Analysis of antimicrobial peptides from porcine neutrophils

Joanna Wessely-Szponder
a,
, Barbara Majer-Dziedzic
b,1
, Anna Smolira
c,2
a
Department of Pathophysiology, Chair of Preclinical Veterinary Sciences, Faculty of Veterinary Medicine, University of Life Sciences, Akademicka 12, 20-033 Lublin, Poland
b
Institute of Biological Bases of Animal Diseases, Sub-Department of Veterinary Microbiology, Faculty of Veterinary Medicine, University of Life Sciences, Akademicka 12,
20-033 Lublin, Poland
c
Institute of Physics, Division of Molecular Physics, Maria Curie Sklodowska University 20-031 Lublin, Poland
a b s t r a c t a r t i c l e i n f o
Article history:
Received 25 March 2010
Received in revised form 2 July 2010
Accepted 5 July 2010
Available online 17 July 2010
Keywords:
Antimicrobial peptides
Cathelicidins
Neutrophils
Pig
Cationic host defence peptides are important components of innate immunity in pigs and other
mammalians. Most of these peptides have a direct antimicrobial activity and they also have a broad
spectrum of effects on the host immune system, which may be taken into account in the introduction of
novel therapeutics. Our method permits simultaneous isolation of six antibacterial peptides, i.e. prophenin-1,
prophenin-2, PR-39, and protegrins 1-3 from a porcine neutrophil crude extract and characterisation of
them. Among the obtained peptides the greatest bactericidal activity expressed as MBC was seen in
protegrins (10 g/ml), whereas in the other studied peptides MBC was on the level of 20 g/ml. Minimal
inhibitory concentrations (MIC) reached 10 g/ml for protegrins 13 and 20 g/ml for prophenins, and PR-
39. Within the bactericidal range all isolated peptides didn't show cytotoxicity on cell lines used in our
experiment.
2010 Elsevier B.V. All rights reserved.
1. Introduction
The increasing bacterial resistance to commercial antibiotics
induces a search for new sources of antibacterial agents. Among the
promising reservoirs of such efcient antibacterial agents currently
under study are antimicrobial peptides derived from neutrophils
(Cheung et al., 2008; Hancock and Sahl, 2006; Ramanathan et al.,
2002; Wieczorek et al., 2010).
Previous studies on mammalian antimicrobial peptides established
that pigs have the most diverse collectionof cathelicidins of any species.
Apart from three myeloid antimicrobial peptides (PMAP-23, PMAP-36,
and PMAP-37) there are porcine cathelicidins puried fromneutrophils
(PMN), i.e. prolinephenylalanine-rich prophenin-1 (PF-1) and pro-
phenin-2 (PF-2), proline-arginine-rich 39-amino-acid peptide (PR-39),
and cysteine-rich protegrin-1 to 5 (PG-1 to PG-5)(Sang and Blecha,
2009).
Prophenins 1 and 2 are two cathelicidins puried from porcine
leukocytes (Zang et al., 2000). They were also isolated from porcine
lung tissue (Wang et al., 2004). These 79-residue cathelicidins are
effective against Gram-negative bacteria (Zang et al., 2000). They have
extended-helical structure and high contents of proline (53%) and
phenylalanine (19%) (Ramanathan et al., 2002).
PR-39 manifests antibacterial activity against Gram-negative
bacteria, and some Gram-positive (Ramanathan et al., 2002). It is
known that PR-39 stops DNA and protein synthesis in Escherichia coli
(Vunnam et al., 1997). Moreover, it participates in wound repair
mechanisms, has chemoattractant properties and interacts with LPS.
It also inhibits the NADPH oxidase reactivity, which has the direct
effect of reducing local tissue injury by reactive oxygen species (ROS)
(Ramanathan et al., 2002; Zang et al., 2000).
Protegrins (PG-1 to PG-5), as active and abundant porcine
cathelicidins were found at bactericidal concentrations in neutrophil
secretions and the abscess uid (Han et al., 2007). They share
common features such as small size (1618 amino acids), -sheet
structure, cationic charge and an amphipathic nature (Steinberg et al.,
1997). The best studied protegrin 1 an 18-amino-acid peptide isolated
from porcine leukocytes displays a broad-range antimicrobial activity
spectrum and has potential for therapeutic use. The cationic nature of
this peptide allows its interaction with the lipid matrix of bacterial
membranes containing negatively charged lipids (Han et al., 2007).
Apart fromsynthetic peptides also naturally obtained cathelicidins
from the neutrophil extract may be used for analysis and possible
therapeutic usage (Anderson et al., 2004; Anderson and Yu, 2008).
Although some papers describing methods of isolation of ovine
cathelicidins have appeared recently (Anderson and Yu, 2003, 2008),
there are few publications about the isolation of porcine cathelicidins.
Most of the available reports concern studies of synthetically obtained
porcine peptides (Jacobsen et al., 2007; Mangoni et al., 1996;
Robinson et al., 2005; Shi et al., 1996). Others describe methods that
allow obtaining only a single kind of peptides. For example,
Kokryakov et al. (1993) described the analysis of protegrin isolated
with ultraltration and vacuumcentrifugation and with the use of fast
atom bombardment (FAB)-mass spectrometric analyses. Prophenin
Journal of Microbiological Methods 83 (2010) 812
Corresponding author. Tel.: +48 814456774; fax: +48 814456024.
E-mail address: joanna.wessely@up.lublin.pl (J. Wessely-Szponder).
1
Tel.: +48 814456016.
2
Tel.: +48 815376130.
0167-7012/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2010.07.010
Contents lists available at ScienceDirect
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j our nal homepage: www. el sevi er. com/ l ocat e/ j mi cmet h
was obtained with ultraltration and characterised by LC-ESI-MS
(Hartwig et al., 1995). The method of separation of PR-39, with AU-
PAGE for identication, used by Shi et al. (1994) is not sufcient for
peptides with lowmolecular masses (Hortin, 2006; Shi et al., 1994). In
this paper we describe a simple and effective method for isolation of
antibacterial peptides from a neutrophil extract. It is a combination of
previously reported techniques used for neutrophils from different
species. This method permits isolation of cathelicidins without the
preliminary step of ultraltration, therefore the method is simple and
cost-effective. Moreover, we have used matrix assisted laser desorp-
tion/ionization time of ight mass spectrometry (MALDI-TOF MS)
without previous trypsin digestion for identication of antibacterial
peptides on the basis of molecular weight. Our modied method
allows for simultaneous isolation of six different bactericidal peptides
from porcine neutrophil extraction.
2. Materials and methods
2.1. Crude extraction
Fresh porcine blood was collected with anticoagulant 3.8% citrate
at an abattoir. A crude extraction fromblood neutrophils was obtained
after red blood cells were lysed by the addition of 0.83% ammonium
chloride at the ratio of 3:1. White blood cells (7585% PMN) were
collected by centrifugation (700g, 15 min, 4 C) and resuspended in
modied phosphate buffer saline (PBSX) buffer (137 mM NaCl,
2.7 mM KCl, 0.5 mM MgCl
2
, 8.1 mM Na
2
HPO
4
, 1.5 mM KH
2
PO
4,
pH
7.4)(Treffers et al., 2005). The cells were then homogenised with DIAX
900 Heidolph (12.5 rpm, 15 min) to release the neutrophil granules.
These granules were collected (25,000g, 40 min, 4 C), suspended in
10% acetic acid and stirred overnight at 4 C to extract the
antimicrobial peptides. Total protein was assessed by Lowry's method
with bovine albumin as a standard (Romeo et al., 1988). The solution
containing the peptides was separated from the granules (25,000g,
20 min, 4 C) lyophilised and stored at 20 C.
2.2. Peptide purication
Gel ltration chromatography was used to separate the compo-
nents present in the crude extraction according to their sizes. The
extract was passed through a Sephadex G-50 (Fine, Sigma-Aldrich)
column, using a running buffer of 5% acetic acid at 0.5 ml/min. The
absorbance of the eluate (every 0.5 ml) was monitored at 280 nm and
fractions were pooled of 5.0 ml (Anderson and Yu, 2003). The
concentration of protegrins and other cathelicidins in every fraction
was assessed using the extinction coefcient (Kokryakov et al., 1993).
The active fractions were lyophilised, resuspended in a solvent
(0.1% triuoroacetic acid [TFA] in water v/v) and were loaded onto a
C18 RP-HPLC column (2504 mm, particle size 5 m, LiChrospiner
100, Merck, Germany) with the owrate of 0.7 ml/min. Peptides were
eluted with gradient 060% acetonitrile in 0.1% TFA and monitored for
absorbance at 225 nm.
2.3. MALDI-TOF analysis
MALDI-TOF analysis was performed on the reectron time of ight
mass spectrometer (RTOF MS built in the Institute of Physics,
Division of Molecular Physics, UMCS Lublin, Poland, with an ion
source MALDI) with parameters described in detail previously (Guch
et al., 2001; Gruszecka et al., 2008).
A matrix, -cyano-4-hydroxycinnamic acid (CCA, 189.2 Da, Sigma-
Aldrich), used without further purication, was diluted in portion of
0.03 g in 3 ml of mixture of acetonitrile and 0.1% TFA (1:1,v/v). Next, an
equal volume of the peptide solution (from 1 ml of total volume of 20
50 g dissolved in 0.1% TFA, 60% acetonitrile) and of the matrix solution
were put on a stainless steel sample holder and dried in air at room
temperature. After evaporation of solvents a visible and apparently
homogenous mixture spot of ~1 mm diameter appeared. The mass
spectra of the investigated peptides were obtained in the linear mode of
the spectrometer, collected at the nitrogen laser (337 nm) in the
positive polarity of the ion source.
2.4. Bacterial strain
A strain of E. coli (25922; obtained fromthe American Type Culture
Collection ATCC) was used for evaluation of the antibacterial
activity of the isolated peptides. It was stored in 10% glycerol at
80 C. The bacterial cultures were cultivated in MuellerHinton
broth (MHB) and MuellerHinton agar (MHA) (Biocorp Warsaw
Poland)(Shi et al., 1994).
2.5. Minimal inhibitory concentrations
The method used was based on the broth dilution assay. Briey,
bacteria were grown in MHB (one colony in 10 ml) pH 7.2, for 20 h at
37 C. The culture for the assay was prepared by diluting the overnight
E. coli culture 1:100 in fresh MHB, and then 1 ml of diluted suspension
of bacteria in 20 ml MHB was stirred for 1 h at 37 C. Equal volumes of
4.610
5
CFU/ml bacteria (150 l) were mixed with a crude extraction
or with each obtained gel ltration fraction (150 l). All samples were
incubated for 20 h at 37 C. Then the mixtures were diluted 100-fold
in PBS and 0.1 ml of every dilution was plated in triplicate on MHA.
After incubation for 20 h at 37 C, the colonies were counted and the
numbers of CFU/ml were calculated and compared with the CFU
values of controls of E. coli prepared in the same conditions. The active
samples were used for determination of minimal inhibitory concen-
tration (MIC). The lyophilised peptides were diluted in two-fold serial
dilutions in MHB from160 to 2.5 g/ml and standardised suspensions
of 4.610
5
CFU/ml E. coli were added and the mixture was incubated
for 20 h at 37 C. Then the inhibitory properties of the examined
samples were estimated by visual comparison with the control
suspension. The smallest concentrations of peptides causing inhibi-
tion of E. coli growth (the broth remained clear) were determined as
MIC (g/ml)(Shi et al., 1994; Skerlavaj et al., 1996).
2.6. Evaluation of bactericidal activity (MBC)
For the bactericidal activity (MBC) determination 100 l of
solution from the last tube where the growth of bacteria was
observed and from subsequent four clear tubes were transferred
onto the plates with MHA. After incubation for 20 h at 37 C the
minimal bactericidal concentration was identied as the lowest
concentration of peptides that did not permit any visible growth on
the surface of the agar. Every assay was done in triplicate (Shi et al.,
1994; Steinberg et al., 1997).
2.7. Cytotoxicity assay
Minimal Essential Medium (MEM), Medium 199, and Hams F-12
(PAA-Immuniq) were used for evaluation of cytotoxicity of active
peptides on the cell culture in vitro. The assay was carried out on two
cell lines, i.e. a feline lung cell line tissue culture (FLF-3) and a rat
hepatocyte cell line (ECACC Salisbury, Wiltshire). MEM with 10%
neonatal calf serum and penicillin (100 U/ml) with streptomycin
(100 g/ml) was used as the growing medium. Medium 199 with
antibiotics was used as medium maintenance. These peptides were
diluted 1:10 and 1:20, and 0.2 ml of each dilution was added to 6
tubes (for dilution) with a tissue culture. The tubes with cell lines
without addition of peptides were used as a control. The tubes were
kept for 10 days at 37 C and were controlled microscopically every
day (Kowalska-Pyka et al., 2001; Shi et al., 1994).
9 J. Wessely-Szponder et al. / Journal of Microbiological Methods 83 (2010) 812
3. Results
3.1. Purication of porcine neutrophil cathelicidins
The crude extraction obtained from each batch by overnight
extraction of porcine neutrophil granules in 10% acetic acid was
lyophilised in portions of about 250 g, as indicated by Lowry's
method. One representative portion from each batch was resus-
pended in MHB and subjected to the initial antibacterial assay. All
tested crude extractions were bactericidal. Subsequently, portions of
crude extractions from active batches containing peptides were
passed through a Sephadex G-50 column in concentrations of about
500 g/ml. Every fraction of 0.5 ml was monitored spectrometrically
at 280 nm and these fractions were pooled of 5.0 ml. The initial
purication of porcine antibacterial peptide extract from Sephadex
G-50 column is shown in Fig. 1.
Every 5.0 ml fraction was tested for antibacterial action against E.
coli and fractions F3, F4, F11 and F13 appeared active. All active
fractions were assessed by HPLC analysis, which permitted the
resolution of several distinct peaks.
3.2. Peptide characterisation
Fractions which showed antibacterial activity against E. coli were
assessed by MALDI-TOF MS analysis. Molecular weight was analyzed by
the ExPASy MW/pI tool (http://www.expasy.ch/tools/pi_tool.htlm).
3.3. Characterisation of prophenin 1
The molecular mass of peptide obtained in fraction F3 was 8683 Da
and was consistent with the predicted PF-1 molecular mass. This
peptide was active against E. coli 25922 ATCC at concentration of 20 g/
ml (Table 1). There were no cytotoxic effects on any cell line in any
studied concentration up to 20 g/ml. We didn't observe changes in cell
size and shape. The cytoplasmic vacuoles and dying cells didn't appear
duringobservationperiod. The microscopic picture was the same for the
control cells and cells with addition of peptide. After 10 days the
degeneration of the cells appeared both in the control and treated lines.
3.4. Characterisation of prophenin 2
Using MALDI TOF technique we estimated that an antimicrobial
peptide with MIC of 20 g/ml and with MBC equal 100% of MIC against
E. coli (Table 1) was present in fraction F4 and was recognised as PF-2
with a molecular mass of 8807 Da (Fig. 2). We didn't observe any
morphological changes and degenerative alterations in either the FLF-
3 tissue culture or the rat hepatocyte cell line during the 10 days of the
experimental period caused by these peptides at examined concen-
trations up to 20 g/ml.
3.5. Characterisation of PR-39
Fig. 3 shows the resulting chromatograph of fraction F11. The pick
shown at this chromatogram had the molecular mass of 4716 Da, as
revealed in MALDI TOF analysis and corresponded to PR-39 (Fig. 4).
PR-39 had antimicrobial activity against E. coli with MIC of 20 g/ml
and with the same MBC (Table 1). The highest dose of PR-39 that was
tested (20 g/ml) was not cytotoxic to the FLF-3 tissue culture or the
rat hepatocyte cell line. The microscopic picture was the same for the
control and tested cells.
3.6. Characterisation of protegrins PG- 1 to PG- 3
Protegrins PG-1 to PG-3 were present in fraction F13. Their
obtained masses were as follows: 2154.5 Da for PG-1, 1955.6 Da for
PG-2, and 2055.5 Da for PG-3 (Fig. 5). Moreover, we noticed a
peptide with the molecular mass of 1307 Da in this fraction but
without antibacterial and cytotoxic properties. The antibacterial
activity of PG-1 to PG-3 was described as MIC 10 g/ml and MBC at
the same level (Table 1). We didn't observe any morphological
changes and degenerative alterations in either the FLF-3 tissue
culture or the rat hepatocyte cell line during the 10 days of the
observation of these peptides at concentration up to 10 g/ml. After
this time degenerative changes of the cells in all groups were
observed.
Fig. 1. Gel ltrationchromatogramfor crude extraction applied ona Sephadex G-50 columnin5%acetic acidand monitoredat 280 nm. Fractions were collected intubes (0.5 ml per tube).
Table 1
Antibacterial activity of studied antimicrobial peptides against E. coli (strain 25922
ATCC) tested with the broth dilution technique.
Fraction Peptide MIC (g/ml) MBC (g/ml)
F 3 Prophenin 1 20 20
F 4 Prophenin 2 20 20
F11 PR-39 20 20
F13 Protegrin (1-3) 10 10
10 J. Wessely-Szponder et al. / Journal of Microbiological Methods 83 (2010) 812
4. Discussion
The increasing number of virulent, antibiotic-resistant strains of
bacteria has created a pressing need for alternative therapies for
infection control (Jacobsen et al., 2007). Moreover, the possibility of
using the immunomodulatory properties of antibacterial peptides as a
basis for therapy has appeared recently (McPhee and Hancock, 2005).
Our experimental method permits isolation of PF-1, PF-2, PR-39,
and PG-1 to PG-3 in an active bactericidal form in quantities sufcient
for further analysis. Porcine neutrophils lack defensins and cathe-
licidins are the only antibacterial peptides there (Sang and Blecha,
2009). These cathelicidin peptides from neutrophil secretion
remained stable in inammatory uids for days in contrast to the
reactive oxygen and nitrogen intermediates (Shi and Ganz, 1998).
Among porcine cathelicidins protegrins were identied as the major
stable antimicrobial substances released by neutrophils. The imma-
ture forms of peptides are stored in porcine neutrophils as inactive
proprotegrins and neutrophil elastase is required for in vitro
activation of protegrins (Cole et al., 2001). However, as in the study
conducted by Anderson and Yu (2003), an extraction process used in
our experiment did not involve the addition of elastase, so the
cleavage was probably carried out by neutrophil elastase naturally
present in the neutrophil extraction.
As in experiment described by Anderson and Yu (2003) we also
used gel ltration method for initial separation of peptides according
to decreasing molecular weight (Fig. 1). However, in our study the
crude extract was passed through a Sephadex G-50 column, which
was previously used by Wang et al. (2008) instead of Biogel P10
column. Treffers et al. (2005), in turn, applied ion-exchange
chromatography for evaluation of antimicrobial peptides from deer
neutrophils.
Polypeptides smaller than 7000 Da are not detected by the usual
techniques of 2-dimensional electrophoresis because they are below
the limits of size resolution, and small components may not be xed in
gels and produce lower staining intensity per mole of peptide.
Consequently MALDI-TOF MS has served as a tool to open up
peptidomic analysis in the mass range from 1000 to 7000 (Hortin,
2006). In our study the MALDI TOF technique permitted assessing the
obtained porcine cathelicidins as PF-1, PF-2, PR-39, and PG-1 to PG-3
with molecular masses which are consistent with the predicted
molecular masses.
Prophenin 1 with the molecular mass of 8683 Da was active
against E. coli 25922 ATCC at concentration of 20 g/ml. According to
Hartwig et al. (1995) PF-1 demonstrated potent bactericidal activity
against E. coli. In our experiment PR-39 had antimicrobial activity
against E. coli with MIC of 20 g/ml and with the same MBC. Sang and
Blecha (2009) showed signicant efcacy of this peptide against
Fig. 2. Positive ion MALDI time of ight mass spectrum of prophenin 2 (8807 Da)
obtained in linear mode of the spectrometer.
Fig. 3. HPLC chromatogram of fraction F11 obtained in gel ltration. The fraction
resuspended in 0.1% triuoroacetic acid [TFA] in water (v/v) was loaded onto a C18 RP-
HPLC column (2504 mm, particle size 5 m, LiChrospiner 100, Merck, Germany) with
a ow rate of 0.7 ml/min. Peptides were eluted with gradient 060% acetonitrile in 0.1%
TFA and monitored for absorbance at 225 nm.
Fig. 4. Positive-ion MALDI mass spectrum of PR-39 with -cyano-4-hydroxycinnamic
acid (CCA) used as a matrix. The spectrum is an average of 256 laser shots.
Fig. 5. Positive-ion MALDI mass spectrum of protegrins obtained at 17 kV accelerating
voltage in linear mode of the time of ight mass spectrometer.
11 J. Wessely-Szponder et al. / Journal of Microbiological Methods 83 (2010) 812
Gram-negative bacteria with MIC about 20 g/ml. Protegrins belong to
elastase-activated polypeptides, which are the predominant antibac-
terial factors in porcine neutrophil secretions generated during
phagocytosis (Shi and Ganz, 1998). In our study the bactericidal
activity of PGs against E. coli was on the level of 10 g/ml. Cole et al.
(2001) estimated that two concentrations (20 and 200 g/ml) of PG-1
completely cleared the wound of S. epidermidis in pigs. According to
Ramanathan et al. (2002) protegrins kill many bacteria at concentra-
tions of 15 g/ml. Sang and Blecha (2009) found that PG-1 is effective
against 14 Gram-negative bacteria with most MIC less than 5 mM.
Apart from these we also established the presence of a peptide with
the molecular mass of 1307 Da. It would be the result of enzymatic
cleavage of prophenin (Hartwig et al., 1995). We didn't observe
antibacterial activity of this peptide.
We didn't observe cytotoxic effect within tested concentrations of
all studied peptides. The cytotoxicity assay was based on the
occurrence of morphological changes. Although it is not a quantitative
method, it allows a relatively fast cytological screening of tested
substances. The use of culture tubes increases precision because it
gives the possibility of obtaining a greater number of cells, enabling
more accurate determinations (Kowalska-Pyka et al., 2001).
The use of cationic peptides mostly involved topical treatment of
difcult infections because of the potential toxicity associated with
systemic drug usage. Unfortunately, the majority of infections that are
life threatening, especially those due to multidrug resistant bacteria,
require systemic treatment (McPhee and Hancock, 2005). Although
our studies revealed that the studied cathelicidins didn't show
cytotoxicity within bactericidal range, literature data underline that
the systemic usage of cathelicidins is restricted because of their
cytotoxic potential, especially in the case of protegrin 1. As Robinson
et al. (2005) estimated, PG-1 is toxic to the mammalian cell at
concentration of 100 g/ml. On the other hand, according to Shi et al.
(1996) PR-39 is not cytotoxic to MadinDarby bovine kidney (MDBK)
cells up to 50 M.
An alternative to the microbicidal effect approach would involve
utilising the immunomodulatory activity of cationic peptides as a
basis for therapy. The stimulation of innate immunity while not
inducing or even suppressing harmful proinammatory responses is
an attractive alternative because of the fact that such immunomodu-
lators would act on the host cells rather than attack bacteria directly.
This approach should prevent any resistance. Therefore it may be an
effective treatment for the increasing number of individuals with
multidrug resistant bacterial infections (Hancock and Sahl, 2006;
McPhee and Hancock, 2005).
As mentioned by Anderson and Yu (2008), in the case of ovine
antibacterial peptides they are signicantly more active in combina-
tion. Therefore it is suggested that better therapeutic results could be
obtained if isolated peptides were used synergistically. Our method of
separation leads to obtaining six different antibacterial peptides from
fresh porcine blood with activity maintained.
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