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HAL‹Ç UNIVERSITY
‹STANBUL
FACULTY OF ARTS AND SCIENCES
1998
Correspondence Address:
The Editorial Office Editorial Board
J o u r n a l o f C e l l a n d M o l e c u l a r Biology
Çimen ATAK
Haliç Üniversitesi, Fen-Edebiyat Fakültesi,
Ahmet Vefik Pafla Cad., No: 1, 34280, Atok OLGUN
F›nd›kzade, ‹stanbul-Turkey
P›nar ÖZKAN
Phone: 90 212 530 50 24
Fax: 90 212 530 35 35 Nihal BÜYÜKUSLU
E-mail: jcmb@halic.edu.tr Kürflat ÖZD‹LL‹
J o u r n a l o f C e l l a n d M o l e c u l a r Biology is Damla BÜYÜKTUNÇER
indexed in EBSCO database.
Özge EM‹RO⁄LU
Summaries of all articles in this journal are
available free of charge from www.halic.edu.tr Mehmet Ali TÜFEKÇ‹
ISSN 1303-3646 Merve ALO⁄LU
printed at yaflar printing house Asl› BAfiAR
Advisory Board
Igor ALEXANDROV, Dubna, Russia Ünal EGEL‹, Bursa, Turkey
Çetin ALGÜNEfi, Edirne, Turkey Candan JOHANSEN, ‹stanbul, Turkey
Aglaia ATHANASSIADOU, Patros, Greece As›m KADIO⁄LU, Trabzon, Turkey
fiehnaz BOLKENT, ‹stanbul, Turkey Valentine KEFEL‹, Pennsylvania, USA
Nihat BOZCUK, Ankara, Turkey Göksel OLGUN, Edirne, Turkey
‹smail ÇAKMAK, ‹stanbul, Turkey U¤ur ÖZBEK, ‹stanbul, Turkey
Adile ÇEV‹KBAfi, ‹stanbul, Turkey Zekiye SULUDERE, Ankara, Turkey
Beyaz›t ÇIRAKO⁄LU, ‹stanbul, Turkey ‹smail TÜRKAN, ‹zmir, Turkey
Ayfl›n ÇOTUK, ‹stanbul, Turkey Mehmet TOPAKTAfi, Adana, Turkey
Zihni DEM‹RBA⁄, Trabzon, Turkey Meral ÜNAL, ‹stanbul, Turkey
Mustafa DJAMGOZ, London, UK Mustafa YAT‹N, Boston, USA
Aglika EDREVA, Sofia, Bulgaria Ziya Z‹YLAN, ‹stanbul, Turkey
Journal of Cell and
M o l e c u l a r Biology
Volume 2/2003
Haliç University
Faculty of Arts and Sciences
‹stanbul-TURKEY
Journal of Cell and
M o l e c u l a r Biology
Haliç University
Published by Haliç University
Faculty of Arts and Sciences Faculty of Arts and Sciences
J o u r n a l o f C e l l a n d M o l e c u l a r Biology
Founder Editor
Prof. Dr. Gündüz GED‹KO⁄LU
Atilla ÖZALPAN
President of Board of Trustee
Rights held by
Prof. Dr. Ahmet YÜKSEL Associate Editor
Rector Narç›n PALAVAN ÜNSAL
Correspondence Address:
The Editorial Office Editorial Board
J o u r n a l o f C e l l a n d M o l e c u l a r Biology
Çimen ATAK
Haliç Üniversitesi, Fen-Edebiyat Fakültesi,
Ahmet Vefik Pafla Cad., No: 1, 34280, Atok OLGUN
F›nd›kzade, ‹stanbul-Turkey
P›nar ÖZKAN
Phone: 90 212 530 50 24
Fax: 90 212 530 35 35 Nihal BÜYÜKUSLU
E-mail: jcmb@halic.edu.tr Kürflat ÖZD‹LL‹
J o u r n a l o f C e l l a n d M o l e c u l a r Biology is Damla BÜYÜKTUNÇER
indexed in EBSCO database.
Özge EM‹RO⁄LU
Summaries of all articles in this journal are
available free of charge from www.halic.edu.tr Mehmet Ali TÜFEKÇ‹
ISSN 1303-3646 Merve ALO⁄LU
printed at yaflar printing house Asl› BAfiAR
Advisory Board
Igor ALEXANDROV, Dubna, Russia Ünal EGEL‹, Bursa, Turkey
Çetin ALGÜNEfi, Edirne, Turkey Candan JOHANSEN, ‹stanbul, Turkey
Aglaia ATHANASSIADOU, Patros, Greece As›m KADIO⁄LU, Trabzon, Turkey
fiehnaz BOLKENT, ‹stanbul, Turkey Valentine KEFEL‹, Pennsylvania, USA
Nihat BOZCUK, Ankara, Turkey Göksel OLGUN, Edirne, Turkey
‹smail ÇAKMAK, ‹stanbul, Turkey U¤ur ÖZBEK, ‹stanbul, Turkey
Adile ÇEV‹KBAfi, ‹stanbul, Turkey Zekiye SULUDERE, Ankara, Turkey
Beyaz›t ÇIRAKO⁄LU, ‹stanbul, Turkey ‹smail TÜRKAN, ‹zmir, Turkey
Ayfl›n ÇOTUK, ‹stanbul, Turkey Mehmet TOPAKTAfi, Adana, Turkey
Zihni DEM‹RBA⁄, Trabzon, Turkey Meral ÜNAL, ‹stanbul, Turkey
Mustafa DJAMGOZ, London, UK Mustafa YAT‹N, Boston, USA
Aglika EDREVA, Sofia, Bulgaria Ziya Z‹YLAN, ‹stanbul, Turkey
J o u r n a l o f C e l l a n d M o l e c u l a r Biology
Dedication
Review articles
P o l y a m i n e s i n p l a n t s : An overview
Bitkilerde poliaminler: Genel bir bak›fl
R. Kaur-Sawhney, A.F. Tiburcio, T. Altabella, A.W. Galston 1-12
P h e n o l i c c y c l e i n p l a n t s a n d e n v i ro n m e n t
Bitkilerde fenolik döngü ve çevre
V. I. Kefeli, M. V. Kalevitch, B. Borsari 13-18
R e s e a rc h p a p e r s
The short-term effects of single toxic dose of citric acid in mice
Farelerde sitrik asidin tek toksik dozunun k›sa süreli etkileri
T. Aktaç, A. Kabo¤lu, E. Bakar, H. Karakafl 19-23
C h a r a c t e r i s a t i o n o f R P P 7 mutant lines of the col-5 ecotype of Arabidopsis thaliana
Arabidopsis thaliana’n›n col-5 ekotipinden elde edilen mutant hatlardan RPP7
geninin karakterizasyonu
C. Can, M. Özaslan, E. B. Holub 25-30
The effect of meta - t o p o l i n o n p r o t e i n p r o f i l e i n r a d i s h c o t y l e d o n s
Meta-topolinin turp kotiledonlar›nda protein profiline etkisi
S. Ça¤, N. Palavan-Ünsal 31-34
H o n o r s : Elected to Phi Beta Kappa; Phi Kappa Phi; Sigma Xi; American Academy of Arts and Sciences, National
Sigma Xi Lecturer, 1966; National Phi Beta Kappa Visiting Scholar, 1972-1973; Award of the New York Academy
of Sciences, 1979; William Clyde De Vane Medal for lifelong teaching and scholarship, Yale University, 1994;
Honorary LL.D, 1980 Iona; Honorary Ph. D., Hebrew University of Jerusalem, 1992.
E x p e r i e n c e : Plant Physiologist, Emergency Rubber Project, California Institute of Technology 1943-1944; Instuctor
in Botany, Yale University, 1946-1947; Senior Research California Institute of Technology, 1947-1950; Associate
Professor of Biology, California Institute of Technology, 1951-1955. Professor of Plant Physiology, Department of
Botany, Yale University 1955-1961; Chairman, Department of Botany, 1961-1962; Director, Division of Biological
Sciences, Yale University, 1965-1966; Professor of Biology, 1962-1973; Eaton Professor of Botany, 1973-;
Chairman, Department of Biology 1985-1988; Eaton Professor Emeritus, 1990.
Fellow of the John Simon Guggenheim Memorial Foundation, Stockholm and Sheffield, 1950-1951; Fulbright
Fellow, Canberra, Australia, 1960-1961; National Science Foundation Faculty Fellow, London 1967-1968; Albert
Einstein Fellow and Visiting Professor, Hebrew University of Jerusalem, 1980; Visiting Fellow Wolfson College,
Cambridge, England, 1983; Visiting Scientist, RIKEN Institute, Wako, Saitama, Japan, 1988-1989.
Secretary, American Society of Plant Physiologists, 1955-1957; Vice President, 1957-1958; President, 1962-1963.
Secretary-Treasurer, International Association for Plant Physiology, 1962-1967. Vice-President
Botanical Society, 1967-1968; President 1968-1969; Award, 1970. Member, Commitee on Space Biology and
Medicine, National Research Council; Member Life Sciences Advisory Committee, NASA; also Long Range
Strategic Planning Committee in Life Sciences Advisory Committee, NASA; Member, NASA Disciplinary Working
Group for CELLS (Controlled Ecological Life Support Sytem).
F o r m e r M e m b e r : Metabolic and Regulatory Biology Panels, National Science Foundation; Executive Committee,
Growth Society; Life Science Advisory Committee, NASA; and Governing Boards, Biological Sciences Curriculum
Study, Commission on Undergraduate Education in the Biological Sciences and AIBS.
First American scientist to visit the People’s Rebuplic of China, 1971.
Books: ‘Principles of Plant Physiology’ (with J. Bonner), Freeman, 1952. ‘Life of the Green Plant’, Prentice Hall,
1961, 2nd Ed., 1964, 3rd Ed., 1980 (with P. J. Davies and R. L. Satter). ‘Control Mechanisms in Plant Development’, (with
P. J. Davies), Prentice Hall, 1970. ‘Daily Life in People’s China’, Crowell, 1973; Simon and Schuster, 1975. ‘Green
Wisdom’ Basic Books, Inc. NY, 1981; Putnam, 1983. ‘Life Processes in Plants’, Freeman (Scientific American
Library), 1994. ‘New Dimensions in Bioethics’, Arthur W. Galston and Emily G. Shurr, eds. Kluwer Academic
Publishers, Boston/Dordrecht/London, 2001.
More than 320 articles in referred scientific journal; approximately 60 general articles on problems of science and
society.
Journal of Cell and Molecular Biology 2: 1-12, 2003. 1
Haliç University, Printed in Turkey.
P o l y a m i n e s i n p l a n t s : An overview
Ravindar Kaur-Sawhney1*, Antonio F. Tiburcio2, Teresa Altabella2, and Arthur W. Galston1
1
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT,
06520-8103, USA; 2Laboratori de Fisiologia Vegetal, Facultad de Farmacia, Universitat de Barcelona,
Spain (* author for correspondence)
Abstract
This article presents an overview of the role of polyamines (PAs) in plant growth and developmental processes. The
PAs, putrescine, spermidine and spermine are low molecular weight cations present in all living organisms. PAs and
their biosynthetic enzymes have been implicated in a wide range of metabolic processes in plants, ranging from cell
division and organogenesis to protection against stress. Because the PA pathway has now been molecularly and
biochemically elucidated, it is amenable to modulation by genetic approaches. Genes for several key biosynthetic
enzymes namely, arginine decarboxylase, ornithine decarboxylase and S-adenosyl methionine decarboxylase have
been cloned from different plant species, and antibodies to some genes are now available. Both over-expressed and
antisense transgenic approaches to PA biosynthetic genes have provided further evidence that PAs are required for
plant growth and development. However, molecular mechanisms underlying PA effects on these processes remain
unclear. Analysis of gene expression by using DNA microarray genomic techniques should help determine the precise
role of these compounds. The potential of proteomics to unravel the role of PAs in particular cellular processes has
also been examined. The extensive use of the two-hybrid system and other proteomic approaches will provide new
insights into the role of PAs in signal transduction. Furthermore, there is evidence that proteomics provides an
excellent tool for determining supramolecular organizations of PA metabolic enzymes which may help in
understanding homeostatic control of this metabolic pathway.
Özet
Bu makalede poliaminlerin (PA) bitki büyüme ve geliflme olaylar›ndaki rolüne genel bir bak›fl yap›lmaktad›r. PA ler
putresin, spermidin ve spermin, düflük molekül a¤›rl›kl› ve tüm canl› organizmalarda mevcut olan maddelerdir. PA
lerin ve bunlar›n biyosentetik enzimlerinin bitkileri strese karfl› korumaya yönelik olarak hücre bölünmesinden
organogeneze kadar de¤iflen genifl bir metabolik olaylar zincirinde yer ald›¤› ortaya konmufltur. Günümüzde PA yolu
moleküler ve biyokimyasal yönden aç›kl›¤a kavufltu¤u için genetik yaklafl›mlarla düzenlenmeye uygundur. Çeflitli
anahtar biyosentez enzimleri, arginin dekarboksilaz, ornitin dekarboksilaz ve S-adenozil metiyonin dekarboksilaz›n
genleri farkl› bitki türlerinde klonlanm›flt›r ve günümüzde baz› genlerin antikorlar›n› elde etmek mümkündür. PA
biyosentezi genlerine hem over-ekspres ve hem de antisens transgenik yaklafl›mlar PA lerin bitki büyüme geliflmesi
için gereklili¤ini daha da ortaya koymufltur. Bununla birlikte bu olaylardaki PA etkilerinin moleküler mekanizmas›
hala aç›kl›¤a kavuflmam›flt›r. DNA mikroarray genom teknikleri kullan›larak yap›lan gen ekspresyon analizleri bu
bilefliklerin rollerini kesin olarak belirlemeye yard›mc› olacakt›r. PA lerin özellikle hücresel olaylardaki rolünü ortaya
koymaya yönelik olarak proteomi¤in potansiyeli de araflt›r›lm›flt›r. ‹ki-hibrit sistemi ve di¤er proteomik yaklafl›mlar›n
yo¤un kullan›m›, PA lerin sinyal iletimindeki rolüne yeni bir bak›fl aç›s› getirecektir. Bundan baflka proteomi¤in, PA
metabolik yolunun homeostatik kontrolünü anlamaya yard›mc› olabilecek, PA metabolizma enzimlerinin
supramoleküler organizasyonunun belirlenmesinde çok önemli bir araç oldu¤u konusunda veriler mevcuttur.
Ornithine Arginine
ODC ADC
Methionine DFMA
DFMO
Agmatine
MGBG Putrescine
S - adenosylmethionine dSAM Spdsynthase
Spermidine
SAMDC
ACC synthase Spmsynthase
AVG
ACC Spermine
ACC oxidase
Ethylene
F i g u re 1: Polyamine biosynthetic pathway and its linkage to ethylene biosynthetis. Biosynthetic enzymes are ADC, ODC and
SAMDC and the inhibitor DFMA, DFMO and MGBG.
the obstacles in understanding their biological role. 3. Polyamines in plant growth and development
Recent studies have shown that PAs are present in the
cell wall fractions, vacuole, mitochondria and The availability of specific inhibitors of PA
chloroplasts (Torrigiani et al., 1986; Slocum, 1991b; biosynthesis has helped in investigating the
Tiburcio et al., 1997). The biosynthetic enzymes, mechanisms involved in PA interactions to some extent,
ODC, SAMDC, and Spd synthase have been reported providing a partial understanding of their physiological
to be localized in the cytoplasm, whereas ADC is role in plant growth and development. Clearly, PAs are
localized in the thylakoid membrane of chloroplast involved in many plant developmental processes,
(Borrell et al., 1996; Tiburcio et al., 1997) and PAO in including cell division, embryogenesis, reproductive
the cell wall (Kaur-Sawhney et al., 1981). ODC organ development, root growth, tuberization, floral
activity has also been observed in the nucleus initiation and development, fruit development and
(Slocum, 1991b). However, these findings have to be ripening as well as leaf senescence and abiotic stresses
interpreted with caution because various procedural (reviewed by Evans and Malmberg, 1989; Galston et
problems can mask the results. Despite these advances al., 1997; Bais and Ravishankar, 2002; Tiburcio et al.,
in understanding the metabolic processes involving 2002). Changes in free and conjugated PAs and their
PAs and their localization in plant cells, the precise biosynthetic enzymes, namely ADC, ODC, and
role of PAs in plant morphogenesis remains elusive. SAMDC have been found to occur during these
developmental processes. Earlier experiments had
shown that increases in PAs and their biosynthetic
enzymes are associated with rapid cell division in many
plant systems e.g., carrot embryogenesis (Montague
4 Ravindar Kaur-Sawhney et al.
and Koppenbrink, 1978; Feirer et al., 1984), tomato Sawhney,1995). Thus, PAs which may or may not be
ovaries (Heimer and Mizrahi, 1982), tobacco ovaries mobile in plants (Young and Galston, 1983; Bagni and
(Slocum and Galston, 1985), and fruit development Pistocchi, 1991) can serve as intracellular mediators of
(reviewed in Kakkar and Rai, 1993). Similar results hormone actions (Galston and Kaur-Sawhney, 1995).
have been reported for many other plant species Supporting evidence for this hypothesis has been
(reviewed in Bais and Ravishankar, 2002). In contrast, obtained in experiments using specific inhibitors of PA
several other studies have suggested that correlations biosynthesis (Bagni et al., 1981; Egea-Cortines and
between PAs and their biosynthetic enzymes and plant Mizrahi, 1991; reviewed in Galston et al., 1997; Bais
growth processes, especially somatic embryogenesis, and Ravishankar, 2002).
are not universal and may be species specific (reviewed Of the major plant hormones, ethylene has been
in Evans and Malmberg, 1989; Galston et al., 1997; most intensively investigated with respect to PA
Bais and Ravishankar, 2002). metabolism. The two metabolites, PAs and ethylene,
In general, cells undergoing division contain high play antagonistic roles in plant processes. While PAs
levels of free PAs synthesized via ODC, and cells inhibit senescence of leaves (Kaur-Sawhney et al.,
undergoing expansion and elongation contain low 1982), cell cultures of many monocot and dicot species
levels of free PAs synthesized via ADC (see review by (Muhitch et al., 1983) and fruit ripening (Kakkar and
Galston and Kaur-Sawhney, 1995). High levels of Rai, 1993), ethylene promotes these processes. The
endogenous PAs and their conjugates have also been most commonly held view is that PAs and ethylene
found in apical shoots and meristems prior to regulate each other’s synthesis, either directly or
flowering (Cabbane et al., 1981) and flower parts of through metabolic competition for SAM, a common
many plants (Martin-Tanguy, 1985). Our experiments precursor for their biosynthesis (Figure 1). PAs inhibit
using callus cultures derived from thin layer explants ethylene biosynthesis, perhaps by blocking the
of pedicels from tobacco inflorescence show that conversion of SAM to ACC and of ACC to ethylene
endogenous Spd increased more rapidly than other (Apelbaum et al., 1981; Suttle, 1981; Even-Chen et al.,
PAs in floral buds than in vegetative buds. Addition of 1982; Furer et al., 1982). Ethylene, on the other hand,
CHA, an inhibitor of Spd synthesis, to the culture is an effective inhibitor of ADC and SAMDC, key
medium reduced flower formation in a dose dependent enzymes in PA biosynthetic pathway (Apelbaum et al.,
manner and such inhibition was correlated with a 1985; Icekson et al., 1985). Thus, PAs may affect
switch to initiation of vegetative instead of flower senescence and fruit ripening by modulating PA and
buds. This inhibition was reversed by the addition of ethylene biosynthesis.
exogenous Spd (Kaur-Sawhney et al., 1988). More Apparently, PAs are essential members of an array
recently, we have found that higher levels of of internal metabolites required in many plant
endogenous PAs occur in flowers and siliques when developmental processes, but their precise role in these
compared with their levels in leaves and bolts of processes has yet to be established. Whereas, specific
certain strains of Arabidopsis. Addition of the PA PAs at specific concentrations may be required at
biosynthetic inhibitors, DFMA and CHA to the culture critical stages of growth and morphogenetic events, no
medium, at time of seed germination, inhibited bolting definitive data are available to establish their role as
and flower formation and this was partially reversed plant hormones.
by addition of exogenous Spd (Applewhite et al.,
2000). These results clearly show that Spd is involved
in flower initiation and development. Similar results 4. Manipulation of the polyamine pathway
have been reported in other plants also (reviewed by
Galston et al.,1997; Bais and Ravishankar, 2002). The PA pathway is ubiquitous in living organisms and
Many plant growth and development processes is relatively short (see Section 2) in terms of the
known to be regulated by plant hormones, such as number of enzymes involved. Most of the genes
auxins, 2,4-D, GA and ethylene, have also been coding for enzymes involved in the pathway have been
correlated with changes in PA metabolism. These cloned from different sources (Kumar et al., 1997;
changes occur in both endogenous levels of PAs and Walden et al., 1997; Galston et al., 1997; Tiburcio et
their biosynthetic enzymes and appear to be tissue al., 1997; Malmberg et al., 1998; Kumar and Minocha,
specific (reviewed by Galston and Kaur- 1998; Panicot et al., 2002b). Thus, the PA pathway
Polyamines in plants 5
represents an excellent model to test various which may correspond to the two gene copies
hypotheses and to answer fundamental biological encoding ADC, ADC1 and ADC2 (Watson et al.,
questions derived from pathway manipulation (Thu- 1998). The mutations have not been mapped and
Hang et al., 2002; Bhatnagar et al., 2002). therefore it cannot be excluded that other functions,
Initially, approaches to manipulate the PA pathway i.e. regulatory elements, are affected (Soyka and
made use of suicide inhibitors, but the effects of Heyer, 1999). More recently, Hanzawa et al. (2000)
DFMO and DFMA on ODC and ADC respectively, are reported that the inactivation of the Arabidopsis
variable in different plant systems, ranging from ACAULIS5 (ACL5) gene causes a defect in the
inhibition to stimulation or no effect and depending on elongation of stem internodes by reducing cell
the concentration, plant system tested and the expansion. It was suggested that ACL5 encodes a Spm
existence of compensatory mechanisms (Slocum and synthase, but the possibility that ACL5 may exhibit
Galston, 1987). Therefore, alternative approaches to broad amine substrate specificities and be involved in
manipulate polyamine metabolism have been the synthesis of other polyamines could not be
developed during the recent years. excluded (Hanzawa et al., 2000).
Thus far the only well characterized plant
4.1. Mutants polyamine biosynthetic mutant has been generated by
using reverse genetics. The availability of mutant
Mutants deficient in PA biosynthesis have been collections generated either by transposon or T-DNA
isolated from several biological systems. Hafner et al. tagging now facilitates the identification of knockouts
(1979) isolated PA mutants in Escherichia coli in any gene of interest using PCR-based mutant
showing decreased growth and increased sensitivity to screening techniques (Ferrando et al., 2002). By using
paraquat (Milton et al., 1990). Yeast mutants these techniques, Soyka and Heyer (2000) isolated an
presenting ODC as the sole pathway, show reduced Arabidopsis thaliana mutant line carrying an insertion
growth and altered sporulation on PA deficient of the En-1 transposable element at the ADC2 locus
medium (Cohn et al., 1980; Whitney and Morris, which should be regarded as a complete loss-of-
1978). Chinese hamster ovary cells lacking ODC function or knockout mutation. The ADC2 knockout
activity do not grow in medium lacking PA (Steglich mutant shows no obvious phenotype change under
and Scheffler, 1983) and a moderately reduced brood normal growth conditions, but is completely devoid of
size was observed in a Caenorhabditis elegans ODC ADC induction by osmotic stress. As ADC1 gene
deletion mutant (Macrae et al., 1995). Mutations in expression was not affected in the mutant, it was
genes affecting Spd and Spm biosynthesis have also concluded that ADC2 is the gene responsible for
been isolated in yeast. The spe3 Spd synthase mutation induction of ADC and PA biosynthesis under osmotic
causes a growth arrest, which can be complemented stress (Soyka and Heyer, 2000). More recently, Pérez-
with externally added Spd (Hamasaki-Katagiri et al., Amador et al. (2002) have shown that ADC2 gene
1997), while the yeast spe4 mutant is defective in Spm expression is induced in response to mechanical
biosynthesis (Hamasaki-Katagiri et al., 1998). wounding and methyl jasmonate treatment in
Less is known about mutants affecting PA Arabidopsis thaliana. All these observations appear to
metabolism in plants. Mutants with high levels of indicate that ADC2 is a key gene involved in the PA
ADC activity have been identified in petunia because response to abiotic stress in Arabidopsis. We envisage
of their abnormal morphology (Geerats et al., 1988), that the extensive use of functional genomics and
but the basis of the mutation is still not known. reverse genetic studies will facilitate the isolation of
Screening for resistance to the SAMDC inhibitor novel knock-out mutants affected in other PA
MGBG (Malmberg and Rose, 1987) or to inhibitory biosynthetic genes.
concentrations of Spm (Mirza et al., 1997), yielded
mutants that showed reduced sensitivity to the 4.2. Transgenic plants
respective agent, but these mutants have not been
further exploited for the analysis of PA function. With the availability of most of the genes involved in
Watson et al. (1998) isolated EMS mutants of A. PA metabolism, it has become possible to manipulate
thaliana that are reduced in ADC activity. The mutants this metabolic pathway using sense and antisense
fall into two complementation groups, spe1 and spe2, transgenic approaches. Thus, cellular PA content has
6 Ravindar Kaur-Sawhney et al.
been modulated by overexpression or down regulation SAMDC gives rise to smaller potato tubers without
of the key genes ODC, ADC or SAMDC (Kumar et al., affecting tuber yield (Rafart-Pedros et al., 1999). The
1997; Walden et al., 1997; Malmberg et al., 1998; distribution of tuber weights is of agronomic
Kumar and Minocha, 1998; Capell et al., 1998; Rajam importance, and generally a reduction of tuber-size
et al.,1998; Roy and Wu, 2001; Bhatnagar et al., 2002). variation is economically advantageous, so that more
Most of the studies have used the constitutive 35S tubers fall into a given size grade either for seed or
promoter, but only few of them were successful in ware (Rafart-Pedros et al., 1999). Similarly, fruit-
using either inducible (Masgrau et al., 1997; Panicot et specific expression of heterologous SAMDC in tomato
al., 2002a; Mehta et al., 2002) or tissue-specific resulted in ripening-specific accumulation of Spd and
promoters (Rafart-Pedros et al., 1999). Overexpression Spm which led to an increase in lycopene, prolonged
of heterologous ODC or ADC cDNAs generally causes vine life, and enhanced fruit juice quality (Mehta et al.,
the production of high levels of Put (DeScenzo and 2002). Besides the agronomic interest of this finding,
Minocha, 1993; Bastola and Minocha, 1995; Masgrau this latter study constitutes one of the most striking
et al., 1997; Capell et al., 1998; Bhatnagar et al., 2002; evidence regarding the in vivo involvement of
Panicot et al., 2002a), but in most cases only a small polyamines in a particular developmental process, i.e.
increase or even no change in Spd and Spm has been fruit ripening (Mehta et al., 2002).
observed. This indicates that elevated levels of Put
resulting from genetic manipulation of a single step
located upstream of the PA biosynthetic pathway (i.e. 5. U n d e r s t a n d i n g t h e role of polyamines
ODC or ADC) are not accompanied by an increase in
subsequent biosynthetic reactions (i.e. Spd and Spm Phenotypic analyses of mutants and transgenic plants
biosynthesis) (Bhatnagar et al., 2002). In contrast, with altered PA levels gives further support to the
overexpression of genes located downstream of the previous physiological studies (see Section 3) with
pathway (i.e. SAMDC or SPDS) generally lead to regard to the involvement of these compounds in
increased levels of Spd or Spm or both (Thu-Hang et several plant processes (reviewed by Tiburcio et al.,
al., 2002; Mehta et al., 2002). Taken together these 2002). These include somatic embryogenesis (Bastola
results suggest that the levels of Spd and Spm in the and Minocha, 1995), stem elongation and flowering
cells are under a tight homeostatic regulation (Gerats et al., 1988; Masgrau et al., 1997; Hanzawa et
(Bhatnagar et al., 2002), which possibly could be al., 2000; Panicot et al., 2002a), root growth (Watson
related to a supramolecular organization of some of et al., 1998; Cordeiro et al., unpublished), tuber
these enzymes (see Section 5). development (Kumar et al., 1996; Rafart-Pedrós et al.,
Discrepancies observed among different studies 1999), fruit ripening (Mehta et al., 1997; 2002), abiotic
may have several causes. These include: transgene stresses (Minocha and Sun, 1997; Soyka and Heyer,
source, positional effects, recipient plant system, plant 1999; Roy and Nu, 2001). However, most of these
material analyzed and type of promoter used. A mutants and transgenic plants have not been further
hierarchical accumulation of polyamines in different exploited for the analysis of PA function. Application
transgenic tissues/organs has been observed (Lepri et of advanced genomic and proteomic approaches will
al., 2001). In general, less metabolically active tissues help to elucidate the role of PA in particular plant
accumulate higher levels of polyamines (Lepri et al., processes.
2001). These results are in line with experiments in
which metabolites such as vitamin A and 5.1. Genomic approaches
pharmaceutical antibodies accumulate at high levels in
seeds of different species. It is reasonable to assume The availability of complete genome sequences
that dormant or less metabolically active tissues permits the use of approaches to explore gene
provide a conducive environment for the accumulation expression variations on a large genome scale. Either
of transgenic products (Thu-Hang et al., 2002). In this cDNAs or large oligonucleotide collections are
regard, it should be stressed that the most remarkable attached on surfaces to create a microarray. The
results have been obtained by controlled expression of hybridisation of the microarray with fluorescent
transgenes using inducible or tissue-specific labelled RNA or cDNA yields an overall image of gene
promoters. For example, tissue-specific expression of expression or ‘transcriptome’ (Lockhart and Winzeler,
Polyamines in plants 7
2000). The global examination of gene expression (DBD) or the transcriptional activation domain (AD),
should reveal the coincidence of spatial and temporal is translationally fused to proteins of interest X or Y,
transcript expression profiles that may reflect a generating respectively the hybrid proteins X-DBD
requirement of co-ordinated gene product expression (bait) and Y-AD (prey). A powerful aspect of the yeast
in response to different type of signals. The technology molecular genetics involves the facility to isolate the
developed for the Arabidopsis genome has been corresponding cDNAs coding for proteins X or Y,
accelerated in the recent years both by public funding introduced in the form of plasmid DNA. This latter
through the Arabidopsis Functional Genomics feature immediately favored the use of this system to
Consortium in the USA and the GARNet in the UK, identify interacting partners for a given bait protein X
and also by private initiatives like Monsanto, using cDNA libraries as a prey (reviewed by Walhout
Affymetrix or Synteny/InCyte (Wisman and Ohlrogge, et al., 2000). The number of studies that have used
2000). proteomics in our field is still scanty. Here we will
Although there are already many examples in the provide two examples that demonstrate the potential of
literature showing the utility of this approach for these techniques to (i) unravel the role of PA in
unraveling complex plant responses and signal transcription; and (ii) to identify PA metabolons (see
transduction processes (Schena et al., 1995; Schaffer et below).
al., 2000), the use of this technology in our field is Although the potential role of PAs in affecting gene
unfortunately in its infancy. So far, DNA microarray expression had already been reported, the molecular
analysis has been used to reveal the induction of ADC mechanisms underlying their effects were unknown
genes during drought stress (Ozturk et al., 2002) or in (Wang et al., 2002). The identification of a polyamine
response to wounding and methyl jasmonate treatment responsive element and corresponding transacting
(Sasaki et al., 2001; Pérez-Amador et al., 2002). protein factors that respond to polyamines has opened
We envisage that global analysis of gene up an exciting new area to study the function of these
expression in well characterized mutant and transgenic compounds in transcription (Wang et al., 1999). By
plants with altered polyamine metabolism will provide using the two-hybrid system, it was recently found that
novel clues in the near future for understanding the the human homologue of the Arabidopsis subunit
molecular mechanisms underlying polyamine effects COP9 signalosome complex binds to such transacting
on plant growth and development. protein factors with the potential to directly affect gene
expression (Wang et al., 2002). Remarkably, the COP9
5.2. Proteomic approaches signalosome proteins were first identified in
Arabidopsis and have been demonstrated to form a
Proteomics’ uses biochemical approaches aimed at regulatory complex involved in light-activated
systematically characterizing the ‘proteome’ or the development and playing a role in intracellular
‘protein complement of the genome’ (Wasinger et al., signalling (Deng et al., 2000). We envisage that similar
1995) in a given organism, tissue, cell or subcellular type of experiments will be performed in the plant PA
compartment. The means of proteome characterization field that hopefully will provide new insights into the
include protein localization, expression and most role of PAs in plant signal transduction.
importantly protein interaction maps. A plethora of Increasing number of reports document that many
innovative procedures has been employed in recent metabolic reactions are catalysed by complexes of
years for the large-scale analysis of protein signalling sequentially acting enzymes that show highly ordered
pathways, including the yeast two-hybrid system structural organization (reviewed in Srere, 1987). In
(Fields and Song, 1989), protein purification methods such multienzyme complexes the metabolites pass
linked to detection by mass spectrometry (Neubauer et from one active enzyme site to the next through a
al., 1997; Verma et al., 2000); protein localization process termed ‘substrate channeling’. The
(Ferrando et al., 2000; 2001; Farràs et al., 2001), and supramolecular arrangement of enzymes involved in
protein microarray techniques (Zhu et al., 2001). such metabolic reactions is referred to as ‘metabolon’.
The yeast two-hybrid system is a genetic tool to Metabolons are multienzyme complexes in both
describe in vivo protein interactions using the yeast prokaryotes and eukaryotes that represent highly
cell as a test tube. Each separated module of the GAL4 organized assemblies of sequential enzymes in a
transcription factor, either the DNA binding domain metabolic pathway and are thought to provide
8 Ravindar Kaur-Sawhney et al.
increased metabolic efficiency and higher substrate DNA replication, transcription of genes, cell division,
selectivity. Metabolons may also help to coordinate the organ development, fruit development and ripening,
activities of enzymes by sharing intermediates in a leaf senescence and abiotic stresses. Despite ample
given pathway, as well as to ensure protection of labile evidence of their involvement in these processes, their
substrates and sequestration of toxic intermediates precise role in these specific processes remains to be
(Sugumaran et al., 2000). In addition, the formation of established. Recent developments of PA-deficient
multienzyme metabolon complexes may enhance mutants and transgenic plants as well as of
enzyme stability, improve enzymatic performance and molecular genetic investigations should further our
provide a means for adaptation to alterations of input understanding of their role in plant growth and
of metabolic reactions, especially during demanding development.
physiological conditions (Abadjieva et al., 2001). The polyamine pathway is now amenable to
The relevant information about intrinsic properties modulation by genetic approaches because it has been
of ‘metabolon’ formation can be acquired by studies of elucidated molecularly and biochemically in plants.
protein-protein interactions using modern proteomic Reverse genetics has identified an Arabidopsis
approaches (Ferrando et al., 2002). In this regard, our knockout mutation of ADC2 gene which reveals
laboratory has recently analyzed possible interactions inducibility by osmotic stress. Extensive use of
between the SPDS and SPMS enzymes of polyamine functional genomics and reverse genetics studies will
biosynthetic pathway in the yeast two-hybrid system facilitate the isolation of novel knockout mutants
(Panicot et al., 2002b). Using the Arabidopsis affected in other polyamine metabolic genes. Sense
spermidine synthase as bait, two similar proteins were and antisense transgenic approaches have revealed the
identified to interact with SPDS2 that were named feasibility of modulating cellular PA contents.
SPDS1 and SPMS. Yeast and bacterial mutant Generally, genetic manipulation of single steps located
complementation tests revealed that SPDS1 encodes a upstream of the PA pathway (i.e. ODC or ADC) lead to
novel spermidine synthase, whereas SPMS displays elevated levels of Put, but no changes occur in the
spermine synthase activity. The heterodimerization higher PAs, Spd and Spm. By contrast, overexpression
capabilities of enzymes catalyzing the two last steps of of genes located downstream of the pathway (i.e.
polyamine biosynthesis were also demonstrated in vivo SAMDC or Spd synthase) generally leads to increased
by co-immunoprecipitation using epitope tagged levels of Spd and Spm, indicating that the levels of Spd
SPDS1, SPDS2 and SPMS proteins (Ferrando et al., and Spm are under a tight homeostic cellular control.
2000; Ferrando et al., 2001). Immunoaffinity Phenotypic analyses of mutants and transgenic plants
purification and size fractionation of SPDS and SPMS affected in polyamine metabolism further support
enzymes labeled with different HA and c-Myc previous physiological evidence, but the molecular
epitopes revealed that the SPDS and SPMS proteins mechanisms underlying PA effects on plant growth and
co-purify with large multiprotein complexes of 650 to development remain to be elucidated. Global analysis
750 kDa. Further analysis of subunits of isolated of gene expression by using the available DNA
SPDS-SPMS metabolon(s) by mass spectrometry is microarray genomic techniques will help to understand
expected to yield important information about yet the role of these compounds. The potential of
unknown regulatory subunits of SPDS-SPMS proteomics to unravel the role of polyamines in
metabolon in the PA biosynthesis pathway. The particular cellular processes is also examined. We
available data support the conclusion that Spd envisage that the extensive use of the two-hybrid
synthesized by SPDS is effectively channeled to system and other proteomic approaches will provide
SPMS to control the formation of the end-product Spm new insights into the role of PAs on plant signal
thereby regulating the synthesis of high molecular transduction. Furthermore, we provide evidence that
weight polyamines (Panicot et al., 2002b). proteomics is an excellent tool to unravel
supramolecular organizations of PA metabolic
enzymes which may help to understand homeostatic
6. Conclusions control of this metabolic pathway.
Mehta RA, Cassol T, Li N, Ali N, Handa AK and Mattoo AK. Roy M and Wu R. Arginine decarboxylase transgene
Engineered polyamine accumulation in tomato enhances expression and analysis of environmental stress
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Milton KW, Tabor H and Tabor CW. Paraquat toxicity is Awai K, Amagai M, Kuwata C, Tsugane T, Masuda T,
increased in E. coli defective in the synthesis of Shimada H, Takamiya K, Ohta H and Tabata S.
polyamines. P roc Natl Acad Sci USA. 87: 2851-2855, Monitoring of methyl jasmonate-responsive genes in
1990. Arabidopsis by cDNA macroarray: self-activation of
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(abstract no. 1552). Plant Physiol. 114: S-297, 1997. 161, 2001.
Mirza JI and Iqbal M. Spermine-resistant mutants of Schaffer R, Langraf J, Pérez-Amador MA and Wisman E.
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Plant Growth Regul. 22:151-156, 1997. Opin Biotechnol. 11: 162-167, 2000.
Montague MJ, Koppenbrink JW and Jaworski EG. Schena M, Shalon D, Davis RW and Brown PO. Quantitative
Polyamine metabolism in embryogenic cells of Daucus monitoring of gene expression patterns with a
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diamines and polyamines on the senescence of plant Slocum RD, Kaur-Sawhney R and Galston AW. The
suspension cultures. Plant Cell Rep. 2: 82-84, 1983. physiology and biochemistry of polyamines in plants.
Neubauer G, Gottschalk A, Fabrizio P, Seraphin B, Arch Biochem Biophys. 325: 283-303, 1984.
Luhrmann R, and Mann M. Identification of the proteins Slocum RD and Galston AW. Changes in polyamine
of the yeast U1 small nuclear ribonucleoprotein complex biosynthesis associated with post-fertilization growh and
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Ozturk ZN, Talame V, Deyholos M, Michalowski CB, Slocum RD and Galston AW. Inhibition of polyamine
Galbraith DW, Gomukirmizi N, Tuberosa R and biosynthesis in plants and plant pathogenic fungi. In:
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and Kumria R. Genetic engineering of polyamine and Sugumaran M, Nellaiappan K, Amaratunga C, Cardinale S
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12 Ravindar Kaur-Sawhney et al.
P h e n o l i c c y c l e i n p l a n t s a n d e n v i ro n m e n t
Valentine I. Kefeli1, Maria V. Kalevitch2* and Bruno Borsari3
1
Slippery Rock Watershed Coalition, 3016 Unionville Rd., Cranberry Twp., PA 16066, USA; 2Robert
Morris University, 881 Narrows Run Rd., Moon Township PA 15108, USA; 3Slippery Rock University,
101 Eisenberg Bldg., Slippery Rock PA 16057, USA (* author for correspondence)
Abstract
Phenolic substances are synthesized in plants and in the soil. They exist in the form of polymers and monomers. The
latter group of phenolics is assembled within the chloroplasts of plant cells, whereas soil phenolics are associated
with the process of humus formation on the alumino-silicate matrix of the soil micelle. As plants grow, phenolics
accumulate in cell vacuoles, or polymerize into lignin, which strengthens the secondary cell walls. In addition to this,
phenolics possess also some physiological functions as they regulate cell elongation. When they are excreted from
plant root systems they exert inhibitory growth function within adjacent rhizospheres. This work presents the latest
experimental evidence of phenolic synthesis and transformation in the environment, while providing an
understanding of their effect in plant-soil relations.
B i t k i l e r d e f e n o l i k d ö n g ü v e ç e v re
Özet
Fenolik maddeler bitkilerde ve toprakta sentezlenir. Bunlar polimerler ve monomerler fleklinde bulunurlar.
Fenoliklerin monomer grubu bitki hücresinin kloroplastlar›nda biraraya gelirken, toprak fenolikleri toprak
misellerinin alumino-silikat matriksi üzerinde humus oluflum olay› ile uyumluluk gösterir. Bitki büyürken hücre
vakuollerinde fenolikler birikir veya sekonder hücre çeperlerine sa¤laml›k kazand›ran ligninlere polimerize olurlar.
Bunlara ilave olarak fenolikler hücre uzamas›n› düzenleyerek baz› fizyolojik ifllevlere de sahiptirler. Bitki kök
sistemlerinden sal›nd›klar› zaman hemen yak›n›ndaki rizosferlerde büyümeyi inhibe edici etki meydana getirirler. Bu
çal›flma fenolik sentezlerinin en son deneysel verilerini ve çevredeki dönüflümlerini sunarken, bitki-toprak
iliflkilerindeki etkilerini anlamam›za yard›m etmektedir.
HO 3´
biosynthesis of the entire flavonoid structure within O O6H10O5
Isosalipurposide
plastids has not been explained, nor the complete 1 2´, 4´, 6´, 4-tetroxychalcone-
enzymatic package of their biosynthesis has been -2´-glucoside of chalconarin-
O H genin (chalcone)
discovered yet. Lack of direct eveidence of flavonoids HO 8 1 C
2´ 3´
1´ 4´ OH
7 2
transport within the cell and through the whole plant 6
5 4
3
CH2
6´ 5´
Hence, molecular genetics becomes a tool, which may excreted substances had an allelopathic nature and
help to regulate the level of secondary metabolites in were involved in developing ecological relationships
plants. Therefore, there is a need continue the search with adjacent plants of different species.
for botanical herbicides as a rise of ecological During the composting process water extracts
concerns has clearly identified the environmental contain many inhibiting substances that might form
impact of herbicides of synthesis. toxic exudates (Kefeli et al., 2001). Paper
Root exudates affect the germination of seeds of chromatography reveals the presence of phenolic acids
different crops: monocots and dicots (Table 1 and and coumarins in water extracts. The highest
Table 2). However, it must be pointed out that only concentrations of these inhibitors was measured in
some phenolics were studied in the exudates of willow abscised leaves of red maple (Acer rubrum L.). One
roots (1) which have no analogues in the roots (2) and gram of dry leaves was mixed in 29 ml of water to
leaves found among the common allelopathogens. prepare the extracts. The pH of the solution was
Although some of these substances could be retained between 5.4 and 5.6 and the extracts were incubated
by willow roots, others where excreted into an external for a week at room temperature while the pH raised to
medium. Chromatography of these water exudates and 7.2. Further observations revealed that during
a subsequent investigation of their chromatograms composting the amount of phenolics was drastically
with UV-B light showed that most of these substances reduced. Seed germination tests were performed with
are polyphenols such as coumarin, or phenolic acids. these water extracts and pure water (control) on lettuce
The phenolic substances retained by cells had different and wheat seeds. Germination rate and seedling
chemical properties than those located in the root lengths were measured to demonstrate that phenolics
exudates. Thus, the data confirm the hypothesis that decreased inhibiting properties after dilution, or after
Tap water 29 23 18 25
Spider plants (Chlorophytum) exudates 15 21 14 2
Willow (Salix vitaminalis) exudates 7 18 13.5 3.5
Table 2: Biological activity of willow root exudates after paper chromatography (Biological activity in % to control (water)).
Clover Lettuce
0 Blue 91 76 90 64
0.14 Blue 94 68 98 58
0.3 Violet 86 80 93 76
0.5 Blue 56 52 71 76
0.67 Yellow 87 68 89 88
0.88 Yellow 52 56 63 64
Phenolic cycle 17
S o i l - m i c ro b i a l c o m p l e x f o r p h e n o l i c d e c o m p o s i t i o n
is an effective medium usable to facilitate composting Biochem Physiol Pflanzen. 184: 363-369, 1989.
of maple and sumac leaves, contaning nature phenolic Yamamoto T, Yokotani-Tomita K, Kosemura S, Yamada K
compounds. and Hasegava K. Allelopathic substances exuded from a
serious weed. J Plant Growth Reg. 18: 65-67, 1999.
Conclusion
R e f e r ences
Abstract
The effects of LD25 (480 mg/kg.bw.) dose of citric acid, a food preservative, were investigated on body weight, organ
weights (liver, kidney, spleen), creatin kinase (CK), lactate dehydrogenase (LDH), alanine aminotransferase (ALT)
and aspartate aminotransferase (AST) enzymes in the blood serum, and the liver tissue of mice after 10 days. Citric
acid (to experimental groups) and physiological saline (to control groups) were given intraperitoneally. The results
of enzyme activities were evaluated using autoanalyzer as IU/L. Even though significant decreases in the body
weights were noted when compared to those of the control group (p<0.001), generally an insignificant increase were
observed in the organ weights (liver: p>0.05, kidney: p>0.05, spleen: p>0.05) and serum enzyme levels
(CK: p>0.05, LDH,: p>0.05, ALT: p>0.05, AST: p>0.05). Microscopical examination of the liver showed
histopathological changes depending on the citric acid. These changes were tissue degeneration, cytoplasmic
vacuolisations, nuclear membrane invaginations, picnotic nucleus and necrosis of the hepatocytes.
F a re l e r d e s i t r i k a s i d i n t e k t o k s i k d o z u n u n k › s a s ü re l i e t k i l e r i
Özet
Bir besin koruyucu olan sitrik asidin LD25 (480 mg/kg.va.) dozu farelere intraperitoneal yolla uyguland›. 10 gün
sonra hayvanlar›n vücut a¤›rl›klar›, organ a¤›rl›klar› (karaci¤er, böbrek, dalak), kreatin kinaz (CK), laktat
dehidrogenaz (LDH), alanin aminotransferaz (ALT) ve aspartat aminotransferaz (AST) enzimlerinin serum düzeyleri
ile, karaci¤er dokusu üzerinde sitrik asidin etkileri araflt›r›ld›. Otoanalizörde tayin edilen enzim aktiviteleri U/L
olarak de¤erlendirildi. Çal›flmada vücut a¤›rl›klar›nda kontrol grubuna k›yasla anlaml› bir azalma gözlenmesine
ra¤men (p<0.001), organ a¤›rl›klar›nda (karaci¤er: p>0.05 , böbrek: p>0.05, dalak: p>0.05) ve enzim aktivitelerinde
(CK: p>0.05, LDH: p>0.05, ALT: p>0.05, AST: p>0.05) anlaml› olmayan bir art›fl gözlendi. Karaci¤erin mikroskopik
incelenmesinde doku dejenerasyonu, sitoplazmik vakuolizasyon, nükleer zar çöküntüleri, piknotik nukleuslar ve
hepatositlerde nekroz gibi histopatolojik de¤ifliklikler gözlendi.
their economic importance, they can have negative physiological saline to control group mice. 10 days
effects on living organisms. Xenobiotics entering the after the injection, the mice were killed by cervical
organism are held by intestine, kidney and liver cells dislocation and then the necessary studies were
for detoxification. These cells contain important commenced. The livers, kidneys and spleens dissected
detoxification enzymes. During the detoxification of out, weighed, liver samples were seperated for
xenobiotics, free radicals are produced in microscopical examination. Blood samples were also
oxidation/reduction reactions, and these radicals can taken for enzyme assays. The serum levels of enzymes
have destructive effects on tissues. were determined using a Merck Mega 600
The toxic effects of many food preservatives on autoanalyser with the aid of Diasis Kits. Data were
living organisms have been studied by many analyzed by M.Whitney U test for multiple
researchers (Makoveç and Sindelar, 1984; Daniel, comparisons for the differences between the control
1986; Cabel et al., 1988; Kagan et al., 1990; Jung et and treated groups. For histological examination, liver
al.,1992; Nijhoff and Peters, 1992; Fujitani, 1993; samples were fixed with 10% buffered formalin,
Weemaes et al., 1997; Mc Farlene et al., 1997; Safer processed and stained hematoxylin-eosin.
and Nughamish, 1999; Kabo¤lu and Aktaç, 2002;
Aktaç et al., 2002). Although the citric acid and metal
salts (sodium or potassium citrat) are widely used in Results
food industry, there is no report on more detailed
effects of citric acid (or its salts) in liver. In addition, The effects of citric acid injection on the body weight
soft drinks, cosmetics and drugs, in which citric acid is and liver, kidney, spleen weights was shown Table 1
approved for use, are consumed by most of humans and 2. Although the liver, spleen and kidney weights
every day. were not changed significantly (p>0.05), the body
A way of analysing harmfull effects of foreign weights were decreased significantly (p<0.001). CK,
materials entered to organism is to determine the LDH, ALT and AST activities were not changed
effects of the chemicals on the enzymes. Enzymes statistically (p >0.05) as shown in Table 2. The results
have a very important role in the metabolical process of the microscopic investigation showed that liver
since they are biological catalysts. Thus, their abnormal of mice treated with citric acid has necrotic
serum levels indicates various diseases. Among these changes, compare to the control group (Figure 1-6).
enzymes are, creatine kinase (CK), lactate These changes were slightly degeneration of tissue
dehydrogenase (LDH), alanine aminotransferase (ALT) (Figure 2), cytoplasmic vacuolisation, nuclear
and aspartate aminotransferase (AST) which are the membran invaginations (Figure 3, 4) and picnotic nuclei
most important. Therefore, we studied short term (Figure 5). In addition, we observed degeneration of the
treatment of citric acid (10 days) in mice. In these blood vessel endothelium (Figure 6).
experiments, firstly we tested total body weigths,
organ weights (liver, kidney, spleen), and determined
the serum levels of creatin kinase (CK), lactate Discussion
dehydrogenase (LDH), alanin aminotransferase (ALT)
and aspartat aminotransferase (AST), and secondly the The effects of xenobiotics in living organisms can
liver tissue was investigated histopathologically. investigate in various ways. Among these are, short-
term toxicity tests which are used very commonly. In
these methods, many parameter are used to test the
Material and Methods effects of xenobiotics. Some of these parameters are
body weight, organ weights, blood profile, and
Male mice (Balb/C albino) weighing 25-30 g were histopathological examination. In this study, the short-
used in our experiments. Five mice were used control term effects of citric acid applied intraperitoneally
group and ten mice were used the citric acid-treated were investigated. It was reported that the body weight
group. Animals were fed by pellet baits and water. decreases in mouse (Würtzen, 1990), and in rats
LD25 dose (480 mg/kg.bw.) of citric acid (Merck; in (Nijhoff and Peters, 1992) by the effects of phenolic
physiological saline) were injected intraperitoneally to antioxidant butylated hydroxytoluene (BHT) and
experiment group mice, and the same amount of butylated hydroxyanisole (BHA) in chronic studies. In
Short-term effects of citric acid 21
F i g u re 1: The control group of the liver tissue, bar F i g u r e 2: Citric acid group. Distortion of general
representes 20 µm. histological structure of the liver, v: blood vessel, bar
representes 10 µm.
F i g u re 5: Citric acid group. Picnotic nuclei (arrows) in F i g u re 6: Citric acid group. Degenerated endothelium
hepatocytes, bar representes 10 µm. (arrows) of blood vessel (v), bar representes 10 µm.
contrast, any significant change was seen in body of sodium benzoate) for ten days by Fujitani (1993).
weight in F344 rats (0.2, 2.5 and 3.0 % of sodium Similarly, Kabo¤lu and Aktaç (2002) were determined
benzoate) and in B6C3F1 mice (1.81, 2.09 and 2.4 % that a significant decrease obtained at 3.0 and 4.0 % of
22 Tülin Aktaç et al.
Values are mean ± SD for ten mice of experiment group and five mice of control group.
(*) significant (p<0.001).
(**) not significant (p>0.05).
Table 2: Effects of citric acid on the organ weights and serum enzyme levels in mice.
Organ weight
Liver (g) 1.278 ± 0.085 1.257 ± 0.043 *
Kidney (g) 0.2260 ± 0.033 0.1910 ± 0.089 *
Spleen (g) 0.1620 ± 0.036 0.1250 ± 0.014 *
Serum enzyme levels
CK (IU/L) 572 ± 122 1050 ± 255 *
LDH (IU/L) 1296 ± 100 2245 ± 321 *
ALT (IU/L) 695 ± 6.84 101.0 ± 19 *
AST (IU/L) 177.8 ± 3.2 307.2 ± 46.6*
Abbrevations : CK = creatin kinase; LDH = lactate dehydrogenase; ALT = alanine aminotransferase; AST = aspartate aminotransferase.
Values are mean ± SD for ten mice of experiment group and five mice of control group.
(*) not significant (p>0.05).
sodium benzoate. Also, at the present study we not significant to compare with the control. These
determined a significant decrease of body weight in results were similar with findings obtained in F344 rats
mice by the effect of citric acid (Table 1). and B6C3F1 mice by Fujitani (1993).
Some autors have shown that food preservatives Although the organ weights and serum levels of
had increasing effects to organ weight. The effects of enzymes were not changed significantly, the
BHT and BHA on the increasing of the liver and examination by light microscopy revealed
thyroid weights were demonstrated in mice by pathological changes in liver of mice, such as
Würtzen (1990). Similarly, the effects of BHT on vacuolisation and glassy cytoplasm in the hepatocyte,
increasing of the liver weight in rats was also shown nuclear membrane invaginations, picnotic nuclei.
by Mc Farlene et al. (1997) and Safer and Nughamish Similarly, with the effect of sodium benzoate in the
(1999). Fujitani (1993) was also obtained significant rats and mice, high vacuolisation and glassy
increasing of the liver and kidney by the effects of appearance in hepatocyte cytoplasm was explained
sodium benzoate in male rats. In our previous studies, (Fujitani, 1993). Again, similar findings were obtained
increasing of the total liver weight were seen oral in the rats with oral treatment of BHT (Mc Farlene et
treatment of sodium benzoate (Kabo¤lu and Aktaç, al., 1997; Safer and Nughamish, 1999), and with
2002) and citric acid (Aktaç et al., 2002) but it was not sodium benzoate, benzoic acid and citric acid in mice
significant. Additionally, in the present study, we could (Kabo¤lu and Aktaç, 2002; Aktaç et al., 2002). The
not find any significant change the liver, kidney and results of present study suggested that citric acid has
spleen weights by the intraperitoneal injection of citric hepatotoxic effects and long term exposure may
acid (Table 2). According to our results, serum CK, induce severe damage in liver of mice. However, the
LDH, AST and ALT levels in the treated animals were mechanism of damaging effects of citric acid need to
Short-term effects of citric acid 23
R e f e r ences
Abstract
In this study, phenotypic characterization of RPP7 that confers resistance to Hiks1 isolate of Peronospora parasitica,
deficient mutant lines of Col-5 ecotype of Arabidopsis thaliana was investigated. The Col-5 plants that exposed to
Fast Neutron (FN) were inoculated with 8 different P. Parasitica isolates and symptom development was
investigated. A total of 4 mutant lines were analyzed. It was found that the RPP7 gene present in the Col-5 ecotype
is a unique gene different from the other RPP genes present in Col-5.
Arabidopsis thaliana ’ n › n C o l - 5 e k o t i p i n d e n e l d e e d i l e n m u t a n t h a t l a r d a n R P P 7 g e n i n i n
karakterizasyonu
Özet
Bu çal›flmada, Arabidopsis thaliana’n›n Col-5 ekotipinde bulunan ve Peronospora parasitica’n›n Hiks-1 izolat›na
karfl› dayan›kl›l›¤› sa¤layan RPP7 geninde mutasyon içeren hatlar›n fenotipik olarak belirlenmesi üzerinde
araflt›rmalar gerçeklefltirilmifltir. Fast Nötron (FN) uygulamalar› ile mutasyon meydana getirilmifl Col-5
tohumlar›ndan geliflen bitkiler 8 farkl› P. parasitica izolat› ile inokule edilerek semptom geliflimleri incelenmifltir.
Toplam olarak 4 mutant hatta gerçeklefltirilen analizlerde, RPP7 geninin Col-5 ekotipinde bulunan ve farkl›
P. parasitica izolatlar›na karfl› dayan›kl›l›¤› sa¤layan genlerden ba¤›ms›z olarak fonksiyon gösteren bir gen oldu¤u
belirlenmifltir.
the working on genome analyses, growth regulation, with many isolates (Century et al., 1995; Aarts et al.,
hormons, flowering, disease resistance and 1998). Similarly, in (Ethylene Intensitive) mutant lines
embryogenesis. Arabidopsis and tomato were used to do have the ethylene synthesis. But it was found that
determine the mechanisms of disease resistance the P. syringae f.s.p. tomato resistance continued in
(Thomas et al., 1997; Botella et al., 1998). It is also the this mutant lines. This study showed that ethylene was
host of many pests which attacks to crop plants. Many not important for A. thaliana and bacteria relationships
genes that provides resistance to bacteria and fungi (Bent et al., 1994; Dong, 1998). Lsd (Lesions
disease have been isolated and characterized from A. Simulating Disease resistance response) and acd
thaliana (Dangl and Jones, 2001; Feys and Parker, (Accelerated Cell Death) mutant lines produce
2000). Hypersensitive Resistance (HR) like symptoms
Peronospora parasitica is a causal agent of mildew without a pathogen infection. These symptoms are
disease in the genus cabbage, turnip etc. of cruciferae formed by the influence of external factors like heat
family. R-genes that determines resistance to P. and light (Lam et al., 1999). So, it is accepted that lsd
parasitica (RPP) were isolated and characterized and acd loci are negative regulators for HR formation
(Holub and Beynon, 1997; Parker et al., 1997; Botelli (Dietrich et al., 1994). In general, the presence of
et al., 1998; McDowell et al., 1998; Bittner-Eddy et al., different resistance mechanisms in A. thaliana which
2000). The researches on RPP genes have shown that are directed by RPP genes was found by the
these genes are at the specific regions at certain places characterization of mutant lines (Glazebrook et al.,
of each chromosome called as “Major recognition 1997; McDowell et al., 2000).
complexes-MRC” (Can, 1997; Holub and Beynon, In this study, the mutant lines of the Col-5 ecotype
1997). of A. thaliana were characterized, to understand the
RPP7 gene is present in Col-5 ecotype and mechanisms of RPP7 gene that confers resistance to
recognized by the Hiks-1 isolate of P. parasitica. This Hiks-1 isolate of P. parasitica.
gene was placed onto the first chromosome between
the markers M421 and M213 by using the hybrid lines
of Col-5 and Nd1 ecotypes (Tor et al., 1994; Can et al., Material and methods
1995; Can, 1997). The Hiks1 isolate also recognizes
the RPP1 gene which is present in Nd-1 ecotype, and Plant and fungus material
has an epistatic effect on the RPP7 gene (Tor et al.,
1994). In this study, Col-5 ecotype of A. thaliana lines having
The mutant lines that lack the R-genes were MRC-B, MRC-C and MRC-H regions (Can, 1997;
studied in detail and has a wide area of interest such as Holub and Beynon, 1997) were used as wild type
molecular and classical genetics. However, in order to ecotype. The Fast Neutron (FN) applied mutant lines
study the relationships between A. thaliana and the were obtained commercially and the selections of
RPP genes and to investigate the genome mutant lines were performed by using the Hiks-1
organizations, some mutant lines were used (Parker et isolate of P. parasitica. The Hiks-1 isolate recognizes
al., 1996). The mutant lines lacking the RPP genes the RPP7 gene which is in the MRC-B region of wild
were obtained from Ws-O that contain RPP14 gene Col-5 ecotype, and 7 days after inoculation it induces
exhibiting resistance to No-Co2 isolate by using Ethyl a resistance which is defined with HR. Four mutant
Methane Sulfate (EMS). The lines were then used to lines were used in this study denoted as FN3922,
separate the RPP10 and RPP1 genes, which were FN3928, FN3929 and FN3930. The HR does not occur
allelic to RPP14 that is on the third chromosome. It in mutant lines, and the pathogen completes its life
was found that the WsEDS line was susceptible to all cycle by sexual and asexual sporulation.
P. parasitica isolates tested and that the WsEDS locus
was necessary for the function of the RPP genes Regeneration of Hiks-1 isolate from oospore
(Parker et al., 1996; Bittner-Eddy and Beynon, 2001; population
Falk et al., 1999). The npr (Non expressor of PR
protein) mutant lines of A. thaliana synthesize the The Hiks-1 isolate was regenerated by using oospore
proteins which are related with pathogenesis. So, population. To do this, the seeds of A. thaliana that
systemic resistance is not seen following inoculation were susceptible to the Hiks-1 isolate were sown into
Characterisation of RPP7 gene 27
little plastic pots containing 4:1:1 (torf: perlit: sand) of Microscopic analysis
mixture for 40-50 seeds each. The pots were irrigated
to wet the seeds and 1-2 x 105 oospore/ ml were added Fungal development in plant tissue was examined
to the pots. The containers were held at 4 °C for 1-2 under light and fluorescence microscope. The infected
weeks to break the dormancy. Following this, the leaves were taken and put absolute methanol for 5-6
containers were placed into the climated room at 18-20 hours followed by saturated chloral hydrate solution
°C, 10 h light and 14 hour dark period. Within 10-15 for 4-5 hours. Then, tissues were placed in 50 %
days following seed germination, some seedlings glycerol solution for microscopic analyses.
having sporulation was collected and placed into
eppendorf tubes containing 200 µl dH2O. The DNA analysis
eppendorf tubes were shaked gently to allow the
conidia to pass to water. The conidia suspension was Total plant genomic DNA was isolated with some
used to inoculate 7 days old seedlings of EBH3529 and modifications by using the methods of Ausubel et al.,
Ksk-1 ecotypes, and the plants were placed into the (1994). Five to eight grams of plant material was
climate room. By this way, the regeneration of the grounded in N2 and transferred to the tubes containing
Hiks-1 isolate was done by subculturing 3-4 times. The 15 ml buffers (100 mM Tris-HCl, 50 mM EDTA, 500
conidia were stored at –20 °C and were used when mM NaCl, 10 mM Mercaptoethanol, %25 SDS) with
needed. 100 mg/lt proteinase K. The solution was kept at 55 °C
The same procedure were applied for, Ahco-1, for 1 hour. At the end of this time period, 5 ml of 5 M
Ahco-2, Ahco-7, Wand-1, Cand-5, Hind-2 and Hind-4 potassium acetate was added and held in ice for 20
isolates using Nd-1 and Col-5 ecotypes (Can, 1997). minutes, and the solution was centrifuged at 17000
rpm for 25 minutes. The supernatant was mixed with
Characterization of P. parasitica isolates by using 0,6 volume of isopropanol and held at -20 °C for
different A. thaliana ecotypes minutes and the DNA was precipitated. Phenol-
chloroform was used to wash the DNA and a second
Regenerated P. parasitica isolates were inoculated into precipitation was done. The DNA was dissolved in
Col-5, Ksk-1, Nd-1, Ws-3, Tsu-1, Ler-1, Oy-1 and dH2O and stored at -20 °C. The isolated DNA was
Wei-1 ecotypes in order to do phenotypic diluted in such to 50-100 ng/µl to use in polimerase
characterization. The A. thaliana ecotypes were chain reactions (PCR). For PCR reactions, the closest
obtained from Dr. Eric Holub (HRI- UK) marker to the RPP7 gene was used (Can, 1997). To do
The conidia suspension was adjusted to 4-5 x 104 this, the solution which contains 0.05 mm primer, 2
conidia/ml concentration for plant inoculations. The mm dNTPs, 25 mm MgCl2, 1 x Taq buffer and IU Taq
cotyledons of 7-8 days of the A. thaliana ecotypes DNA polymerase was completed to 25 ml volume. The
were inoculated in such a way that it would be one PCR reactions were performed at 94 °C for 5 min
drop to each cotyledon. The plants were placed into followed by 94 °C for 1 minute, 56 °C for 1 min, 72 °C
climated room with 18-20 °C, 10 h light, 14 h darkness for 13 minute (35 cycles) and 72 °C for 10 min. The
conditions after the inoculation and the plants were samples were electrophoresed at 80 W for 4 hours.
checked at the end of 3. and 7. days. The evaluation
was done regarding the pathogen sporulation and
hypersensitive reaction types (the interaction Results and discussions
phenotypes). Phenotypic reactions were examined
under the fine group as; pitting with no pathogen Characterization of P. parasitica isolates
sporulation (PN), flecking with no pathogen
sporulation (FN), flecks with delate and moderate In order to determine the changes at the RPP7 locus in
pathogen sporulation, 1-20 sporangiophorus per each the mutant lines, Ahco-1, Ahco-2, Ahco-7, Wand-1,
cotyledon (DM), flecking with delate pathogen Cand-5, Hind-2 and Hind-4 isolates were used. Ahco-
sporulation, 5-10 sporangiophorus per each cotyledon 1, Ahco-2, Ahco-7 recognize MRC-B region which is
(FDL), early and heavy pathogen sporulation, 20> located at the first chromosome in the Nd-1 ecotype
sporangiophores per cotyledon (EH), (Holub et al., (Can, 1997), and these isolates were presumed to
1994). recognize RPP7 allele of Nd-1 ecotype. Wand-1,
28 Canan Can et al.
Hiks-1 FN EH PN PN FN DM FDL
Ahco-1 DM FN FDL FN FDL FN FDL
Ahco-2 DM FN FDL FN FN FN DM
Ahco-7 DM FN FDL FN FDL FN EH
Wand-1 FN FN EH FN FN FN DM
Cand-5 FN EH EH FN CN FDL DL
Hind-2 FN FN EH PN FN FN EH
Hind-4 FR FN EH PN EH FN EH
*Necrotic pits (PN), necrotic flecks (FN), cavities (CN), flecks with delate and moderate pathogen sporulation, 1-20
sporangiophorus per each cotyledon (DM), flecking with delate pathogen sporulation, 5-10 sporangiophorus per each cotyledon
(FDL), early and heavy pathogen sporulation, 20> sporangiophores per cotyledon (EH).
Cand-5, Hind-2 and Hind-4 isolates recognize MRC-B Phenotypic characterization of mutant lines
and MRC-C region which present at the second
chromosome in the Col-5 ecotype. The isolates P. parasitica isolates were used to inoculate the Col-5
regenerated from the oospore populations were lines. The results were shown in Table 2.
inoculated on different A. thaliana ecotypes (Col-5, As indicated in Table 1, DM and EH phenotypes
Ksk-1, Nd-1, Ws-3, Ler-1, Oy-1 and Wei-1) to developed, following inoculation of FN3922, FN3928,
determine if they were original. The results are shown FN3929 and FN3930 mutant lines with the Hiks-1
in Table1. isolate. These results revealed that the RPP7 gene is
As it could be seen in Table 1, the isolates not present in the mutant lines. However, Ahco-1,
generated from the oospore populations were found to Ahco-2 and Ahco-7 isolates exhibited the EH
be as original, and there was no variation (Can, 1997). phenotype compared to DM in the wild Col-5 ecotype.
Therefore these isolates were used to inoculate the This result showed that absence of the RPP7 gene
mutant lines recovered through inoculation with the increased susceptibility. The important point in here
Hiks-1 isolate. was that mutation of one R-gene could effect the
resistance in same plant to other isolate. The result
Table 2: Interaction phenotypes exhibited by Col-5 mutant lines following inoculation with different P. parasitica isolates.
Hiks-1 FN PN EH EH DM EH EH
Ahco-1 DM FDL FN EH EH EH EH
Ahco-2 DM FDL FN EH EH EH EH
Ahco-7 DM FDL FN EH EH EH EH
Wand-1 FN EH FN EH FN FN FDL
Cand-5 FN EH EH EH FN FN FN
Hind-2 FN EH FN FN FN FN FN
Hind-4 FR EH FN EH FN FN FN
*Necrotic pits (PN), necrotic flecks (FN), flecks with moderate and late pathogen sporulation, 1-20 sporangiophorus per each
cotyledon (DM), flecking with delate pathogen sporulation, 5-10 sporangiophorus per each cotyledon (FDL), early and heavy
pathogen sporulation, 20> sporangiophores per cotyledon (EH).
Characterisation of RPP7 gene 29
Abstract
Meta-topolin (mT) has been established as an active aromatic cytokinin recently. The present investigation assessed
the effects of mT on radish cotyledon growth and protein content. 0.05 to 1 mM mT increased the cotyledon growth
about 2 fold in fresh weight basis. mT at 0.1, 0.25 and 0.5 mM concentrations caused an increase in soluble protein
levels compared to the control cotyledons almost in the same ratio by 3 %. Compared to control cotyledons analysis
of the soluble proteins displayed different electrophoretic pattern in mT treated cotyledons.
Son y›llarda meta-topolin (mT) aktif aromatik sitokinin olarak saptand›. Bu araflt›rma da mT’in turp kotiledonlar›n›n
büyüme ve protein içeri¤ine etkisi araflt›r›ld›. 0.05-1 mM mT kotiledon büyümesini taze a¤›rl›k baz›nda yaklafl›k 2
kat kadar teflvik etti. 0.05, 0.1 ve 0.25 mM mT çözünür protein düzeylerini kontrole oranla yaklafl›k % 3 oran›nda
artt›rd›. Çözünür proteinlerin analizleri, mT uygulanan kotiledonlarda kontrole oranla farkl› bir elektroforetik dizilim
gösterdi.
documented in radish cotyledons. A new active room temperature. The gels were diffusion-destained
aromatic cytokinin meta-topolin (mT) have been by repeated washing in the solution containing 7.5 %
determined by Strnad et al. (1997) in poplar. The acetic acide, 5 % methanole and 87.5 % distilled water.
sensitivity of the radish cotyledon bioassay to mT has
been established by us before (Palavan-Ünsal et al.,
2002). This study will focus on the effect of mT on Results and discussion
soluble protein contents in radish cotyledons that has
not been studied before. The early observations revealed that cytokinins exert
parallel effects in maintain protein or nucleic acid
levels while inhibiting senescence. Cytokinins
Material and methods stimulate both structural and enzymatic protein
synthesis. They are selectively increasing the levels
Plant material and bioassay of certain enzymes associated generally with
photosynthetic process (Feierabend, 1969). It is not
Radish (Raphanus sativus L.) seeds were germinated in clear whether the enhanced activity is due to greater
darkness for 4 days at 25 °C on moist filter paper in 5 synthesis, inhibition of degradation or activation of the
cm petri dishes. Cotyledons were excised excluding enzymes.
petiole tissues and four cotyledons were placed in each We already observed that new aromatic cytokinin
petri dish after measuring the fresh weight. The mT at 0.25 to 1 mM concentration range delayed
cotyledons were placed with their adaxial sides down the senescence in excised wheat leaf segments
on the paper. They were incubated in a growth chamber (Palavan et al., 2002). This concentration range was
at 25°C ± 2°C and 12 h light-dark photoperiods. Three high for radish cotyledon growth therefore lower
ml mT was applied per petri dish at 0.05, 0.1, 0.25, 0.5 concentrations were examined (0.05 to 1 mM) in
and 1.0 mM concentrations. Cotyledon growth was addition.
measured by determining fresh and dry weights 3 days Cotyledon growth increased with the treatments of
after the application (Letham, 1971) and the data mT significantly (Figure 1). Stimulation of cotyledon
presented here representative of 15 experiments. growth was closely related with increasing
concentrations of mT; 0.05 to 1 mM mT increased the
Measurement of soluble protein content cotyledon growth about two fold in fresh weight basis
(p<0.05), while dry weights of cotyledons during the
Soluble protein content was determined as in Bradford growth were not effected by mT application (Palavan-
(1976) using bovine serum albumin as standard. Each Ünsal et al., 2002).
experiment was repeated four times and each treatment Cytokinins promote cell enlargement in certain
included three replicates. tissues and organs. This effect is most clearly seen in
cotyledons. The expansion of cotyledons is resulted
Electrophoresis for proteins
Acknowledgement
R e f e r ences
Abstract
Many recent studies have focused on the investigation of the biological effects of electromagnetic field. Although
the several types of biological effects of electromagnetic fields have been shown, the molecular mechanisms of these
effects have not been explained yet. Some epidemiological studies have suggested that exposure to ambient, low-
level 50-60 Hz electromagnetic fields increase risk of disease including cancer such as leukemia among children who
live close to power lines or among men whose jobs expose them to electromagnetic field, while others have
suggested that electromagnetic fields exposure could increase both the concentration of free radicals and oscillating
free radicals. Electromagnetic fields are known to affect radical pair recombination and they may increase the
concentration of oxygen free radicals in living cells. In this study, oxidative stress was formed by the oxidation of
ascorbic acid and the effect of 50 Hz, 0.3 mT electromagnetic fields on the oxidative DNA damage has been
investigated. The results of the study showed that extremely low-frequency electromagnetic fields enhanced the
effect of oxidative stress on DNA damage and supported the idea obtained from the previous studies on an increasing
effect of electromagnetic fields on the concentration and the life-time of free radicals.
Key words: Electromagnetic fields, DNA damage, ascorbic acid, vitamin C, oxidative stress
E l e k t ro m a n y e t i k a l a n › n o k s i d a t i f D N A h a s a r › ü z e r i n d e k i e t k i s i
Özet
Günümüzdeki birçok çal›flma, elektromanyetik alan›n biyolojik etkilerinin araflt›r›lmas› üzerinde odaklanm›flt›r.
Elektromanyetik alan›n biyolojik etkilerinin baz› türlerinin gösterilmifl olmas›na ra¤men, bu etkilerin moleküler
mekanizmalar› henüz aç›klanamam›flt›r. Baz› epidemiyolojik çal›flmalar, 50-60 Hz dolay›ndaki düflük düzeyli
elektromanyetik alana maruz kalman›n yüksek gerilim hatlar›na yak›n yaflamakta olan çocuklarda veya
elektromanyetik alana maruz kalarak çal›flanlarda görülen lösemi gibi kanser vakalar›n› kapsayan hastal›klara iliflkin
riski art›rd›¤›n› öne sürerken, baz› çal›flmalar ise elektromanyetik alan maruziyetinin serbest radikal
konsantrasyonunu ve serbest radikallerin izlenebilirli¤ini art›rabilece¤ini ileri sürmüfltür. Elektromanyetik alan›n
radikal çifti rekombinasyonunu etkiledi¤i bilinmektedir ve bu da, hücrelerdeki oksijene dayal› serbest radikal
konsantrasyonunu art›rabilir. Bu çal›flmada, askorbik asit oksidasyonu ile oksidatif stres oluflturulmufl ve 50 Hz, 0.3
mT düzeyindeki elektromanyetik alan›n, oksidatif DNA hasar› üzerindeki etkisi araflt›r›lm›flt›r. Bu çal›flman›n
sonuçlar›, oldukça düflük frekansl› elektromanyetik alan›n, oksidatif stresin DNA hasar› üzerindeki etkisini art›rd›¤›n›
göstermifl ve önceki araflt›rmalardan elde edilen, elektromanyetik alan›n serbest radikal konsantrasyonu ve yar› ömrü
üzerindeki art›r›c› etkisine dair düflünceleri desteklemifltir.
Anahtar sözcükler: Elektromanyetik alan, DNA hasar›, askorbik asit, C vitamini, oksidatif stres
36 Serkan ‹fller and Günhan Erdem
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
F i g u re 1: The effect of EMF and Cu(II) concentrations on F i g u re 2: The effect of incubation time on oxidative DNA
oxidative DNA damage. All lanes include 0.5 µg DNA. The damage depends in electromagnetic fields. All lanes include
DNA samples in the lanes from 1 to 6 were incubated under 0.5 µg DNA. Incubation times were 30 min from 1 to 4, 20
normal condition, 7 to 12 were incubated in EMF at room min from 5 to 8 and 10 min from 9 to 12. Ascorbic acid
temperature in the presence of 0.25 mM ascorbic acid concentration were 0.25 mM in all lanes. Cu(II)
except the 1 and 7 which were control lanes. Cu(II) concentrations were 2.5 µM in 1, 3, 5, 7, 9 and 11 while
concentrations were 1.25 µM in 2 and 8, 2.5 µM in 3 and 9, 5 µM in 2, 4, 6, 8, 10 and 12. The samples in 1, 2, 5, 6, 9 and
5 µM in 4 and 10, 7.5 µM in 5 and 11, 10 µM in 6 and 12 10 were incubated in EMF. Other samples were incubated at
lanes. Incubation time was 30 min for all samples. normal conditions.
chelating cupric ions and eliminated their oxidative In the presence of EDTA as a cationic metal
effects). chelator, oxidative DNA damage was not observed.
The results of this study showed that the oxidative This result showed that ascorbate oxidation and
DNA damage depends on the incubation time (Figure 2). oxidative DNA damage depend on cupric ions as an
DNA breakages could be observed at the 20th minute of oxidizing agent (Figure 3, lanes 6 and 12). As an
incubation time (Figure 2, lanes 6 and 8). EMF antioxidant, cystein did not block the oxidative DNA
exposure enhances the oxidative DNA damage after damage (Figure 3, lanes 4 and 10). Glutathione
the 20th minute (Figure 2, lanes 2 and 4). reduced the oxidative stress. Therefore, the DNA
damage was formed as aggregation rather than
fragmentation in the presence of glutathione (Figure 3,
1 2 3 4 5 6 7 8 9 10 11 12 lanes 3 and 9). Dithiotreitol (DTT) was the most
effective antioxidant of all investigated but EMF
exposure inhibited the effectiveness of DTT (Figure 3,
lanes 5 and 11).
The oxidative species produced by ascorbate
oxidation in the presence of copper(II) ions damage
the DNA molecules (Figure 1). Previously DNA
damage depending on ascorbate oxidation had been
studied (Erdem et al., 1994; Zareie et al., 1996).
Oxidative DNA damage was observed as
F i g u re 3: The effect of some antioxidants (glutathione, fragmentation or aggregation. The degree of oxidative
cystein, dithiothreitol) and metal chelator (EDTA) on
DNA damage varies in the levels and reactivity of free
excessive oxidative DNA damage in EMF. All lanes include
radicals produced in the reaction medium. In the
0.5 µg DNA. Ascorbic acid and Cu(II) concentrations were
0.25 mM and 7.5 µM in all lanes except the control DNA presence of oxygen, the hydroxyl and peroxyl radicals
lanes 1 and 7, respectively. Lanes 2 and 8 were scission such as superoxide anion and hydroperoxyl radical are
controls. Glutathione (3 and 9), cystein (4 and 10), produced by the reaction between radical form of
dithiothreitol (5 and 11) and EDTA (6 and 12) ascorbic acid (ascorbyl radical) and molecular oxygen
concentrations were 0.5 mM. The samples in 1 to 6 were (Fuchs et al., 1990).
incubated at normal condition. The others were incubated in These radicals attack to electrophilic nuclei on the
EMF. Incubation time was 30 min for all samples. targets and create secondary carbon radicals. At the
38 Serkan ‹fller and Günhan Erdem
C h ro m o s o m e s o f a b a l a n c e d t r a n s l o c a t i o n c a s e e v a l u a t e d w i t h
a t o m i c f o r c e m i c r oscopy
Zerrin Y›lmaz1*, Mehmet Ali Ergun2, Erdal Tan3
1
Department of Medical Biology and Genetics, Baskent University, Faculty of Medicine, 06570,
Maltepe, Ankara, Turkey; 2Department of Medical Biology and Genetics, Gazi University, Faculty of
Medicine, 06510, Besevler, Ankara, Turkey; 3Materials Research Department, Ankara Nuclear Research
and Training Center, 06100, Besevler, Ankara, Turkey (*author for correspondence)
Abstract
A couple was referred to our genetics department for cytogenetic analysis because of two previous abortions. The
cytogenetic analysis of the male was found as 46, XY and the female revealed a balanced translocation; 46, XX,t
(7;12) (p21;q14) and also she had 14 cenh+ as her mother. Atomic force microscopy (AFM) is a useful method for
detecting detailed structures of chromosomes. With the help of this new technique the surface topography of human
chromosomes can be examined. We used AFM in order to analyse the surface topography of derivative chromosomes
of the patients, and found a 0.6 µm gap region. In this study, we aimed to examine the differences between the images
of the derivative chromosomes detected by light and atomic force microscopy analyses.
D e n g e l i t r a n s l o k a s y o n v a k a s › n d a k ro m o z o m l a r › n a t o m i k g ü ç m i k roskobu ile
de¤erlendirilmesi
Özet
Ardarda iki gebelik kayb› nedeniyle departman›m›za yönlendirilen çiftin sitogenetik analizleri yap›lm›flt›r. Erkekte
normal kromozom kuruluflu 46, XY saptanm›fl ancak kad›nda dengeli translokasyonla birlikte
14. kromozoma ait sentromer art›fl› saptanm›flt›r; 46, XX, t (7;12) (p21;q14), 14 cenh+. Proband›n ailesinde yap›lan
sitogenetik çal›flma ile ayn› kromozom kuruluflunun proband›n annesinden kal›t›ld›¤› saptanm›flt›r. Atomik güç
mikroskobu kromozomlar›n yap›sal olarak detayl› incelenmesinde kullan›lmaktad›r. Bu yeni tekni¤in yard›m›yla
insan kromozomlar›n›n yüzey topografisi incelenebilmektedir. Biz de atomik güç mikroskobunu kullanarak derivatif
kromozomun yüzey topografisini araflt›rd›k ve 0.6 µm’lik bir gap bölgesi saptad›k. Bu çal›flmada probanda ait
derivatif kromozom yap›s›n› hem ›fl›k mikroskobu hem de atomik güç mikroskobu ile ayr› ayr› de¤erlendirerek
sonuçlar›m›z› karfl›laflt›rd›k.
uniform unbanded appearance to chromosomes are genitourinary, endocrinological and other organ
referred to solid or covential staining. They can, systems; also laboratory findings were normal.
however, be useful for studies on chromosome
breakage as scoring gaps and breaks can be difficult in Light microscopy analysis
lightly stained chromosome bands. Giemsa banding
(G-banding) has become the most widely used Metaphase chromosome preparation was obtained
technique for the routine staining of human from peripheral blood lymphocytes using standard
chromosomes. The chromosome banding patterns techniques (Verma and Babu, 1995). Conventional
obtained reflect both the structural and functional cytogenetic analysis was carried out using GTG-
composition of chromosomes. Consititutive banding and C-banding techniques (Benn and Perle,
heterochromatin is the structural chromosomal 1992). The chromosome images were captured by
material seen as dark staining material in interphase as computer imaging (Cytovision system, Image
well as during mitosis. It includes both repetetive analysis, Applied Imaging, Saunderland, UK).
DNA, satellit DNA and some non-repetetive DNA. For each patient we analysed 20 metaphases, and
C-banding can be used to demonstrate the repetetive C-banding procedure was performed while
DNA (Benn and Perle, 1992). investigating 14 cenh+.
Atomic force microscopy (AFM) is a diagnostic
tool for detecting detailed structures of the Atomic force microscopy and analysis
chromosomes and the surface topography of human
chromosomes can be examined using this new The AFM used in this study was TopoMetrix
technique (Binning et al., 1986; Musio et al., 1997). TMX2000 Explorer, operating in contact mode and air.
AFM could be considered as a tool for further Throughout the surface analysis, we have used
chromosomal studies. standard pyramidal tip (1520-00) with the radius of
In our previous studies using AFM, we showed curvature of approximately 1000 A°. During the
that, unbanded human metaphase chromosomes surface analysis, the metaphase region was primarily
displayed a banding pattern similar to G-bands, and for determined and addressed by light microscopy. Later,
the first time we have provided an AFM imaging of the region under consideration was scanned via AFM
chromosomes in trisomy 13, 21 and Klinefelter at various scan ranges changing from 150 µm down to
Syndrome patients (Ergun et al., 1999). Besides, G and 10 µm or less to image the chromosomes in a good
C-banding patterns of chromosomes were also manner. The applied force and the image resolution
investigated (Sahin et al., 2000; Tan et al., 2001). were between 1 and 3 nN and 400x400 pixels (or
In this study, we used AFM in order to analyse the higher) respectively for each image acquisition. The
surface topography of derivative chromosomes of a raw data gathered were analysed by using the software
female patient whose daughter was referred to our of the microscopy system in two or three-dimensional
Genetics department with the chief complaints of patterns.
abortions. In our study, the chromosomes of the patient were
spread on glass surface. Then, the metaphase spreads
were analysed by AFM. Line measure analysis was
Materials and methods performed on derivative chromosomes.
Case presentation
Results
In this study we evaluated the chromosomes of a
family. This family was referred to our genetics The karyotype of the male revealed 46, XY, while the
department for cytogenetic analysis because of two cytogenetic analysis of his wife was karyotyped as
previous abortions during the first trimester. They had 46, XX, t (7;12) (p 21;q 14); a balanced translocation.
no live-born children after a marriage of 5 years. The She also had 14cenh+. In order to understand the origin
male was 37 years old and healthy, and his non- of this translocation chromosome, her mother was
consanguineous wife was 36 years old. Her physical karyotyped and she was also found to be a translocation
examination revealed no abnormalities in carrier; 46, XX, t (7;12) (p 21; q14) and 14 cenh+.
AFM in chromosome evaluation 41
Discussion
repetitive regions that are located on the centromeres Uehara S, Sasaki H, Takabayashi T and Yajima A. Structural
of chromosomes 1, 9, and 16 and on the distal arm of aberrations of metaphase derivative chromosomes from
Y chromosome (Burkholder and Duczek, 1980; Cook, reciprocal translocations as revealed by scanning
1995). Our AFM image also helps us to understand electron microscopy. Cytogenet Cell Genet. 74: 76-79,
1996.
that these regions were not belonging to G- banding
Verma RS and Babu A Banding techniques. In: Human
regions, as there was not a banding pattern (Tan et al., Chromosomes Principles and Techniques. Verma RS and
2001). Babu A (Ed). McGraw-Hill Inc. New York. 72-133,1995.
AFM can be considered as a novel technique for
analysing detailed structures of chromosomes for its
line measure analysis and 3-D image capture
capabilities. Reflecting these capabilities, AFM helped
us to investigate the gap region on the derivative
chromosome and this study is also novel by making
new implementations on the mechanism of
translocation. As a conclusion, the capability of AFM
for detecting chromosomal abnormalities will reflect
light into further studies.
R e f e r ences
E f f e c t o f e p i r u b i c i n o n m i t o t i c i n d e x i n c u l t u r ed L-cells
Gül Özcan Ar›can* and Mehmet Topçul
‹stanbul University, Faculty of Science, Department of Biology, 34459 Vezneciler, ‹stanbul, Turkey
(*author for correspondence)
Abstract
Cancer chemotherapy is an additional application to surgical operations and radiotherapy in the treatment of
widespread tumors. An anthracycline-derived antibiotic, epirubicin (EPI) is one of the clinically used antineoplastic
drugs. In this study the cytotoxic effects of EPI in transformed mouse fibroblasts (L-cell) were examined. EPI
concentrations of 0.001 µg/ml, 0.01 µg/ml and 0.1 µg/ml were applied to the cells for 2, 4, 8, 16 and 32 hours. The
results showed that EPI diminished mitotic index of L-cells depends upon time and applied concentrations. This
decrease was found statistically significant in each treatment group when compared to control (p<0.05 - p<0.001).
E p i r u b i s i n i n k ü l t ü r d e k i L - h ü c re l e r i n d e m i t o t i k i n d e k s e e t k i s i
Özet
Kanser kemoterapisi, yayg›n tümörlerin tedavisinde cerrahi uygulama ve radyoterapinin yan›nda gerçeklefltirilen ek
bir uygulamad›r. Antrasiklin türevi bir antibiyotik olan epirubisin (EPI) klinik olarak kullan›lan antineoplastik
ilaçlardan birisidir. Bu çal›flmada, EPI in sitotoksik etkileri, transforme edilmifl fare fibroblastlar›nda (L-hücreleri)
araflt›r›ld›. EPI in 0.001 µg/ml, 0.01 µg/ml ve 0.1 µg/ml konsantrasyonlar› 2, 4, 8, 16 ve 32 saat süresince hücrelere
uyguland›. Sonuçlar uygulanan zaman ve konsantrasyona ba¤l› olarak EPI in L-hücrelerinin mitotik indeks
de¤erlerini düflürdü¤ünü gösterdi. Bu düflüfl kontrol grubu ile karfl›laflt›r›ld›¤›nda, her bir deney grubunda istatistik
olarak anlaml› bulundu (p<0.05 - p<0.001).
Anahtar sözcükler: Epirubisin, L-hücreleri, transforme edilmifl fibroblast, mitotik indeks, in vitro
Mitotic index were studied by the methods of Feulgen. Mitotic index values which obtained from experiments
Before the cells were treated with Feulgen, they were were calculated to evaluate the statistical analysis. The
prepared with 1 N HCl at room temperature for 1 differences between the percentage distrubition of M
minute and then hydrolized with 1 N HCl for 10.5 phase of the various treatment groups and control were
minutes at 60°C. After slides were treated with compared by the Student-t test (n=25).
Feulgen, they were rinsed for few minutes in distilled
water and stained with 10% Giemsa stain solution pH
6.8, for 3 minutes and washed twice in phosphate Results
Table 1: Mitotic index values in cultures of L-cells treated with various concentrations of EPI, given in mean ± Standard devia-
tion (SD).
Control 1.44 ± 0.12 SD 1.93 ± 0.14 3.04 ± 0.08 3.39 ± 0.15 3.84 ± 0.21
0.001 µg/ml 1.35 ± 0.09 a 1.80 ± 0.10 a 2.72 ± 0.07 a 2.96 ± 0.04 b 3.10 ± 0.30 b
0.01 µg/ml 1.29 ± 0.11 a 1.79 ± 0.05 b 2.61 ± 0.06 b 2.70 ± 0.13 b 2.99 ± 0.16 b
0.1 µg/ml 0.94 ± 0.02 c 1.04 ± 0.01 c 1.77 ± 0.09 c 1.85 ± 0.08 c 1.02 ± 0.22 c
a
: p < 0.05, b: p < 0.01, c: p < 0.001
46 Gül Özcan Ar›can and Mehmet Topçul
EPI significantly decreased the mitotic index in Although, in vitro studies with antitumour agents,
cultures of L-cells. The results show that EPI and with anthracyclines in particular, have not shown
decreased the mitotic index at significant level p<0.05 to predict the antitumour activity in vivo (Sinha and
- p<0.01 for lower drug concentrations 0.01 µg/ml and Politi, 1990; Nistico et al., 1999), in some studies,
0.001 µg/ml, at highly significant level p< 0.001 for significant correlations have been detected between the
0.1 µg/ml drug concentration when compared with the in vitro activity of EPI and other anthracyclines against
control. various tumour specimens, and therapeutic response
In addition, the reductions in mitosis of the cells (Bartkowiak et al., 1992; Plosker and Faulds, 1993).
following different treatment times (2, 4, 8, 16 and 32 Anthracyclines, including EPI, appear to result in
hours) with 0.1 µg/ml EPI concentration were maximal cell death in the S and G2 phases of the cell
statistically significant (p<0.001) from each other. cycle, but cytotoxic effects may occur in the G1 and M
However, this level of significance for the different phases at higher drug concentrations (Plosker and
treatment times was not observed with 0.01 µg/ml and Faulds, 1993; Topçul et al., 2002). Maximal lethal
0.001 µg/ml concentrations of EPI, respectively. effects of EPI were demonstrated in the S and G2
phases of the cell cycle in murine and human tumour
cell lines (Hill and Whelan, 1982).
Discussion An important comparison between EPI and
doxorubicin in vitro was carried out by Hill and
Anthracycline antibiotics have been used extensively Whelan (1982). The studies were performed on a wide
in the treatment of wide variety of malignancies, and range of murine and human cell lines: NIL8 (Syrian
are a standard component of many combination golden hamster cells); four human tumor lines (COLO-
chemotherapy regimens. EPI has been used alone or in 206 and LOVO, derived from colon carcinomas; SCC-
combination with other antineoplastic agents in the T/G, derived from a squamous cell carcinoma from the
treatment of a broad range of neoplasms. Studies in tongue; CHP 100, derived from a neuroblastoma);
understanding the mechanism of EPI have suggested L5178Y lymphoblastoid cell sub-lines. In the all cell
that EPI forms a complex with DNA by intercalation lines tested, both drugs showed comparable
between DNA strands, thus inhibiting DNA replication cytotoxicity, which increased exponentially with drug
and transcription (Özcan et al., 1997; Stewart et al., concentration and with duration of exposure. Of high
1997), and it increases DNA strand brekage (Cantoni et interest was the study carried out on NIL8 cells
al., 1990). synchronized by mitotic selection and treated for 1
EPI induced differentiation of human hour with the drug. Results showed that maximum cell
neuroblastoma cells in vitro, possibly related to a kill was observed with both drug in the S phase, some
reduction in the growth of surviving cells, thus kill during early G2, but no kill in G1 and M if low
allowing activation of intrinsic differentiation concentrations were used. Data from flow
mechanisms. Following culture of human microfluorimetry analyses and monitoring of mitotic
neuroblastoma cell lines with EPI 10 or 100 nmol/L for indices suggested population arrest in G2 for
24 hours, outgrowth of neurite processes was doxorubicin (Casazza and Giuliani, 1984).
detectable 3 or 4 days after exposure, and maximal In the present study, treating L-cells with various
morphological differentiation was achieved after 5 or 6 concentrations of EPI for 2, 4, 8, 16 and 32 hours,
days (Rocchi et al., 1987). decreased mitosis. The results showed that EPI
EPI has also been shown to be effective in diminished mitotic index of L-cells, depending upon
inhibiting basement membrane degradation, a property time and applied concentrations. When compared to
deemed necessary to prevent development of the control, this decrease was found statistically
metastases (Plosker and Faulds, 1993). In addition, EPI significant in each group (p<0.05-p<0.001). The values
has been shown to inhibit proliferation of all of mitotic index reached a minimum at EPI
neuroblastoma cell lines by 69 to 78 % relative to concentration of 0.1 µg/ml with increasing treatment
controls (Rocchi et al., 1987), of a human alveolar time. Increased concentrations resulted in a decrease
rhabdomyosarcoma cell clone (Lollini et al., 1989), on the values of mitotic index, being statistically
and of haemopoietic progenitor cells from several significant (p<0.001). Briefly, EPI concentration of 0.1
human leukaemic cell lines in liquid culture (Bagnara µg/ml showed to possess relatively more effect on
et al., 1987). proliferation of L-cells. Thus, the results of our study
Epirubicin effect on mitotic index 47
seem to be concordant with the above mentioned Di marco A. Epirubicin: Mechanism of action at the cellular
studies suggesting that cytotoxic effects of EPI might level. In: Advances in Anthracycline Chemotherapy:
occur in the G1 and M phases at higher drug Epirubicin. Bonadonna G (Ed). Masson, Milano-Italy.
41-47, 1984.
concentration.
Earle WR. Production of malignancy in vitro. IV. The mouse
In our study, decreases in the mitotic index of cells fibroblast cultures and changes seen in the living cells.
with increasing both treatment time and EPI J Nat Cancer Inst. 4: 165-212, 1943.
concentration have confirmed that EPI is an effective El-Mahdy Sayed Othman O. Cytogenetic effect of the
inhibitor of mitosis. anticancer drug epirubicin on Chinese hamster cell line
In conclusion, the results of this study declared the in vitro. Mutation Res. 468: 109-115, 2000.
cell kinetics and cytotoxic effects of the anticancer Haldane A, Finlay GJ, Baguley BC. A comparison of the
drug, EPI, in treated cultures of L-cell line. Although effects of aphidicolin and other inhibitors on
EPI has less systemic and cardiac toxicity than topoisomerase II-directed cytotoxic drugs. Oncol Res.
5 (3): 133-138, 1993.
doxorubicin and other anthracyclines with an
Hill BT and Whelan RDH. A comparison of the lethal and
equivalent spectrum of antitumor action, it still has kinetic effects of doxorubicin and 4’-epidoxorubicin
cytotoxic effects. in vitro. Tumori. 68: 29-37, 1982.
Lollini PL, De Giovanni C, Del Re B. Myogenic
differentiation of human rhabdomyosarcoma cells
Acknowledgment induced in vitro by antineoplastic drugs. Cancer
Research. 49: 3631-3636, 1989.
We would like to thank Prof. Dr. Atilla Özalpan for his Nistico C, Garufic C, Barni S, Frontini L, Galla DA,
Giannaarelli D, Vaccaro A, Dottovio AM, Terzoli E.
kind help and critics.
Phase II study of epirubicin and vinorelbine eith
granulocyte colony-stimulating factor: A high-activity,
dose-dense weekly regimen for advanced breast cancer.
R e f e r ences Ann Oncol. 10 (8): 937-942, 1999.
Özcan G and R›dvano¤ullar› M. The effect of epirubicin on
Bagnara GP, Rocchi P, Bonsi L. The in vitro effects of the cell cycle of L-cells. 13th National Congress of
epirubicin on human normal and leukemic hemopoietic Biology. Istanbul, Turkey. 3: 267-276, 1996.
cells. Anticancer Research. 7: 1197-1200, 1987. Özcan FG, Topçul MR, Y›lmazer N, R›dvano¤ullar› M.
Bartkowiak D, Hemmer J, Rottinger E. Dose dependence of Effect of epirubicin on 3H-thymidine labelling index in
the cytokinetic and cytotoxic effects of epirubicin in cultured L-strain cells. J Exp Clin Cancer Res. 16(1): 23-
vitro. Cancer Chemother Pharmacol. 30 (3):189-192, 27, 1997.
1992. Plosker GL and Faulds D. Epirubicin. A review of its
Cantoni O, Sestili P, Cattabeni F. Comparative effects of pharmacodynamic and pharmacokinetic properties, and
doxorubicin and 4’-epidoxorubicin on nucleic acid therapeutic use in cancer chemotherapy. In: Drugs.
metabolism and cytotoxicity in a human tumor cell line. Chrisps P (Ed). 0012-6667. 45 (5): 788-856, 1993.
Cancer Chemother and Pharmacol. 27: 47-51, 1990. Robert J and Gianni L. Pharmacokinetics and metabolism of
Casazza AM and Giuliani FC. Preclinical properties of anthracyclines. Cancer Surv. 17: 219-252, 1993.
epirubicin. In: Advances in Anthracycline Chemotherapy: Rocchi P, Ferreri AM, Simone G. Epirubicin-induced
Epirubicin. Bonadonna G (Ed). Masson, Milano-Italy. differentiation of human neuroblastoma cells in vitro.
31-40, 1984. Anticancer Research.7: 247-250, 1987.
Chabner BA and Myers CE. Antitumor antibiotics, Sinha BK and Politi PM. Anthracyclines. Cancer chemother
In: Cancer: Principles and Practice of Oncology. De vita apy. Biol Response Modif. 11: 45-57, 1990.
VT (Ed). AJF Lippincott, Philadelphia. 374-385, 1993. Skladanowski A and Konopa J. Interstrand DNA
Di marco A, Casazza AM, Gambetta R, Supino R, Zunino F. crosslinking induced by anthracyclines in tumour cells.
Relationship between activity and aminosugar Biochem Pharmacol. 47 (12): 2269-2278, 1994.
stereochemistry of daunorubicin and adriamycin Stewart DJ, Cripps MC, Dahrouge RGS, Yau J, Tomiak E,
derivates. Cancer Res. 36: 1962-1966, 1976 Huan S, Soltys K, Prosser A, Davies RA. Pilot study of
Di marco A, Casazza AM, Dasdia T, Necco A, Pratesi G, multiple chemotherapy resistance modulators plus
Rivolta P, Velcich A, Zaccara A, Zunino F. Changes of epirubicin in the treatment of resistant malignancies.
activity of daunorubicin, adriamycinand stereoisomers Cancer Chemother Pharmacol. 41: 1-8, 1997.
following the introduction or removal of hydroxyl Topçul MR, Ar›can Özcan G, Erensoy N, Özalpan A. Effect
groups in the amino sugar moiety. Chem Biol Interac. of epirubicin and tamoxifen on labelling index in FM3A
19: 291-302, 1977. cells. J Cell and Mol Biol. 2: 81-85, 2002
48 Gül Özcan Ar›can and Mehmet Topçul
L e t t e r to editor
granin-A (GRN-A). GRN-A is a 49-kilodalton protein such as the amount of tumor angiogenesis, and
that is produced exclusively by endocrine and immunohistochemical levels of various markers,
neurondocrine (NE) cells. It is costored and cosecreted including Ki-67, Bcl-2, p53, and E-cadherin. E-
with the resident hormones of these cells, such as cadherin as a biomarker to predict prognosis in
catecholamines and calcitonin (CT). Although the patients at risk of disease recurrence after radical
function of GRN-A is not known, it can serve as a prostatectomy is warranted.
tissue and serum marker for a variety of endocrine Serum total homocystein: Homocystein (Hcy) as a
cells and tumors. There are several major cancer types tumor marker targets to reveal chemotherapy effects
are characterized by NE differentiation. Recently, the on patients. It is largely derived from cellular
importance of NE differentiation and the attendant methionine, an essential amino acid drawn from
expression of chromogranin-A has become dietary intake. Intracellular homocysteine is normally
appreciated for prostate cancer. Clinical and basic secreted extracellularly, at rapid rates. In the
roles of chromogranin-A in human prostate cancer are circulating blood, the majority of the homocysteine
still studied. Although the function of GRN-A is not binds to albumin, forming a disulfide linkage.
known, several theories have emerged about its role: Approximately 10% to 20% of the Hcy also exists as a
(1) that it participates and perhaps regulates the storage mixed disulfide with cysteine or with homocysteine
and secretion of its coresident hormones in secretory itself . Very little Hcy is present in the circulating
vesicles; (2) that it inhibits proteolytic cleavage blood in a free reduced form (approximately
enzymes; (3) that it binds calcium and thus regulates 1%).Elevated serum tHcy (total homocysteine, free
the biologic effects of this ion; and (4) that it is a and protein-bound) are detectable in patients with
precursor for peptides that have unique biologic effects malignant diseases. Finding increased circulating tHcy
on the function and growth of its resident cells. in tumor cells may also be related to the so-called
Function notwithstanding, the production of GRN-A in ‘‘methionine dependency’’ of many, but not all, tumor
NE prostate cancers has resulted in the availability of cells. Many tumor cells are methionine dependent
a new serum and tissue marker for the tumors. The because of their inability to convert homocysteine
clinical potential of GRN-A as a serum and tumor (Hcy) to methionine by way of the remethylation
marker in prostate cancer. It is now wellestablished reaction. On the other hand, normal cells have no
that GRN-A can be a marker for advanced disease. problem obtaining methionine from homocysteine.
More importantly, GRN-A may be a marker for early Folate is critical to the remethylation reaction. Any
and recurrent disease, even in the absence of abnormal folate deficiency will result in the impairment of
PSA. GRN-A serum levels may also have prognostic function of the remethylation reaction, causing
significance, especially for androgen-independent accumulation of Hcy. Therefore, it was generally
prostate cancer. believed that the rapid proliferation rate of tumor cells,
E - c a d h e r i n : In attempts to determine which cancers such as in prostate cancer and in the so-called
of patients with clinically localized disease who methionine dependency of tumor cells, was due to the
undergo radical prostatectomy will recur, the most depletion of folate by the rapid growing tumor cells
well-characterized and accepted predictors are model and changing levels of fLV (a form of folate) in 24 h
equations that take into account preoperative serum after therapy. In other words, with a better
prostate-specific antigen (PSA), final Gleason score, understanding of the effects of various drugs, the rise
and final pathologic stage. Prediction of regression for and fall of circulating tHcy could be used as a new
the individual patient using these statistical models, tumor marker to monitor cancer patients during
however, is still not precise, and these models could therapy, complementing commonly used tumor
still be improved on. Thus, additional markers are markers. The general impression that elevated tHcy is
needed to more accurately target high-risk patients for detectable in cancer patients derives from the fact that
inclusion in clinical trials involving investigational many cancer patients take anti-folate drugs such as
therapies for locally advanced prostate carcinoma. methotrexate. It is important to know that the level of
Several other approaches show promise in this regard, tHcy reflects the tumor cell proliferation rate.
including nuclear morphometry, where the results have Regardless of the folate status, it is very likely given
been quite consistent. Other more controversial our results and others that the rapid proliferation of
markers include DNA ploidy and other biomarkers, tumor cells is one of the major reasons that elevated
51
Serdar Ar›san
fiiflli Etfal State Hospital
1. Urology Clinics, fiiflli, ‹stanbul
53
Book reviews
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Haliç Üniversitesi,
Moleküler Biyoloji ve Genetik Bölümü
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