7 and C8orf38
protease-sensitive linker regions were identified, one between residues are important during early stages, acting as potential transcriptional
151-153, the other at residue 314. The domain containing residues 159- activators of the mtDNA-encoded Complex I subunit ND1. Pathogenic
314 was implicated in the reaction mechanism despite being distal to mutations in the genes of either of these assembly factors result in a
the C-terminally located active site. Mutations within this domain give translation defect and/or the rapid turnover of ND1, leading to Complex I
rise to the metabolic disorder multiple carboxylase deficiency (MCD), deficiency and patient death. The assembly factor C6orf66 (NDUFAF4)
which is poorly responsive to current therapies. Novel mutations in this also acts at early stages of assembly, however it does not interact
N-terminal region were generated using ‘error prone’ PCR and isolated with any mtDNA-encoded subunits, instead aiding the assembly of
through a genetic screen in bacteria. Residues mutated in these isolates early matrix arm intermediates. During the middle stages of Complex
were identified by DNA sequencing and found to be those normally I biogenesis the assembly factors CIA30 (NDUFAF1) and ECSIT both
highly conserved between species. Using yeast-two hybrid analysis we directly interact with the subunit ND2, suggesting roles for both as
present the first evidence for an interaction between the N-terminal and molecular chaperones. At later stages, CIA30, ECSIT, and the assembly
C-terminal halves of HCS. The region of N terminal domain required factor B17.2L (NDUFAF2) are found in larger Complex I assembly
for interaction was mapped to the domain encompassing residues 159- intermediates of ~830 kDa. However, B17.2L only co-precipitated with
314. We show that mutations in the N-terminal region compromise the ECSIT, suggesting the existence of different ~830 kDa species which
interaction of HCS with its protein substrate, but not the intramolecular contain 1) CIA30 and ECSIT after assembling from smaller intermediates
interaction between the two halves. This provides a new mechanism for or 2) B17.2L and ECSIT following the release of CIA30. Our studies
protein biotinylation as well as a molecular explaination for MCD. have helped to define the function of different assembly factors during
various stages of Complex I assembly, and have also provided insights
into how defects in these proteins disrupt Complex I biogenesis and lead
to mitochondria disease.

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POS-MON-257 POS-TUE-258
ELUCIDATING THE MOLECULAR DETAILS OF RSSB- A MECHANISM OF DNA HOMOLOGY SEARCH, BASED
MEDIATED TURNOVER OF SIGMAS BY THE AAA+ ON THE STRUCTURE OF DNA BOUND TO PROTEINS
PROTEASE, CLPXP THAT PROMOTE HOMOLOGOUS RECOMBINATION
Micevski D., Spall S.K., Truscott K.N. and Dougan D. Masuda T.1, Ito Y.2, Terada T.3, Shibata T.1 and Mikawa T.1, 4
La Trobe Institute for Molecular Sciences, La Trobe University, 1
Cllular & Molecular Biology Unit, RIKEN Advanced Science Institute.
Melbourne 3086, AUSTRALIA. 2
Department of Chemistry, Tokyo Metropolitan University. 3Graduate
School of Agricultural and Life Sciences, The University of Tokyo.
In bacteria, promoter recognition and transcription initiation requires 4
Biometal Science Laboratory, RIKEN SPring-8 Center.
both the RNA polymerase core enzyme and a sigma factor. There are
seven different sigma factors in Escherichia coli, each of which are able Homologous recombination (HR) plays a critical role in genetic diversity.
to recognise a specific subset of genes. The SigmaS (also known as The RecA/Rad51 family of proteins is well known for promoting HR in
RpoS) regulon comprises ~10% of all E. coli genes, and is crucial for an ATP-dependent manner. Recently, the crystal structure of the RecA-
the cells response to a variety of different stresses including glucose ssDNA complex has been determined. In addition, ATP-independent
starvation and DNA damage. As the master regulator of what is often HR proteins with significantly different structures to that of RecA/Rad51
referred to as the “general” stress response it is a key factor in bacterial proteins have been identified. However, the mechanism of HR remains
adaptation. Given this crucial role in bacterial adaptation, the cellular unclear. In this study, we demonstrate a base rotation mechanism
concentration of SigmaS is tightly controlled, not only at the transcriptional for HR based on the structure of ssDNA bound to HR proteins. We
and translational levels, but also most importantly by proteolysis. The determined the structure of ssDNA bound to five evolutionarily distinct
turnover of SigmaS is an ATP-dependent process, which is carried out HR proteins and found that these proteins induced a common ssDNA
by the AAA+ protease, ClpXP. Interestingly an additional factor - RssB structure characterized by long inter-base distances and reduced DNA
(Regulator of SigmaS B) - is also required for SigmaS turnover, however base stacking. Furthermore, the molecular dynamics simulations of
to date the mechanism by which the adaptor protein RssB prepares or the RecA-ssDNA complex indicated that the bases in RecA filament
delivers SigmaS for ClpXP-mediated degradation remains unclear. One rotate significantly, providing a mechanism for the homology search
model suggests that RssB delivers SigmaS to the unfoldase ClpX via a process of HR. Interestingly, RNA was unable to adopt this extended
direct interaction between the C-terminal tail of RssB and the N-terminal conformation. Our results suggest that the extended structure of
domain of ClpX. To dissect the role of RssB, in ClpXP-mediated SigmaS ssDNA is a critical determinant of HR, and have provided DNA with
turnover, we have performed a range of different experiments including an important evolutionary advantage over RNA for the development of
site-directed mutagenesis, half-life and co-immunoprecipitation genomic diversity.
experiments. Collectively our data favours a direct delivery model.

POS-MON-259 POS-TUE-260
DIHYDRODIPICOLINATE SYNTHASE: EBITEIN1, A NOVEL ERK1/2 BINDING PROTEIN
O-AMINOBENZALDEHYDE ASSAY FOR HIGH-
THROUGHPUT CHEMICAL SCREEN AND Miura K. and Imaki J.
CHARACTERISATION OF CHROMOPHORE Department of Developmental Anatomy and Regenerative Biology,
National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama
359-8513, Japan.
Mitsakos V.1, 2, 3, Hutton C.A.1, 2 and Perugini M.A.2, 3
1
School of Chemistry, The University of Melbourne, Parkville, VIC,
Australia. 2Bio21 Molecular Science and Biotechnology Institute, The EBITEIN1 is a novel protein that binds with ERK1/2 that is one of the
University of Melbourne, Parkville, VIC, Australia. 3Department of classical MAP kinases. EBITEIN1 was found abundantly in round
Biochemistry and Molecular Biology, The University of Melbourne, spermatids. During spermatogenesis, EBITEIN1 was first translated
Parkville, VIC, Australia. after meiosis when cells became haploid. Then, the amount of
EBITEIN1 protein gradually increased, reaching a maximum at
Oakberg’s stage 9. The level of EBITEIN1 decreased such that it
In search of a new class of antibacterial agents, the exploration of was undetectable when the flagellum of the spermatozoon was
compounds to target the enzyme dihydrodipicolinate synthase (DHDPS) generated. EBITEIN1 was localized to the cytoplasm on a subcellular
is described in this study. The colourmetric o-aminobenzaldehyde level. Binding experiments using various deletion mutants identified
(o-ABA) assay is an assay suitable for a high-throughput chemical a 40-amino acid minimal sequence for binding ERK2. Furthermore,
screen. Optimisation studies in cuvette format and subsequently 96- binding experiments using substitution mutants indicated the crucial
well format have allowed the derivatisation of kinetic data using the role of arginine residues in this sequence. Based on empirical and
o-ABA assay, with values matching those shown from coupled assay bioinformatic analyses, we proposed two domains in EBITEIN1. One
studies. In addition, the purple chromophore of the o-ABA reaction is EB domain, the minimal sequence for binding ERK2, and the other
has been analysed by UV/Vis-spectrophotometry, NMR and MS and is ECT domain, the EBITEIN1 C-terminal domain. EBITEIN1 bound to
found to be a diazaanthracene. The characterisation of the product has nonphosphorylated and phosphorylated forms of ERK1 and ERK2,
provided confidence that the assay is suitable for quantitative work. A respectively. Phosphorylation and dephosphorylation experiments
high-throughput screen (88,000 compounds) against DHDPS from the indicated that EBITEIN1 is usually phosphorylated in vivo and that it
pathogen B. anthracis has shown several hits in the micromolar range, is a substrate of ERK2. The ERK2-binding domain was required for
which will allow the design of analogues with potent inhibition (nano- phosphorylation of EBITEIN1. Based on these results, it is suggested
picomolar range) against DHDPS. that EBITEIN1 is a phosphoprotein and a downstream interactor of
ERK2 that participates in the intracellular signal transduction pathway,
especially in the morphogenetic development of round spermatids into
spermatozoa. Although EBITEIN1 cDNA was originally cloned from a
mouse brains cDNA library, the functions of EBITEIN1 in the brains
remain to be identified.

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POS-MON-261 POS-TUE-262
ANALYSIS OF THE CONSERVED CX3C MOTIF IN TIM9 PROTEOMIC ANALYSIS OF RICE ROOT CULTURES
AND TIM10 PROTEINS OF THE MITOCHONDRIAL (ORYZA SATIVA L. SUB. INDICA) DURING SODIUM
INTERMEMBRANE SPACE CHLORIDE STRESS
Mooga V.P., Baker M.J., Stojanovski D. and Ryan M.T. Bhorncharoennop C.1, Phaonakrop N.2, Jaresitthikunchai J.2, Uyen U.1,
La Trobe University. Atikankun S.1, Roytrakul S.2 and Wetprasit N.1
1
Department of Biotechnology, Faculty of Science, Ramkhamhaeng
About ~99% of mitochondrial proteins are encoded by nuclear genes, University, Bangkok 10240, Thailand. 2Genome Institute, National
synthesised in the cytosol and are imported into the organelle. Specific Center for Genetic Engineering and Biotechnology, Pathumthani
translocases have evolved in the outer and inner mitochondrial 12120, Thailand.
membranes to sort nuclear encoded proteins to one of four sub
compartments; the outer membrane, the intermembrane space, the Saline soil is one of the major problems that limit rice production in
inner membrane and the matrix. The Tim9-Tim10 complex of the Thailand. In salt stress response in rice, root is the first organ that
mitochondria acts to chaperone the movement of selected hydrophobic perceives salinity. In order to improve the salt-tolerant ability in rice
precursor proteins through the intermembrane space. The crystal cultivars, the differentially expressed proteins in rice root during salt
structure of the yeast Tim9-Tim10 complex revealed that it is structurally stress are necessary to investigate. The roots of 2-month old salt-
similar to its human counterpart. The conserved dual Cx3C motif in sensitive (Pathumthani 1) and salt-tolerant (Hom-Jan) rice seedlings
Tim9 and Tim10 allows the formation of two intramolecular disulfide were cultured in sugar-free liquid MS (Murashige & Skoog) media before
bonds, which stabilise the helix loop helix topology. To analyse the applying the salt stress (513 mM NaCl). The roots were then collected
significance of the disulfide bonds in the biogenesis of Tim9 and Tim10 at different exposure times as 0, 3, 6, 9 12, 24, and 48 h. Total proteins
in vivo, yeast with Tim9 and Tim10 cysteine mutants were generated. were extracted and subjected to SDS-PAGE separation, proteins from
Removal of both disulfide bonds in either Tim9 or Tim10 resulted in a systematically sectioned gel lanes were identified by nano LC-MS/MS
lethal yeast phenotype. However, deletion of the inner disulfide bond of tryptic peptides. The LC-MS data were analyzed using DeCyderMS
in Tim9 or Tim10 (tim9C39S/C55S, tim10C44S/C61S) caused only a 2.0 followed by MASCOT software. Eight proteins were observed
temperature sensitive phenotype, while removal of the outer disulfide as differentially expressed following sodium chloride stress in both
bond (tim9C39S/C55S, tim10C40S/C65S) resulted in minor growth varieties and 4 proteins were identified to be the functional proteins.
defects. Substrate import and assembly studies suggest that a single These proteins involved in oxidative stress, photosynthesis system and
disulfide bond is sufficient for the biogenesis and function of Tim9 and cell wall organization. Of these 8 proteins showed down-regulation in
Tim10, with the inner disulfide bond appearing to be more important root culture of the salt tolerant Homjan rice while 7 proteins exhibited
than the outer disulfide bond. up-regulation in root culture of salt sensitive Pathumthani 1. Only 1
protein in Pathumthani 1 root performed an increasing at first and then
gradually decreasing expression pattern. These results indicated the
extent of the salinity response in rice root cells and suggested a number
of key regulatory proteins and pathways that are involved in modulating
the response of rice root cells to salinity. These will lead to gain more
understanding about salt stress response that could be useful for crop
selection and improvement of salt-tolerant in rice.

POS-MON-263 POS-TUE-264
COLD ADAPTATION OF PROTEINS: A BIOPHYSICAL COMPARATIVE STUDY OF HIV-1 TAT TRANSIENTLY
STUDY OF A PSYCHROPHILIC ALPHA-AMYLASE AND AND TRANSGENICALLY EXPRESSED IN TOMATO
ITS STABILIZED MUTANTS
Cueno M.E., Yamato H., Hibi Y. and Okamoto T.
Cipolla, A.C., Damico, S.D. and Feller, F.G. Nagoya City University, Nagoya City, Aichi, Japan.
Laboratory of Biochemistry, Center of protein Engineering, Institute of
Chemistry B6a, University of Liege, B-4000 Liege, Belgium. Plant-based protein production has grown to be an ideal method for
transient protein production since it is free from animal pathogens and
Permanently cold environments, like polar regions, have been colonized has lower cost of production. Current methods of protein production in
by a great variety of psychrophilic organisms producing enzymes plants include transient expression and transgenic plant development.
adapted to function efficiently at low temperatures. We have investigated In this study, we noted the advantages and disadvantages of Tat
the role of weak interactions in thermal adaptation of proteins by site- protein transiently expressed in tomato calli with Tat protein expressed
directed mutagenesis of the psychrophilc alpha-amylase (AHA) from the in a transgenic tomato plant. Plant-optimized gene constructs was
Antarctic bacterium Pseudoalteromonas haloplanktis. Two stabilized synthesized and introduced into leaf calli through bombardment. One
multiple-mutants (Mut5 and Mut5CC) have been constructed. The set of bombarded calli was maintained in zeatin-containing medium
single mutations were selected by comparison of the presence of weak to allow Tat transient expression. Another set of bombarded calli was
interactions in a mesophilic homolog from pig pancreas, PPA. The three maintained in growth medium to allow Tat-expressing transgenic
enzymes AHA, Mut5 and Mut5CC have been analyzed by differential tomatoes. Immunological assays determined the presence of Tat in
scanning calorimetry, thermal and chemical denaturation. The flexibility both sets of tomatoes. Effects of Tat expression on tomato were visually
has been studied by acrylamide-induced fluorescence quenching. In determined and confirmed by measuring cytokinin levels. Likewise,
order to investigate the kinetic origin of the gain in stability, the kinetics production time and thermostability of Tat from both tomato sets were
of unfolding and refolding in GdmCl have been monitored at 15°C. The compared. Ultimately, Tat produced from both sets were introduced
newly introduced weak interactions stabilized the mutants, protected intradermally to Balb/c mice and immunogenic responses were observed
them against heat and chemical unfolding and also induced an effective through ELISA and ELISPOT. Both tomato sets showed preferential
loss of flexibility. In addition, the two multiple-mutants exhibit an fusion protein expression and thermostable production in room
increased optimum temperature for activity. The first results of kinetic temperature. Transgenically expressed Tat impaired plant development
studies show a similar refolding phase but differences between the by differentially expressing cytokinin oxidase resulting to lower cytokinin
three amylases in the unfolding phase. These results unambiguously levels. Transiently expressed Tat was found to be secreted and absorbed
support the capital role of weak interactions in the balance between from bombarded and naive tomato calli, respectively. Both humoral
activity, flexibility and stability and provide a better knowledge of the and cellular immune responses were induced using either tomato.
adaptation of enzymes to cold temperatures. Interestingly, higher cellular immune response was induced using
transiently expressed Tat as compared to transgenically expressed Tat.
Our results show more advantages in transiently expressing Tat in plant
calli than developing transgenic tomatoes expressing Tat.

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POS-MON-265 POS-TUE-266
EVALUATION OF ALTERNATIVE MECHANISMS FOR DIFFERENTIAL ANALYSIS OF VITAMIN D TREATED
THE EFFICIENT PRODUCTION OF A PLANT-MADE U937 MYELOID LEUKEMIC CELLS
VACCINE
Durand F.M.1, 2 , Levina V.1, 2, Cooke I.1, 2, Coley A.1, 2, Hoogenraad N.2
De Guzman G.M., Walmsley A.M., Webster D. and Hamill J.D. and Talbo G.H.1, 2
Monash University, Clayton, VIC, Australia 3800.
1
CRC for Biomarker Translation, La Trobe University, Melbourne,
Australia. 2:La Trobe Institute for Molecular Science, La Trobe
Using plants as an alternative vaccine production system to current University, Melbourne, Australia.
systems has shown promise with a range of vaccines produced in a
range of plant systems. Plants provide possible advantages for oral Unlike normal progenitor cells, haemopoietic cells from patients with
delivery, reduced cost, large scale production and storage. In this study, acute myeloid leukemia have an apparent inability to differentiate
hairy root cultures were utilized for their rapid growth, high genetic and terminally mature. U937 myeloid leukemic cells can be induced
stability, potential for rhizosecretion, and their ability to be regenerated to differentiate into macrophages by 1α,25-dihydroxyvitamin D3
into whole plants. The B-subunit of the heat-labile toxin from E.coli (1α,25(OH)2D3). Growth arrest of these cells has been shown to
was expressed in hairy root cultures of Tobacco, Tomato and Petunia be blocked at G1 in the cell cycle, happening concomitantly with an
to evaluate LTB production in and between each species. The hairy upregulation of the cell surface membrane proteinaceous monocyte/
root cultures of Tobacco and Petunia expressed LTB antigen at high macrophage-specific markers, CD14 and CD11b. Granulocyte-
concentrations of LTB ~70ug/g FW tissue on average, while tomato macrophage colony-stimulating factor (GM-CSF), when used in
produced low amounts of antigen (~2ug/g FW). Throughout the root conjunction with 1α,25(OH)2D3 has been shown to have a synergistic
growth cycle, LTB was shown to peak after 22 days however, a marked effect on U937 cells resulting in an increased upregulation of these
growth impairment was observed correlating to high expression of LTB, cell surface markers. Three batches of U937 cells were grown for
interestingly petunia growth was shown to be least affected by high 96 hours in media plus vehicle, media plus 1α,25(OH)2D3 and media
levels of LTB. The ability of hairy roots to secrete LTB into the supplied plus 1α,25(OH)2D3 and GM-CSF. Aliquots of cells were subjected to
media was also confirmed in each species; however improvement of our differential proteomics protocol. In short, the glyco moiety of the
secretion methods is required for effectively testing secretion patterns. outer membrane proteins were oxidised by NaIO4, the cells were lysed
The ability of hairy roots to regenerate into whole plants and retain their and the oxidised carbohydrate covalently bound to hydrazide coupled
high antigen production was also shown and the ability for cultures to beads. The covalent binding of the proteins facilitates extensive washing
be re-established form these plants has been also shown This research to remove membrane lipids and other debris. The bead bound proteins
demonstrates hairy roots as a viable system for vaccine production, and were reduced, alkylated and digested by trypsin. The residual tryptic
in particular roots derived from Petunia where high production coupled glycopeptides were released by PNGase F and analysed by LC-ESI-
with low growth inhibition, secretion potential and regeneration ability microTofQ-MS/MS as seen in the poster titled: “Analytical Prerequisites
allow for a very promising production system. for Differential and Quantitative Proteomics”. The known cell surface
proteinaceous marker CD11b was shown to be upregulated on the
1α,25(OH)2D3 as well as the 1α,25(OH)2D3 and GM-CSF treated cells,
while the marker CD14 upregulated on the 1α,25(OH)2D3 and GM-CSF
treated cells, only.

POS-MON-267 POS-TUE-268
NOVEL BIOACTIVE SECONDARY METABOLITES FROM OXIDATION OF TETRAHYDROPTERINES BY
AUSTRALIAN MEDICINAL PLANT ENDOPHYTES QUINONES
Fazendin Y.K.1, Lim K.F.2, Cahill D.1, Rookes J.1, Conlan X.1 and Garcia Molina F.1, Muñoz Muñoz J.L.1, Martinez Ortiz F.2, Varon R.3,
Barnett N.W.1 Tudela J.1, García Canovas F.1 and Rodriguez-Lopez J.N.1
1
Deakin University, Life and Environmental Sciences, Pigdons 1
GENZ: Grupo Investigación Enzimologia.Deparatamento de
Road, Waurn Ponds, Vic, Australia 3217. 2Deakin University, Life Bioquímica y Biologia Molecular A. Facultad de Veterinaria. Campus
and Environmental Sciences, 221 Burwood Highway, Burwood, Vic, de Espinardo. E-30100, Murcia (Spain). 2Grupo de Investigación
Australia 3125. de Electroquímica Teórica y Aplicada.Departamento de Química
Física. Facultad de Química.Universidad de murcia. A. Correos
Plant endophytes are producers of biologically active secondary E-30100. Murcia, Spain. 3Departamento de Química-Física. Escuela
metabolites that have provided new antibiotics, antiparasitics, de Ingenieros Industriales de Albacete. Universidad de Castilla la
antimalarial, and anticancer agents. There has been limited investigation Mancha. Avda. España s/n. Campus Universitario, E-02071, Albacete,
of endophytic microorganisms associated with Australian traditional Spain.
medicinal plants. A range of plant species, incorporating four diverse
families and seven genera, were collected from State and National Tetrahydrobiopterine (6BH4) can diminish the oxidative stress of
Parks around Victoria, Australia. Of the eight plant species selected, keratinocytes and melanocytes by reducing the o-quinones generated
endophytic fungi and bacteria were found in association with the by the oxidation of the corresponding o-diphenols. In this work, we
leaves and stems of Psilotum nudum, Ajuga australis, and Phebalium demonstrate that 6BH4 and its analogues methyltetrahydropterine (MBH4)
squameum. The fungal endophytes were isolated on potato dextrose and dimethyltetrahydropterine (DMBH4) can reduce all the o-quinones
agar (PDA), 10% V8 agar, and purified on malt agar. The ITS-5.8S-ITS2 studied, including 1,2 benzoquinone but not p-benzoquinone. The
rDNA region of each of the endophytic fungi was PCR amplified using formal potentials of different quinone/diphenol pairs obtained by square
universal primers, and gene sequenced. A BLAST search was conducted wave voltammograms indicate that the o-quinones with withdrawing
to identify the fungal isolates to genus. A preliminary co-culture screen groups are more potent oxidants than those with donating groups.
of the fungal endophytes against several fungal plant pathogens showed This work has been partially supported by grants from several Spanish
antifungal activity. A large-scale culture of an endophytic fungus in organizations. Ministerio de Educación y Ciencia (Madrid, Spain)
PDA broth (10 x 500 mL, 5 L) at room temperature (20–25 ° C) was Project BIO2009-12956, from the Fundación Séneca (CARM, Murcia,
undertaken, followed by extraction of mycelia and growth media with Spain) Projects 08856/PI/08 and 08595/PI/08, from the Consejería de
ethyl acetate. The extract is currently being screened for activity against Educación (CARM, Murcia, Spain) BIO-BMC 06/01-0004 and from the
four bacterial strains, including an antibiotic resistant strain of E. coli), Consejería de Salud y Bienestar Social de la Junta de Comunidades
and antioxidant activity using the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) de Castilla La Mancha, Project FISCAM PI-2007/53. JLMM has a
radical scavenging activity test and an acidic potassium permanganate fellowship from the Ministerio de Educación y Ciencia (Madrid, Spain)
(KMnO4) chemiluminescence method. Anticancer activity will also Reference AP2005-4721. FGM has a fellowship from Fundación Caja
be tested on several cancer cell lines. Bioassay-guided fractionation, Murcia (Murcia, Spain).
and a range of spectroscopic techniques will be used to isolate and
characterise the bioactive constituent(s).

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POS-MON-269 POS-TUE-270
CONSERVED BLOCK7 SEQUENCE OF BACILLUS DEVELOPMENT OF ANALYTICAL METHODS FOR
THURINGIENSIS CRY TOXINS. -THE INNOVATIONAL SEQUENCING OLIGONUCLEOTIDES USING MALDI-
PEPTIDE-TAG FOR EFFICIENT PROTEIN PRODUCTION- TOFMS
Hayakawa T., Shimizu Y., Ishida T. and Sakai H. Minohata T.1, Hewetson J.W.2 , Yamazaki Y.1 and Yamada M.1
Graduate School of Natural Science and Technology, Okayama 1
Applications Development Center, Analytical Applications
University, 3-1-1 Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan. Department, Shimadzu Corporation, Kyoto, Japan. 2Shimadzu
Scientific instruments (Oceania) Pty Ltd., Rydalmere, Australia.
We have developed a novel system to prepare proteins effectively
by using peptide-tag derived from the mosquitocidal Cry4Aa toxin of Studies of oligonucleotides containing both RNA and DNA are of great
a soil bacterium, Bacillus thuringiensis. Fusion with this peptide tag, interest in relation to the development of oligonucleotide therapeutics and
designated 4AaCter, facilitates the formation of crystal-like inclusion fundamental research. It has been recognized that various sequences
bodies of glutathione S-transferase in Escherichia coli without loosing of oligonulcleotides indicate antisense effect or RNA interference, which
the enzyme activity. In this study, we analyzed functional motif, in are responsible for effective and specific gene suppressions to regulate
particular Block7, which may work for formation of crystal-like inclusion expressions of unwanted proteins in living system. Although demand
bodies. Block7 is a stretch of amino acid sequence conserved among for a precise sequencing method of short length oligos (20-30 mer) is
Cry toxins. We selected fourteen Cry toxins and synthesized the genes increasing to study biological function, only a few analytical methods
encoding their Block7s. The Block7 peptides were expressed as GST have been reported, such as acid-hydrolysis and nuclease treatment
fusions and formation of crystal-like inclusion bodies in E. coli was in conjunction with mass spectrometry. On the other hand, MALDI-
analyzed. As a result, among the 14 tested Block7 peptides, those from TOFMS has been mainly applied to the detection of oligonucleotide
Cry5Ba, Cry32Aa, and Cry48Aa formed crystal-like inclusion bodies as molecular weight. We will report studies of two sequencing methods
well as that of Cry4Aa. But no inclusion observed for Cry47Aa Block7. using MALDI-TOFMS, including on-plate acid-hydrolysis and in-source
Formation efficiency of crystal-like inclusion body for the remaining 10 decay (ISD) on MALDI-TOFMS. We modified a previous report of acid-
of the tested Block7 peptides was ranging from 39 to 66%. Thus, the hydrolysis for DNA sequencing and investigated favorable conditions
conserved Block7 may be one of factors responsible for crystallization for the reaction of sequencing 19 mer RNA. Hydrolysis was conducted
of Cry toxins, but the role of Block7 may vary with type of Cry toxin. directly on the MALDI plate with a mixture of matrix, strong acid and
In addition, our results suggested the possibility to design a shorter RNA, and then analyzed by MALDI-TOFMS. Conditions were optimized
peptide-tag based on Block7 which form protein crystal efficiently. to generate many ladder signals, between which mass differences
Results of detailed mutational analyses will also be presented. represent constituent units of ribonucleotides, thus the sequence of
the RNA was clearly observed. Notably, it was possible to apply these
conditions to the analysis of 2-O-methylation of siRNA. Whilst ISD,
which is a degradation reaction inside the ion source of MALDI, has
been applied to protein sequencing, this method has been rarely applied
to RNA analysis. We will report successful results of the modified
siRNA by using ISD, and introduce newly developed software for the
interpretation of results.

POS-MON-271 POS-TUE-272
EFFICIENT PRODUCTION OF THE RECOMBINANT PROTEIN STRUCTURE DETERMINATION FROM
CYSTATIN C USING INNOVATIONAL PEPTIDE-TAG PSEUDOCONTACT SHIFT
DERIVED FROM BACILLUS THURINGIENSIS CRY
TOXIN Schmitz C.1, Vernon R.2, Otting G.3, Baker D.2 and Huber T.3
1
Bijvoet Center for Biomolecular Research, Utrecht University, The
Iwamoto S.1, Fan K.2, Sato S.1, 3, Hayakawa T.3, Sudo S.1 and Sakai H.3 Netherlands. 2Department of Biochemistry, University of Washington,
1
Japan Lamb CO.,LTD, Department of Bioscience, Development Seattle, USA. 3Research School of Chemistry, Australian National
Division, Okayamadai-incubator 108, 1-1-1 Tsushima-Naka, Okayama University, Australia.
700-0082, Japan. 2JSR Corp., 25 Miyukigaoka, Tsukuba-shi,
Ibaraki, 305-0841, Japan. 3Graduate School of Natural Science and The pseudocontact shift (PCS) effect, induced by a bound paramagnetic
Technology, Okayama University, 3-1-1 Tsushima-Naka, Kita-ku, lanthanide ion, is becoming widely used in protein nuclear magnetic
Okayama 700-8530, Japan. resonance (NMR) spectroscopy as it yields a complementary
combination of orientational and (long range) distance restraints. This
Cystatin C is a potent inhibitor of lysosomal proteinases and produced by versatile effect can be accurately determined with highly sensitive
all human cells with a nucleus. Since Cystatin C is a biomarker of kidney NMR experiments and has been successfully used (i) to automatically
function, the recombinant protein is highly demanded in the market of assign NMR resonances, (ii) to determine the structure of protein-
diagnosis agent. In this study, we present an innovational production protein complexes and protein-ligand complexes, and (iii) to refine NMR
system of the recombinant Cystatin C using a peptide-tag derived from structures. However, it has been speculated whether or not PCS data
Bacillus thuringiensis Cry toxin. Fusion with this peptide tag, designated as the only experimental restraints are sufficient for de novo structure
4AaCter, facilitates the formation of alkali soluble protein inclusion in determination of a protein. Here we show that 3D structures of proteins
Escherichia coli. The recombinant Cystatin C fused with 4AaCter at the can reliably be determined using PCS data from a single metal binding
N-terminal end was successfully expressed and accumulated as protein site combined with backbone chemical shifts. We present results
inclusion in E. coli. The yield of 4AaCter-Cystatin C was 40mg/L culture from a statistically meaningful number of proteins with different folds
and was five fold higher than that of Cystatin C without the tag. The ranging in size from 56 to 186 amino acids and show that PCS restraints
inclusion of 4AaCter-Cystatin C was solubilized in denaturing buffer and implemented in the fragment assembly step of the ROSETTA software
purified by tag-based affinity chromatography. The purity of the final is highly efficient in biasing the sampling of the conformational space
product was more than 90% and the final yield was 8mg/L culture. towards the correct target structure. We further show that the best
Western blotting revealed that the recombinant protein reacted with structures computed have a backbone RMSD from the native structure
anti-Cystatin C antibody as well as that of native Cystatin C. In addition, as low as 1.0 Ångstrom. Finally, the question whether we are performing
latex agglutination assay showed that the reactivity of the recombinant structure prediction assisted by experimental data, or truly de novo
Cystatin C by our method was higher than that of the native one. The structure determination is discussed.
novel system we have developed using 4AaCter is very simple and
enables efficient production of the recombinant Cystatin C with superior
quality in low cost.

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POS-MON-273 POS-TUE-274
INNOVATIVE STRATEGY FOR EFFICIENT PROTEIN FUNCTIONAL MICROPATTERNS TO CONTROL CELL
PRODUCTION USING A NOVEL PEPTIDE TAG DERIVED BEHAVIORS
FROM CRY TOXIN
Ho C., Kumar G. and Co C.
Sakai H.1, Sato S.1, 2, Iwamoto S.2, Sudo S.2, Sakamoto Y.1, Uchida M.1, University of Cincinnati.
Matsushima K.1, Kashino Y.1 and Hayakawa T.1
1
Graduate School of Natural Science and Technology, Okayama Microfabrication techniques are widely used in the electronic industry
University, 3-1-1 Tsushima-Naka, Okayama 700-8530, Japan. 2Japan to generate small features with size between 1-100 μm. This size
Lamb CO., LTD, Department of Bioscience, Development Division, range is on the same order of a single cell, thus, these microsystems
Okayamadai-incubator 108, 1-1-1 Tsushima-Naka, Okayama 700- are well suited for studying cell behaviors. This talk will demonstrate
0082, Japan. some examples of using micropatterned surfaces to control local
environment around the cell. Micropatterned surfaces were used to
We are often confronted with difficulties that some proteins cannot be control the cell size and shape. Endothelial cells patterned within the
efficiently produced by ordinary recombinant DNA techniques based on 20 µm grooves formed capillary tube-like structure containing a central
cloned genes. The difficulties may be because the recombinant protein lumen. Capillary networks embedded in other tissue specific cell types
products are unstable or have deleterious effects on the host cell. To can be formed on the biomaterials for creating vascularized tissue.
overcome the difficulties in producing these difficult-to-produce proteins, Patterned biomaterials can be applied to guide neurons to extend axon
some innovative recombinant DNA techniques for heterologous protein and neurite for creating neuronal networks. We have also devised a
production are eagerly desired. We developed an innovative procedure completely novel microarray-based technique to amplify the natural
to produce proteins efficiently by using a novel peptide tag derived from directional persistence of migrating cells (MANDIP). Using MANDIP,
the insecticidal Cry toxin of a soil bacterium, Bacillus thuringiensis. Fusion we can amplify this directional persistence to coerce the migration of
with this peptide tag, designated 4AaCter, facilitates the formation of cells indefinitely along arbitrary paths in one preset direction without
crystal-like inclusion bodies of glutathione S-transferase in Escherichia chemoattractants, gradients in substrate adhesiveness, or external fields.
coli without loosing the enzyme activity. For all the target proteins tested Potential applications of MANDIP include cell-sorting, drug screening,
to date, it was proved that the recombinant proteins were accumulated tissue engineering, wound healing, and mechanistic studies of cell
in the crystal-like inclusion bodies and their biological activities were migration, cell-cell interactions, and other cellular processes requiring
also maintained. Application of 4AaCter to the production of syphilis temporal and spatial regulation. We have devised and are seeking to
antigens, TpN15, TpN17 and TpN47 from Treponema pallidum, yielded commercialize a simple in vitro migration assay that offers significant
excellent results, including a dramatic increase in the production level, improvements in reliability and ease of implementation compared to
simplification of the product purification and high reactivity with syphilis traditional “wound healing” assays. Instead of mechanically wounding
antibody. The use of 4AaCter may provide an innovational strategy for cells, which leads to interferences caused by dead cell debris that block
the efficient production of difficult-to-produce proteins. cell movement and molecules released by wounded cells that alter
artificially the rate of cell migration, confluent groups of cells, initially
confined within patterns of cell-resistant polyelectrolyte, are released
by electrostatic adsorption of a second, cell adhesive polyelectrolyte.

POS-MON-275 POS-TUE-276
CHLAMYDOMONAS REINHARDTII. - AN ALGAL FLUOROMETRIC APPROACH TO SELECTIVITY
FEEDSTOCK FOR LIQUID BIOFUELS SCREENING OF MODIFIED OLIGONUCLEOTIDE
HYBRIDIZATION PROBES
James G., Hocart C., Hillier W. and Djordjevic M.
Plant Science Division, Research School of Biology, The Australian Kabilov M.R. and Pyshnyi D.V.
National University, Canberra, Australia. Institute of Chemical Biology and Fundamental Medicine SB RAS.

C. reinhardtii is a model algal system of intense research focus as Today various approaches to the detection of point mutations in DNA
a feedstock for the production of biodiesel and the processing of are used. A number of them are based on principles of allele-specific
hydrocarbons to jet fuel (Wang et al., Eukaryot. Cell, Dec 2009; 8: 1856- hybridization (ASH). To improve the efficiency in discrimination of
1868, Moellering et al., Eukaryot. Cell, Jan 2010; 9: 97-106, Yanto et al., imperfect duplexes, there is a reason to look for oligonucleotide
Metabolic Engineering, doi:10.1016/j.ymben.2010.02.002,. C. reinhardtii analogues and derivatives with higher hybridization efficiency as
usually stores excess carbon as starch when nutrient deprived with compared to their native precursors. In most studies, however, there
only limited production of neutral lipids (triacylglycerols). Mutations are no detail characteristics of the influence of modified probes on the
in the starch biosynthetic pathway cause carbon to be redirected into selectivity of the analysis because this problem requires the in-depth
lipid biosynthesis and storage triacylglycerols that are suitable for liquid thermodynamic analysis. Properly speaking, the efficiency of mismatch
biofuels. The fatty acid profiles of wild-type and starch mutants were discrimination should be characterized by comparable thermodynamic
quantified by gas chromatography mass spectrometry. We found C. parameters (enthalpy, entropy, and free energy) for the formation of
reinhardtii starch mutants produce significantly elevated levels of 16:0, perfect and mismatched complexes. Just these values determine the
18:1 and 18:3 fatty acids that are suited for producing biofuels. Fluorescent formation efficiency (the extent of association) of duplexes composed
spectroscopy and fluorescent activated cell sorting was used to develop by an oligonucleotide probe under given conditions. The use of the
high-throughput methods for screening neutral lipid accumulation in standard real-time PCR detection system allows one to detect the
cells. Global transcriptional profiling by RNA-sequencing is underway change of the fluorescence level when heating samples. The advantage
on the Illumina platform to identify a putative gene set associated with of this system is the opportunity of the parallel analysis of a large number
the lipid biosynthetic pathway and to understand the molecular basis for of probes at rather low concentrations. We have considered various
how cells switch to lipid synthesis. We conclude that C. reinhardtii is an options of the fluorescent labeling of both probes and DNA templates,
alga that can be manipulated to produce high levels of triacylglycerols. which should ensure the optimal level of the signal. We attempted to
design a system, which could answer the question “is the modified
probe more selective than native one?” by the analysis of melting
curves. The parallel examination of the same oligonucleotide probes
using the UV melting technique confirmed our results. The results
demonstrate a possibility of the massive parallel analysis of selectivity
of modified oligonucleotide probes based on the thermal denaturation
of fluorescent DNA complexes.

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ROLE OF CARBOHYDRATE BINDING DOMAIN IMMUNOLOGICAL MEASUREMENT OF TROPONIN I IN
ON EXPRESSION, ACTIVITY AND STABILITY OF SERA FROM THE PATIENTS WITH HEART DAMAGE
XYLANASE C OF CLOSTRIDIUM THERMOCELLUM DISEASES
Khan M.I.M., Sajjad M. and Akhtar M.W. Kim H.J.1, Kim T.K.1, Wang H.1, Oh S.W.2 and Choi E.Y.1
School of Biological Sciences, University of the Punjab, Lahore-54590, 1
Department of Biomedical Science, Hallym University, Chuncheon,
Pakistan. South Korea 200-702. 2Department of Biology Education, Institute of
Fusion Science and Science Education, Chonbuk National University,
Xylanase (XynC), a major component of Clostridium thermocellum Jeonju, South Korea, 561-756.
cellulosome, was cloned and expressed with and without carbohydrate
binding domain. Four constructs with the gene encoding only the A cardiac-specific iso-form of Troponin I (cTnI) has been known as
catalytic domain (xynC CD), the catalytic domain with binding domain at a marker of heart damage for more than 20 years. At present cTnI is
N-terminus (xynC CBD-CD), the catalytic domain with binding domain at considered to be one of the most specific and sensitive markers of
C-terminus (xynC CD-CBD) and the catalytic domain with binding domains myocardial cell death. For the preparation of antigen, we produced a
at both termini of the catalytic domain were produced (xynC CBD-CD-CBD). human cardiac troponin I recombinant protein. The cardiac toponin
The XynCCD, XynCCBD-CD, XynCCD-CBD and XynCCBD-CD-CBD expressed at I clones were transformed into BL21 cell for expression and the
levels ~45%, ~30%, ~30% and ~33% of the total E. coli cell proteins after recombinant cTn I protein was purified by affinity chromatography.
induction with lactose, as analyzed by photodensitrometric scanning of The cTnI protein was confirmed to be an expected size on an SDS-
SDS-PAGE gels. The overall activities produced by XynCCD, XynCCBD-CD, PAGE gel. To generate monoclonal antibodies against the protein,
XynCCD-CBD and XynCCBD-CD-CBD were 1,488, 3,489, 3,860 and 5,732 U l-1 recombinant cTnI was injected into BALB/C mice. From the fusion
OD600 -1 against oat spelt xylan, respectively. All these enzymes with and experiments, several hybridomas were selected by Western blotting and
without non-catalytic domains were found to be quite stable over a broad further screened by ELISA method. With the monoclonal antibodies, we
pH range (pH 4 - 9). XynCCBD-CD was more thermostable as it retained developed a POCT type (point of care of test) immunoassay system
70% activity on incubation at 70ºC for 2 hrs whereas XynCCD lost total and conducted performance evaluation for measuring cTnI in human
activity under these conditions. XynCCD-CBD retained 56% and XynCCBD- serum. The linearity fell in the range 0-10ng/ml of cTnI and the analytical
CD-CBD
retained 80% of its activity under these conditions. Km values detection limit was 0.5ng/ml of cTnI. The precision of intra- and inter-
for XynCCD, XynCCBD-CD, XynCCD-CBD, and XynCCBD-CD-CBD as determined assay was CVs<6% and CVs<8%. Also, we found that the amount of
on soluble xylan as substrate were 3.12, 3.57, 3.12 and 1.64 mg ml-1, cTnI protein mass in the Heart damage patients was much higher level
respectively. Thus presence of carbohydrate binding domain on both than that in normal population. The performance test result indicated
terminals leads to the 4 fold increase in specific activity as compared to that newly developed immunoassay system using fluorescence bead
the native enzyme. and lateral-flow chromatography is a simple, fast method for quantifying
the cTnI concentration in human blood. Currently we development of
an alternative method enables to measure the low amount of cTnI more
accurately.

POS-MON-279 POS-TUE-280
CHARACTERISATION OF THE GRAIN SPECIFIC TELOMERASE ACTIVITY AND ITS USE IN MONITORING
HOMEODOMAIN TRANSCRIPTION FACTORS FROM BLADDER CANCER
WHEAT
Lalla M.1, Dezfouli S.1, Mendez R.1, Tian P.2, Elwood N.2 and Macreadie I.1
Kovalchuk N., Wu W., Bazanova N., Singh R., Ismagul A., Eliby S.,
1
Sienna Cancer Diagnostics Ltd., Bio21 Institute, VIC 3010, Australia.
Johnson A., Hrmova M., Langridge P. and Lopato S.
2
Cord Blood Stem Cell Research Institute, Murdoch Children’s
Australian Centre for Plant Functional Genomics, Hartley Grove, Research Institute, VIC 3052, Australia.
Urrbrae, South Australia 5064, Australia.
The shortening of chromosomes is a normal part of the aging process,
Two HDZip class IV genes, designated Triticum aestivum GLABRA like however, cancer cells are immortal and overcome lethal chromosome
7 and 9 (TaGL7 and TaGL9), were isolated using Y1H to screen a cDNA shortening. Most cancer cells maintain chromosomes through the activity
library from developing wheat grains (0-6 DAP) with the palindromic of telomerase, a DNA polymerase that extends the ends of chromosomes
repeat -CATTAAATG- as bait. A predicted molecular model of TaGL9 through the addition of (TTAGGG) repeats. The activity of telomerase
suggested that at least five independently folded domains could be is therefore a key biomarker in monitoring cancer. Assays to monitor
present, although their spatial distribution is hypothetical at this stage. telomerase activity include the in vitro extension of an oligonucleotide
The identified protein domains are currently being used as baits in the substrate with (TTAGGG) repeats. Typically such assays involve the
Y2H screen of a wheat endosperm library. The 3’UTR of TaGL7 and generation of a few attomoles of telomeric extension products (TEPs)
TaGL9 were used as probes to isolate genomic clones of orthologues/ which are amplified by the polymerase chain reaction (PCR) and then
homologues from a Triticum durum BAC library. Full length genes analysed on gels. Such PCR reactions are highly unusual since they
containing 3 kb-long promoter regions were designated TdGL7 and generate products even without TEPs. A characteristic to be looked
TdGL9. Spatial and temporal expression patterns of TdGL7 and TdGL9 for in the telomeric repeat amplification protocol (TRAP) is a telomeric
were examined in transgenic wheat, barley and rice expressing promoter- ladder on gels. Further refinements include measurements using real
GUS fusion constructs. Whole-mount and histochemical GUS staining time PCR with amplifluors, however, once again products are generated
patterns were analyzed in large numbers of independent transgenic without TEPs. Because of the high signal in telomerase negative
lines. Although very low activity of the TdGL7 promoter was detected controls, it is considered that PCR-based assays have limitations that
in most plant tissues, much stronger GUS expression was observed in may prohibit their utility for clinical assays. Work at Sienna is focused
the female gametophyte before fertilization, and later in the syncytial on the development of non-PCR procedures to measure telomerase
and starchy endosperm. The TdGL9 promoter was active only in grain. activity in bladder cancer using exfoliated cells in urine so as to avoid
GUS activity was initially observed on the 5th day after pollination and these effects. Significant challenges are the low cell numbers in urine,
persisted in the embryo until grain harvest. Small differences were the harsh environment and non-epithelial cell types. Our aim is to assay
observed in the spatial patterns of GUS expression in wheat, barley and for telomerase activity in around 100 cells, each of which may have only
rice. Wheat transgenic plants with constitutive overexpression of TaGL7 20 molecules of telomerase.
and also with antisense RNA expressed under the TdGL7 promoter
were generated and are currently being analysed.

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ANALYTICAL PREREQUISITES FOR DIFFERENTIAL RAPID RESPONSE OF THE HUMAN NUCLEOLAR
AND QUANTITATIVE PROTEOMICS PROTEOME TO SERUM STIMULATION
Levina V.1, 2 , Cooke I.1, 2, Durand F.M.1, 2, Boyd S.E.2, 3, O’Connor L.2, 3, Li Z.F.1, Liang Y.M.1, Wang X.2 and So L.K.Y.3 , Lam Y.W.1
Hoogenraad N.2 and Talbo G.H.1, 2
1
CRC for Biomarker Translation, La Trobe University, Melbourne, VIC,
1
Dept. of Biology and Chemistry, City University of Hong Kong.
Australia. 2La Trobe Institute for Molecular Science, La Trobe University,
2
College of Medicine, Zhejiang University, Hangzhou, China. 3Dept. of
Melbourne, VIC, Australia. 3AgriBio, the Centre for AgriBioscience, Biochemistry, Hong Kong University.
Melbourne, VIC, Australia. The nucleolus is the most prominent organelle in the cell nucleus. It is
The molecular composition of the cell surface plasma membrane and its responsible for synthesizing and assembling 60S and 40S ribosome
dynamic changes determine how a cell interacts with its environment. As cell subunits, and plays a crucial role in the regulation of cell proliferation
surface proteins confer specific cellular functions, and are accessible, these and growth. The responses of the nucleolus upon external stimulations
are the proteins of greatest interest to modern diagnostic methodology and is therefore of high interest. The high density and structural stability of
specifically as biomarkers. Many membrane surface proteins are glycosylated the nucleolus makes it possible to purify this nuclear organelle, and to
and the chemically distinct properties of carbohydrates from those of proteins, analyse the dynamics of the nucleolar proteomes. However, the existing
have led to the development of bead based protein isolation approaches, nucleolar isolation method, developed in 1963, involved relatively tedious
which target cell surface glycoproteins. Plasma membrane proteins isolated procedures that tend to obscure momentous biochemical changes
by this approach are identified by tandem mass spectrometry. Despite some in this organelle. Here we developed a new and simplified nucleolar
success most cell surface proteins remain undetected due to the quality of the isolation method in which cells were rapidly harvested and lysed,
methodologies currently available for the isolation of cell surface glycoproteins. thus the nucleolar proteome could be “frozen” at precisely controlled
This is highlighted by the relatively small number, approximately 340, of cluster time points. The purity of nucleoli obtained using this protocol was
determinant (CD) cell surface protein markers1 that have been identified to date,
compared with the number of predicted human transmembrane proteins being comparable to those using the classical method, as judged by electron
greater than 13,0002. Potentially, even more important than the identification microscopy and Western blotting. The complete proteomes of nucleoli
of the plasma membrane glycoproteins is determination of the relative prepared by these two methods were also compared using SILAC-
abundance of those proteins in diseased compared with normal states of the based quantitative proteomics.To further demonstrate the applications
cell. Therefore, we set out to develop a protocol encompassing a quantitative of this new method, we performed a time-lapse nucleolar proteomics
workflow from “Cell to Protein Identification and Relative Protein Amount.” after serum stimulation. HeLa cells were serum starved for 24 hours and
In short, the glyco moiety of the outer membrane proteins was oxidised by then serum re-stimulated, and the nucleolar proteome dynamics was
NaIO4, the cells were lysed and the oxidised carbohydrate covalently bound followed in the first 10 minutes of serum replenishment. This approach
to hydrazide coupled beads. The covalent binding of the proteins of interest reveals for the first time that some nucleolar proteins responded to
facilitates the extensive washing required to remove membrane and other serum stimulation within as short as the first 5 min. Subsets of small
debris. The bead bound proteins were reduced, alkylated and digested by and large ribosomal proteins, histones and heterogeneous nuclear
trypsin followed by washing. The residual tryptic glycopeptide was released ribonucleoproteins family increased 20% to even 100% within 10 mins.
by PNGase F and analysed by LC-ESI-microTofQ-MS/MS and MALDI-tof/ Interestingly, the eukaryotic translation initiation factor 6 (EIF6) and the
tof-MS. The results were reproducible; peak intensities greater than 2000 proliferation marker Ki-67 were found to be quickly accumulated in the
showed less than 12 % CV across 30 replicate samples. 1) Nicholson, IC, nucleolus within 10 mins. To the best of our knowledge, this is the first
Mavrangelos, C, Fung, K, Ayhan, M, Levichkin, I, Johnston, A, Zloa, H and study demonstrating these “quick triggering proteins” in the nucleolus.
Hoogenraad, NJ. J.Immunol. Methods, 305, 84-93, 2005 2) Zola and Swart, The underlying mechanisms and the roles of these very early events in
2003.Trends Immunol.24,353, 2003. cell growth will be discussed.

POS-MON-283 POS-TUE-284
DISCOVERY OF NOVEL PHAGE PROTEINS FOR SIGNIFICANT IMPROVEMENT OF THE THERMO-
BIOTECHNOLOGY BY HIGH THROUGHPUT STABILITY AND OPTIMUM PH RANGE OF CATALYTIC
SEQUENCING OF PHAGE DNA FROM SOUTH AFRICAN ACTIVITY OF A LACTONE-SPECIFIC ESTERASE BY
DEEP GOLD MINES RANDOM MUTAGENESI
Mabizela N.B. and Litthauer D. Ishizuka M.1, Tsukimura W.1, Namiki K.1, Akanuma G.1 and Ushio K.2
Metagenomics Platform, Department of Microbial Biochemical and 1
Department of Applied Chemistry, Chuo University, Tokyo, Japan.
Food Biotechnology, University of the Free State. Nelson Mandela 2
Department of Applied Chemistry and Biotechnology, Niihama
Drive, Bloemfontein, South Africa. National College of Technology, Niihama, Japan.

Shotgun sequencing of metagenome libraries from South African mines Stable and high stereo-selective lipase (EC 3.1.1.3) and esterase (EC
revealed untapped phage communities in the deep subsurface. The 3.1.1.1) have attracted much attention from viewpoints of industrial
majority of the clones from four mines shared no similarity to known usage. Pseudomonas-like bacterium isolated in our laboratory from
proteins. Pyrosequencing was used to assess the metagenomic wastewater sample produces a thermo stable lipase by the addition of
diversity of phage DNA from Beatrix Mine in South Africa. Annotated fatty alcohols (stearyl alcohol and palmityl alcohol) as the most effective
data showed that approximately 75% of the proteins had no homology to super-inducers. The addition of fatty alcohols brought about more than
any known proteins in public databases. About 40% of the proteins that several hundred-fold enhancement of the lipase activity compared to
were assigned to a specific function were of viral origin. Most of the hits the case with no additive. This means several dozen-fold enhancement
were from the Enterobacteria phages and Acanthamoeba polyphaga of lipase activity compared with olive oil grown case. Gram per litter
mimivirus. Seven prophage regions of bigger than 4 kb were identified lipase production has been capable. On the other hand, carboxylic
using Prophage Finder. Novel viral proteins with biotechnological ester esterase is not induced by the addition of fatty alcohols. In this
functions were identified. In this study a phage DNA ligase protein was study, we report a lactone-specific esterase expression in Escherichia
identified among the hundreds of genes obtained. Sequence analysis coli and significant improvement of the thermo stability and wide
indicated that the protein is novel showing 46% similarity to its closest optimum pH range of catalytic activity of the mesophilic esterase by
relative. In addition the second, most conserved motif, SLRFPRFIRIR random mutagenesis and evolutionary engineering. Thermal stability
was not detected; only that containing the catalytic lysine. The gene of a purified mutant esterase (R29C, Q82L, R96H, N212D, R286H)
encoding this protein was cloned and expressed in E. coli. The protein is obtained by the error-pron PCR (EP-PCR), DNA shuffling, and thermal
41 kDa in size, is ATP-dependent and ligated cohesive and blunt ended selection (three times) rises 12 °C in temperature (10 fold long in half-life
λ-DNA fragments, the former being ligated more efficiently. Maximal period) compared with the stability of the purified wild-type esterase. As
ligation of the blunt ends was achieved with added polyethelene glycol a result of site-specific mutagenesis, Contribution of R96H and N212D
(10%). The enzyme is active at 4 ºC, 16 ºC and 22 ºC. BamHI cut pUC19 for thermal stability are 5 °C and 4 °C, respectively. Additionally, wide
plasmid could be ligated at 30, 40, 50, 60 and 70 ºC. optimum pH range (7-9) of the mutant catalytic activity and stability were
obtained without the loss of the lactone-specificity (γ-Butyrolacton,
δ-Valerolactone, and ε-Caprolactone).

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EFFECTIVENESS OF PEER DISCUSSION FOR ACQUISITION OF SCIENTIFIC WRITING SKILLS
STUDENT UNDERSTANDING OF ACID-BASE THROUGH ACTIVITIES EMBEDDED INTO BIOLOGICAL
EQUILIBRIA LABORATORY COURSES
Watters D.J. and Love C.A. Tonissen K.F.1, Lee S.E.1, Woods K.J.1 and Osborne S.A.1, 2
School of Biomolecular and Biophysical Sciences, Griffith University, 1
School of Biomolecular and Physical Sciences, Griffith University,
Nathan Campus, Brisbane, Australia 4111. Nathan, Qld, Australia 4111. 2CSIRO Livestock Industries, St Lucia,
Qld, Australia, 4067.
Students frequently have difficulty in understanding the central concepts
of acid-base chemistry and buffers. The term, troublesome knowledge, Scientific writing skills are important for a science career, yet specific
as defined by Meyer and Land, accurately describes this problem since training can be difficult to integrate effectively into a University program.
students are able to perform superficial tasks such as titrations, and Rather than design specific courses solely focused on writing, we
apply the Henderson Hasselbalch equation without understanding embedded writing activities into two project style third year biological
the basic concepts (ritual knowledge). They also fail to transfer their science laboratory courses where students wrote about their own data.
understanding of simple acids and bases to the structure and function Students were expected to complete the writing exercises during breaks
of proteins or other complex biological molecules. Thus pH could fit into in their experimental procedures and were given feedback during the
the category of a threshold concept which is core to the understanding laboratory session. These activities were focussed on preparing figures,
of many aspects of chemistry, cell biology, physiology and biochemistry. tables, figure legends, and writing results and discussion paragraphs.
We investigated the use of concept questions in class with the use of These exercises provided practice and a model to assist students in
clickers to identify student misconceptions, as well as peer discussion, writing the remainder of the report. We probed student opinions regarding
in an effort to improve student learning of these topics. Peer discussion scientific writing and the use of the exercises by anonymous pre- and
has previously been shown to have a positive effect on student learning. post-course surveys using a combination of closed and open questions.
Our results show that, in the case of threshold concepts such as acid- In the first course student confidence towards scientific writing and
base equilibria, peer discussion is not effective and merely propagates performing simple writing tasks significantly improved after experiencing
misconceptions. These findings suggest that substantial groundwork the writing activities. Therefore students commenced the second
needs to be done to enable students to gain the necessary understanding course with a higher confidence level. They related that undertaking
of the fundamental concepts of acid-base chemistry. writing activities in more than one class helped them consolidate their
writing skills and challenged them to improve further. Students also
commented that they thought the activities helped them develop skills
relevant to future scientific careers. Independent assessors evaluated
the standard of students’ written reports that originated from the same
course held in years before and after writing activities were incorporated
into the curriculum. There was a significant improvement in scientific
writing quality that correlated with the increase in student confidence
and attitudes towards writing.

POS-MON-287 POS-TUE-288
HOW RESEARCH INFORMS AND ENHANCES CONSTRUCTING A GENERALIST BIOCHEMISTRY
LEARNING USING STUDENT PROJECT CASES IN THE COURSE AROUND CORE CONCEPTS: BUILDING
MONASH MBBS PROGRAMME THE BIG PICTURE FOR A LARGE MIXED-LEARNER
COHORT
Samarawickrema N.A. and Macaulay J.O.
Department of Biochemistry and Molecular Biology, Monash Rowland S.L., Gillam E., Hamilton S., Harrinson J., Wright T. and
University, Clayton, VIC 3800. Ward L.
University of Queensland.
There is a strong synergistic connection between teaching and research
because (1) research fertilises teaching with new topics and methods The University of Queensland has recently revised the BSc, and
(2) research provides teachers with personal engagement and (3) consequently we now offer a single, second-year, undergraduate
academic staff research guarantees connections with developments in biochemistry course. This course, BIOC2000, must service over 500
the research arena. In turn, students perceive their courses to be up to students from almost 20 different programs of study. The students
date and intellectually stimulating. Here we report the results of a study have many different backgrounds, goals, and abilities. This is a radical
of undergraduate student engagement in a research tutored curriculum departure from our smaller, more “science-centric” cohorts of previous
based on the current research of the tutor. We used Student Project years. In its first year of delivery, BIOC2000 uncovered serious problems
Cases (SPC) offered in second year of the Monash University medical in understanding of basic chemistry concepts for many of the cohort as
curriculum, which comprises a teamwork activity that emphasises evidenced by the answers to a chemical concept inventory test that we
interdisciplinary learning, where the students research and present a delivered to the students, and also by the final exam results. We decided
medical disease. The co-topics required for this SPC were specified and to rebuild the course around core chemical and biochemical concepts,
the students were required to research into these and prepare a written rather than around content, and use a “Backwards design” approach to
and an oral presentation as part of the SPC assessment process. At the the new curriculum. Developing student understanding of fundamental
end of the SPC, students completed a questionnaire which examined chemistry concepts and their application to the molecules and structures
their exposure to research and research culture, their experiences on of life is the primary learning objective of the new curriculum. We surveyed
the preparation of this SPC and participated in a Focus group. The over 80 working scientists to determine the “fundamental concepts” of
students stated that the knowledge that the SPC topic being a current biochemistry. We will present our data regarding (i) what these concepts
research interest of an academic staff member involved with the SPC are, (ii) how we determined if the students understood them before the
process had a positive impact on their attitude towards the topic. They course started, (iii) how we integrated these concepts into learning and
stated that being aware that the research was happening now made it assessment activities, and (iv) how student understanding of these
exciting to study about the topic and therefore a motivational factor. concepts changed during the course. This project provides a roadmap
for generalist course design around the fundamental guiding principles
of a discipline, rather than around content. The presentation should be
useful to all course designers.

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IMI HO’OLA POST-BACCALAUREATE PROGRAM AT STUDENT ENGAGEMENT IN 1ST YEAR BIOSCIENCES
THE UNIVERSITY OF HAWAII SCHOOL OF MEDICINE
Nott M.W. and Poronnik P.
Ha C., Lee W.M. and Judd N. RMIT University, School of Medical Sciences, Melbourne.
University of Hawaii School of Medicine, 651 Ilalo Street, MEB306,
Honolulu, HI 96813, USA. Students typically fail to fully realise the relevance of core preliminary
subjects, and in particular the generic skills they need to develop in
Imi Ho’ola (Hawaiian meaning “Those who seek to heal”) is a post 1st year bioscience studies: chemistry, mathematics and statistics,
baccalaureate program established in 1972 to prepare students from and even biology. In Pharmaceutical Sciences at RMIT University we
socially, educationally, or financially disadvantaged backgrounds for have devised a series of interventions that require 1st year students to
the entry into the rigors of medical school education. Each year 10 draw on and integrate their learning in each core subject. For the first
students are selected to participate in the one-year program and upon intervention, staff from each discipline agreed on the common theme:
matriculation students are guaranteed acceptance into the University of Malaria and its Drug Treatment. The theme fulfilled criteria aimed
Hawaii School of Medicine as regular first year medical students. The to enhance student engagement and their awareness of relevance of
curriculum of the Imi Ho’ola program spans over 12 months and consists each subject. Thus the theme is contemporary, given that many of
of three phases: 1. Summer orientation and assessment phase to obtain our students or their families come from malaria-endemic countries;
base-line data on students’ knowledge in the sciences, reading, and media-rich, given that malaria (and dengue) is increasingly in the
learning skills, Phase 2. Enrichment phase to improve students’ critical news because of global warming and the need for new strategies for its
thinking skills in the content areas of biology, medical biochemistry, treatment (eg, artemisinins) and eradication (eg, genetically engineered
scientific basis of medicine, and speech and ethics in healthcare, flightless mosquitoes); and research-relevant, given that Melbourne is
Phase 3. Prematriculation phase to help students’ smooth transition into a centre for immune studies approaches. The theme allowed seamless
regular medical school curriculum with introduction of clinical skills by integration of learning materials in chemistry (which stressed molecular
shadowing physicians. For the past 30 years, Imi Ho’ola program has structures of antimalarials and their binding to protein targets); maths
provided educational opportunities to disadvantaged minority students and statistics (which stressed epidemiology and vector population
pursuing a career in medicine. Out of 204 graduates of the program studies) and biology (which stressed drug-susceptible stages of the life
and medical school, 96% provide health services to disadvantaged or cycle of plasmodia). Students worked in teams to create and defend
underserved populations in the state of Hawaii and the Pacific islands posters, thus honing their visual and oral presentation skills as they
and 72% provide primary care services such as internal medicine, family worked on 9 topics around the central theme. In doing so, students were
medicine, and pediatrics. Imi Ho’ola program is a successful educational able to integrate their first year studies as well as get an early start
model that contributes to the development of a diverse healthcare on developing professional capabilities according to RMIT University’s
workforce that provides services for the underserved communities of Graduate Attributes: 1) work ready; 2) environmentally aware and
Hawaii and the Pacific region. responsive; 3) global in outlook and competence; 4) culturally and
socially aware; 5) life-long learners; 6) active learners; 7) innovative.

POS-MON-291 POS-TUE-292
GENE EXPRESSION PROFILING IN CIRCULATING CHARACTERISATION OF PUTATIVE PLASMODESMATA
CELLS OF BREAST CARCINOMA PATIENTS PROTEINS OF THE ARABIDOPSIS CALNEXIN FAMILY
Zima T1, Mikulova V1, Tesarova P1, Kolostova K 2, Kubecova M2, Liu D.Y.T.1, Smith P.M.C.1, Day D.A.1, 2 and Overall R.L.1
Rusnakova V3 and Kubista M3 1
School of Biological Sciences, The University of Sydney, Sydney
1
General University Hospital and First Faculty of Medicine, Charles NSW 2006 Australia. 2Present address: Flinders University, Adelaide
University in Prague. 2 General University Hospital Královské SA 5042 Australia.
Vinohrady and Third Faculty of Medicine, Charles University in Prague.
3
Institute of Biotechnology, Academy of Sciences, Czech Republic. Plasmodesmata are plasma membrane-lined channels spanning the
cell wall, connecting the cytoplasm and endoplasmic reticulum (ER)
Background: Tumor cell dissemination is an early process in breast of adjacent cells. Plant survival depends upon the proper function and
cancer (BC) and circulating tumor cells (CTCs) are considered potential regulation of plasmodesmata, since developmental signals, nutrients,
surrogate marker for the detection and characterization of minimal and even viruses move through these channels. However, only a few
residual disease. Here we monitored hematogenous micrometastasis protein constituents of plasmodesmata have been discovered. In this
in BC patients by gene expression profiling of CTCs based on CTC- study, we extend a comparative proteomics analysis of Chara (Faulkner
abundance in blood and expression of 35 oncomarkers at the mRNA et al., Proteomics 5: 2866) by identifying and characterising Arabidopsis
level by multimarker quantitative PCR. Methods: A total of 87 patients proteins with sequence similarity to characean peptides isolated from
with diagnosed BC at stage I to III and 115 metastatic patients were plasmodesmata-rich cell fractions. These proteins were screened for
enrolled into a prospective study. Immunomagnetic enrichment of plasmodesmatal localisation in Arabidopsis and Nicotiana benthamiana
CTCs from the 5ml of whole blood followed by cells characterization using green fluorescent protein (GFP) tagging. Calnexin, one of the
using gene expression analysis for the presence of tumor associated proteins identified, is a type I membrane protein localised in the ER with
genes HER2, MUC-1 and GA 733-2 were processed using AdnaTest the main catalytic domain lying within the ER lumen where it may function
BreastCancer® kit. If possible bone marrow samples were obtained for as a chaperone. The Arabidopsis calnexin protein family consists of two
disseminated tumor cells detection using Epimet® Kit. RNA from FFPE members, AtCNX1 and AtCNX2, with 83% amino acid identity. Both
tumor tissue (n=85) has been isolated. All obtained cDNA molecules proteins colocalise with aniline blue-induced fluorescence of callose
have been gene-specifically pre-amplified for multimarker qPCR analysis at plasmodesmata, although colocalisation is reduced upon deletion
measured on Biomark® microfluidic chip. Results: 286 CTC samples of the signal peptide. No significant morphological differences were
have been analyzed in total. The analysis has shown that the gene observed in single or double homozygous Arabidopsis T-DNA knock-
expression profiles of CTCs in primary breast cancer patients correlated out mutants of AtCNX1 and AtCNX2. However, cell-to-cell diffusion of
to those measured on the primary tumor, while CTCs of metastatic BC GFP as well as deposition of callose at plasmodesmata were affected
patients had significantly different gene expression profiles. Analyzing in mutants, suggesting some role for calnexin in plasmodesmatal
the gene expression data from CTC-positive patients in comparison to physiology. Quantitative real-time PCR data suggested that in knock-out
CTC negative patients and FFPE samples we have revealed several mutants other chaperones may provide redundancy for CNX, including
genes that were differentially expressed (p<0,05) (e.g. CK19, GA7332, calreticulin, an ER-lumenal homolog of calnexin. The role of calnexin at
AURKA, MLF1IP, SATB1, PTEN). plasmodesmata will be discussed.

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CRITICAL ROLE OF C-ZYME ON P53IG-MEDIATED PHOSPHORYLATION OF PTP-PEST AT SER-39 IS
APOPTOSIS REGULATED THROUGH THE BINDING OF PP1 ALPHA
Kang M.Y. Palmer H.E.F., Nakamura K. and Mashima K.
Department of Bio-Materials Engineering, Graduate School and Department of Life Science, Rikkyo University.
DNA Repair Research Center, Chosun University, 375 Seosuk-dong,
Gwangju 501-759, Korea. PTP-PEST is expressed in a wide variety of cell types and is an efficient
regulator of integrin-medicated signaling in adherent cells and antigen
A number of genes involved in control of the cell cycle and apoptosis receptor-mediated signaling in lymphocytes. PTP-PEST-associating
are regulated by p53-induced genes (p53IGs). A series of p53IG have molecules are important in elucidating the function of PTP-PEST.
been identified that are predicted to encode proteins that could generate The major phosphorylation site of PTP-PEST at Ser-39, regulates
or respond to oxidative stress. C-zyme, an anti-oxidative enzyme is an the PTP activity. Herein, we have identified protein phosphatase 1α
important scavenger of ROS related to p53IG activation. We found that (PP1α) as a novel PTP-PEST binding protein, and tried to elucidate the
purified p53IG coimmunoprecipitated recombinant C-zyme suggesting