Elastin peptides prepared from piscine and mammalian elastic tissues inhibit collagen‐induced platelet aggregation and stimulate migration and proliferation of human skin fibroblasts

We obtained pure elastin peptides from bovine ligamentum nuchae, porcine aorta, and bonito bulbus arteriosus. The inhibitory activity of these elastin peptides on platelet aggregation induced by collagen and the migratory and proliferative responsivenesses of human skin fibroblasts to these elastin peptides were examined. All of bonito, bovine, and porcine elastin peptides found to inhibit platelet aggregation, but bonito elastin peptides showed a higher inhibitory activity than bovine and porcine elastin peptides did. All elastin peptides enhanced the proliferation of fibroblasts 3.5‐ to 4.5‐fold at a concentration of 10 µg/ml. Bovine and porcine elastin peptides stimulated the migration of fibroblasts, with the optimal response occurring at 10^?1 µg/ml, while maximal response was at 10² µg/ml for bonito elastin peptides. Furthermore, pretreatment of fibroblasts by lactose depressed their ability to migrate in response to all elastin peptides, suggesting the involvement of elastin receptor in cell response. These results suggest that both mammalian and piscine elastin peptides can be applied as useful biomaterials in which elasticity, antithrombotic property, and the enhancement of cell migration and proliferation are required. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Mechanisms of interaction between human skin fibroblasts and elastin: differences between elastin fibres and derived peptides.

3H-Labelled kappa-elastin peptides (kE:75 kDa molecular weight) were shown to bind to confluent human skin fibroblast (HSF) cultures in a time-dependent and saturable manner. Scatchard analysis indicated the presence of high affinity binding sites with kD = 2.7 x 10(-10) M and 19,000 sites per cell. Binding of kE to its receptor on HSF accelerates and intensifies the adhesion of insoluble elastin fibres (iE) to confluent HSF. Optimal effect was attained for a kE concentration of 0.3 x 10(-9) M close to kD. This stimulatory effect of kE on the binding of iE to HSF could be inhibited by neomycin, retinal and pertussis toxin, substances which act at different levels of the transduction mechanism following the activation of the receptor and the subsequent triggering of cell biological events (chemotaxis, modification of calcium fluxes). The stimulation of iE adhesion to HSF induced by kE as well as kE binding to the cells could be inhibited by lactose and laminin but not by Arg-Gly-Asp-Ser(RGDS) peptides. This indicates that the elastin peptide receptor on HSF possesses lectin-like properties and shares homology with the laminin receptor as also shown for other cell types. None of the substances tested, that is inhibitors of the transduction mechanism, lactose, laminin and Arg-Gly-Asp-Ser(RGDS) peptides were shown to interfere significantly with the binding of iE (in the absence of added kE) to confluent HSF. The proteins adhering strongly to elastin fibres were isolated by a sequential extraction procedure and the final hydrochloride guanidinium-DTT extract was analysed by SDS-PAGE under reducing conditions, Western blots using specific antibodies against several connective tissue proteins and affinity for [3H]-kE following nitrocellulose electro-transfer of proteins. Fibronectin, vitronectin, tropoelastin(s), and a 120 kDa cysteine rich glycoprotein previously designated as elastonectin were identified. Among these proteins, [3H]-kE was found to bind exclusively to a 65 kDa protein that could be eluted selectively from elastin fibres with a neutral buffer containing 100 mM lactose. Therefore the elastin peptide receptor on human skin fibroblasts shares properties with the elastin receptor characterized from other cell types. Conformational differences between elastin peptides and elastin fibres could explain the differences in the mechanisms of interactions between elastin fibres and elastin peptides with HSF in culture. The stimulatory effect of elastin-derived peptides on the adhesion of elastin fibres to HSF could have implications in the oriented biosynthesis of elastin fibres.     

Elastin synthesis by ligamentum nuchae fibroblasts: effects of culture conditions and extracellular matrix on elastin production

Fetal bovine ligamentum nuchae fibroblasts maintained in culture synthesized soluble elastin but were unable to form the insoluble elastic fiber. Secreted elastin precursors accumulated in culture medium and were measured using a radioimmunoassay for elastin. When elastin production was examined in ligament tissue from fetal calves of various gestational ages, cells from tissue taken during the last trimester of development produced significantly more elastin than did cells from younger fetal tissue, with maximal elastin synthesis occurring shortly before birth. Soluble elastin was detected in ligament cells plated at low density until proliferation began to be density inhibited and the cells became quiescent. Also, soluble elastin production per cell declined with increasing population doubling or with age in culture. Cells grown in the presence of 5% fetal calf serum produced approximately four times as much soluble elastin as cells grown in serum-free medium. The addition of dexamethasone (0.1 microM) and bleomycin (1 microgram/ml) increased soluble elastin production by cultured cells 180% and 50%, respectively, whereas theophylline (5 micrograms/ml) depressed production 50% and antagonized stimulation by dexamethasone. Ascorbate (50 micrograms/ml), soybean trypsin inhibitor (1 mg/ml), insulin (100 microunits/ml), and aminoacetonitrile (50 micrograms/ml) had no effect, but cycloheximide at 10(-4) M completely inhibited soluble elastin production. In contrast to cells in culture, ligament tissue minces (ligament cells surrounded by in vivo extracellular matrix) efficiently incorporated soluble elastin precursors into insoluble, cross-linked elastin. In addition, soluble elastin production per cell (per microgram of DNA) was higher in tissue minces than elastin production by cells maintained on plastic. These results suggest a role for extracellular matrix in formation of the elastic fiber and in stabilizing elastin phenotypic expression by ligament fibroblasts. Fibroblasts from the bovine ligamentum nuchae present an excellent model for in vitro studies of elastin biosynthesis.     

Growth Stimulation of Human Skin Fibroblasts by Elastin-Derived Peptides

Elastin-derived peptides (kappa-elastin: KE, mean molecular mass: 75 kDa), either coated onto plastic dishes or added to culture media (0.26 to 1.33 nM) stimulated the growth of human skin fibroblasts (HSF) strains obtained from different donors and tested at different cell passages (4 to 12). Coated 44.4 μg/cm²insoluble elastin (iE) exhibited the same action; coated iE or KE significantly modifies the HSF morphology: after 5-6 days of culture, HSF are more elongated, and at preconfluence state, formation of HSF clusters surrounding iE were observed. Increased ³H thymidine incorporation and proliferative effect of HSF by KE (1.3 to 2.2 fold as compared to control cells) was observed after a lag phase period which raised with initial HSF density. Optimal proliferative effect was obtained at KE 8.5 10^-10M, a value close to the dissociation constant (k_D= 2.7 10^-10M) of KE to HSF. Valine-glycine-valine-alanine-proline-glycine (VGVAPG), but not valine-glycine-valine (VGV) or Valine-glycine-valine-valine-glycine-alanine (VGWGA) also significantly stimulated, optimally at 7.0 10^-10M, HSF proliferation. It was concluded that the stimulatory influence of elastin derived peptides on HSF proliferation was mediated through a binding to plasmalemmal receptor of HSF.     

Effect of elastin peptides on the activities of antioxidant enzymes in fibroblasts.

The effect of elastin peptides (kappa-elastin) was investigated on murine fibroblasts. The data indicate that elastin peptides increase the activities of antioxidant enzymes detoxifying free radicals and increase the lipid peroxide concentration within the cell. These results suggest that the influence of elastin peptides on oxygen metabolism may be related to their activities in vivo following elastin degradation and can contribute to their role in the pathogenesis of atherosclerosis.     

Growth Stimulation of Human Skin Fibroblasts by Elastin-Derived Peptides

Elastin-derived peptides (kappa-elastin: KE, mean molecular mass: 75 kDa), either coated onto plastic dishes or added to culture media (0.26 to 1.33 nM) stimulated the growth of human skin fibroblasts (HSF) strains obtained from different donors and tested at different cell passages (4 to 12). Coated 44.4 μg/cm²insoluble elastin (iE) exhibited the same action; coated iE or KE significantly modifies the HSF morphology: after 5-6 days of culture, HSF are more elongated, and at preconfluence state, formation of HSF clusters surrounding iE were observed. Increased ³H thymidine incorporation and proliferative effect of HSF by KE (1.3 to 2.2 fold as compared to control cells) was observed after a lag phase period which raised with initial HSF density. Optimal proliferative effect was obtained at KE 8.5 10^?10M, a value close to the dissociation constant (k_D= 2.7 10^?10M) of KE to HSF. Valine-glycine-valine-alanine-proline-glycine (VGVAPG), but not valine-glycine-valine (VGV) or Valine-glycine-valine-valine-glycine-alanine (VGWGA) also significantly stimulated, optimally at 7.0 10^?10M, HSF proliferation. It was concluded that the stimulatory influence of elastin derived peptides on HSF proliferation was mediated through a binding to plasmalemmal receptor of HSF.     

Venom from Anemesia species of spider modulates high voltage-activated Ca(2+) currents from rat cultured sensory neurones and excitatory post synaptic currents from rat hippocampal slices

The actions of crude venom from Anemesia species of spider were investigated in cultured dorsal root ganglion neurones from neonatal rats and hippocampal slices. Using mass spectrometry (MALDI-TOF MS), 10-12 distinct peptides with masses between about 3 and 10kDa were identified in the crude spider venom. At a concentration of 5 microg/ml crude Anemesia venom transiently enhanced the mean peak whole cell voltage-activated Ca(2+) current in a voltage-dependent manner and potentiated transient increases in intracellular Ca(2+) triggered by 30mM KCI as measured using Fura-2 fluorescence imaging. Additionally, 5-8 microg/ml Anemesia venom increased the amplitude of glutamatergic excitatory postsynaptic currents evoked in hippocampal slices. Omega-Conotoxin GVIA (1 microM) prevented the increase in voltage-activated Ca(2+) currents produced by Anemesia venom. This attenuation occurred when the cone shell toxin was applied before or after the spider venom. Anemesia venom (5 microg/ml) created no significant change in evoked action potentials but produced modest but significant inhibition of voltage-activated K(+) currents. At a concentration of 50 microg/ml Anemesia venom only produced reversible inhibitory effects, decreasing voltage-activated Ca(2+) currents. However, no significant effects on Ca(2+) currents were observed with a concentration of 0.5 microg/ml. The toxin(s) in the venom that enhanced Ca(2+) influx into sensory neurones was heat-sensitive and was made inactive by boiling or repetitive freeze-thawing. Boiled venom (5 microg/ml) produced significant inhibition of voltage-activated Ca(2+) currents and freeze-thawed venom inhibited Ca(2+) transients measured using Fura-2 fluorescence. Our data suggest that crude Anemesia venom contains components, which increased neuronal excitability and neurotransmission, at least in part this was mediated by enhancing Ca(2+) influx through N-type voltage-activated Ca(2+) channels.     

Concentration-dependent conformational transition of alpha-elastin in aqueous solution

Circular dichroism measurements on aqueous solutions of alpha-elastin have given evidence of beta-bend structure at elevated concentrations. When Ca2+ was added, the concentration-dependent conformational transition was somewhat inhibited and the binding of the metal ion was shown, by means of equilibrium dialysis, to be essentially independent from alpha-elastin concentration in the range of 1 to 10 mg/ml. The results obtained are discussed in connection with the elasticity and calcification of elastin.     

Effect of PGE2 and indomethacin on human fibroblast collagen production

Fibroblasts can synthetize prostaglandins (particularly PGE2) "in vitro" but it still remains unclear what role they play in the regulation of fibroblast proliferation and collagen production. We report here the effect of PGE2 and indomethacin on collagen synthesis by cultured human dermal fibroblasts. PGE2 (range: 1-300 pmoles/ml) and indomethacin (range: 0.0025-1.0 micrograms/ml) did not significantly affect fibroblast collagen production, when added for 24 hours at 37 degrees C to the cultures, in comparison to controls (fibroblasts incubated for 24 hours at 37 degrees C in medium only). Prostaglandins probably modulate collagen synthesis, as described in a previous report, by means their effect on cell proliferation. It appears they do not affect the intracellular mechanism of collagen production.     

The effect of pH on biological activity of plant cytotoxin cauloside C

The effect of plant carboxyl-containing glycoside cauloside C upon eucaryotic cells has been studied. The glycoside interacts with cells as a pH-dependent cytotoxin and increases K+ leakage and Ca2+ uptake with strong action in acidic media Cell viability after glycoside action at acidic pH may be recovered by the shift of medium pH from 5.6 to 7.4. Directed transport of low molecular weight effectors such as cAMP and Ca2+ to human embryo fibroblasts under the action of cauloside C has been demonstrated. Calcium uptake is accompanied by about a twofold stimulation of fibroblast proliferation in serum-free medium. The manifestation of the effect depends on the strictly determined time of the 'open' state of the membrane permeability (2 min) and upon concentration of glycoside in the medium (1 ng/ml) Cauloside C-stimulated Ca-transport is not blocked by Ca-channel blockers such as verapamil, diltiasem, and nitrendipine (all at a concentration of 1 x 10(-6) M) but these blockers inhibit cauloside C-stimulated proliferation of fibroblasts. We conclude that stimulation of fibroblast proliferation is caused by activation of membrane associated Ca-channels at the expense of calcium, incorporated into cells with cauloside C. The use of cauloside C as a new biochemical tool for cell permeabilisation is suggested.     

Retinyl acetate inhibits platelet-derived growth factor-induced Ca2+ signals in C3H 10T1/2 fibroblasts

Mitogenic stimulation of density-arrested C3H 10T1/2 mouse fibroblasts by serum or purified platelet-derived growth factor (PDGF) was potently inhibited by retinyl acetate (RAc; IC50 = 0.1 microgram/ml, 0.3 x 10(-6) M) when administered during the first 2 hours of mitogen exposure. This inhibitory effect of RAc coincided with a period early in the cell growth-division cycle when density-arrested C3H 10T1/2 cells stimulated by PDGF were found to require physiological levels of extracellular Ca2+ for the transition from G0 to G1 of the cell cycle. To determine if the inhibitory effect of RAc was mediated through alterations in the Ca2+ signaling pathway induced by mitogens, we examined Fura-2-loaded fibroblasts for changes in the Ca2+ response elicited by PDGF. Addition of PDGF (5 ng/ml) induced a transient increase in the [Ca2+]i that was not significantly effected by the extracellular Ca2+ concentration. Treatment of cells with RAc caused a concentration- and time-dependent inhibition of this PDGF-stimulated Ca2+ flux (IC50 = 0.45 microgram/ml or 1.5 x 10(-6) M; t1/2 = 15 min), whereas release of intracellularly stored Ca2+ by thrombin was unaffected by RAc (1.2 micrograms/ml, 4 x 10(-6) M). Treatment with RAc did not significantly affect PDGF binding to cell surface receptors or the generation of inositol phosphates. These results suggest that the mechanism by which RAc inhibits PDGF- or serum-induced mitogenesis is through modulation of the Ca2+ signal stimulated by PDGF, and thereby depriving the cell of a rise in intracellular Ca2+ necessary for progression through the cell cycle.     

Improvement of antioxidant activity of peptides with molecular weights ranging from 1 to 10 kDa by PEF technology

Egg white protein powder was hydrolyzed with Alcalase to produce antioxidant peptides. Then, the peptides were fractionated with ultrafiltration membranes. The peptides (1-10 kDa) were further treated by pulsed electric field (PEF) to investigate its effect on the antioxidant activity of the peptides. Antioxidant activity was evaluated using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical inhibition assay. The results indicated that optimal electric field intensity and standing times of PEF can enhance the antioxidant activity of the peptides. Therefore, a Box-Behnken design (BBD) with three independent variables including concentration, electric field intensity and pulse frequency was used to establish the regression equation of second-order response surface. The optimal conditions were as follows: concentration 8 mg/ml, electric field intensity 10 kV/cm and pulse frequency 2400 Hz. Under these conditions, the peptides antioxidant activity was 62.64% ± 0.98%. The present study demonstrated that the antioxidant activity of peptides (1-10 kDa) could be improved using PEF.     

Cleavage of the matricellular protein SPARC by matrix metalloproteinase 3 produces polypeptides that influence angiogenesis

SPARC, a matricellular protein that affects cellular adhesion and proliferation, is produced in remodeling tissue and in pathologies involving fibrosis and angiogenesis. In this study we have asked whether peptides generated from cleavage of SPARC in the extracellular milieu can regulate angiogenesis. Matrix metalloproteinase (MMP)-3, but not MMP-1 or 9, showed significant activity toward SPARC. Limited digestion of recombinant human (rhu)SPARC with purified catalytic domain of rhuMMP-3 produced three major fragments, which were sequenced after purification by HPLC. Three synthetic peptides (Z-1, Z-2, and Z-3) representing motifs from each fragment were tested in distinct assays of angiogenesis. Peptide Z-1 (3.9 kDa, containing a Cu2+-binding sequence KHGK) exhibited a biphasic effect on [3H]thymidine incorporation by cultured endothelial cells and stimulated vascular growth in the chick chorioallantoic membrane (CAM). In contrast, peptides Z-2 (6.1 kDa, containing Ca2+-binding EF hand-1) and Z-3 (2.2 kDa, containing neither Cu2+-binding motifs nor EF hands), inhibited cell proliferation in a concentration-dependent manner and exhibited no effects on vessel growth in the CAM. Reciprocal results were obtained in a migration assay in native collagen gels: peptide Z-1 was ineffective over a range of concentrations, whereas Z-2 or Z-3 stimulated cell migration. Therefore, proteolysis of SPARC by MMP-3 produced peptides that regulate endothelial cell proliferation and/or migration in vitro in a mutually exclusive manner. One of these peptides containing KHGK also demonstrated a concentration-dependent effect on angiogenesis.     

促卵泡激素结合片段通过P13K／Akt—cyclinD1通路抑制卵巢癌细胞增殖活性

目的：研究促卵泡激素（FSH）结合片段对卵巢上皮性细胞癌细胞株hey增殖活性的影响。方法：应用卵巢上皮性细胞癌细胞株hey作为实验对象,分别加入FSH、FSH结合片段、FSH＋FSH结合片段。用四甲基偶氮唑蓝（MTT）法检测卵巢癌hey细胞的生长状况,Westernblot检测cyclinD1、Akt、pAkt分子的表达。结果：FSH对卵巢癌hey细胞的生长有明显的促进作用,细胞生长活性提高21％。FSH结合片段对卵巢癌细胞的生长有明显抑制作用,对细胞平均抑制率为22％,且能下调cyclinD1的表达,在2．5×10^-9Mol／L达70％。FSH结合片段对FSH有竞争抑制作用,细胞抑制率为18％,能抑制FSH上调cyclinD1的作用,抑制率为61％。FSH能上调pAkt的表达,对Akt的袁达没有明显影响,在40mlU／ml的浓度时pAkt／Akt上调率达224％。FSH结合片段对Akt的表达没有明显影响,但能明显下调pAkt的表达,且呈时间依赖性（在15分钟时达66％）和剂量依赖性（在2．5×10^-9Mol／L达31％）。FSH结合片段能抑制FSH上调pAkt的作用,抑制率为80％。结论：FSH结合片段可通过抑制FSH诱导的P13FdAkt-cycl—inD1信号通路进而抑制上皮性卵巢癌hey细胞的增殖活性,     

Physiological Ca2+ level and Ca2+-induced Permeability Transition Pore control protein phosphorylation in rat brain mitochondria

Phosphorylation of several low molecular mass proteins (3.5, 17, 23 and 29kDa) was observed in rat brain mitochondria (RBM) at ATP concentration close to that in the mitochondrial matrix. Furthermore, regulatory effects of Ca2+ on phosphorylation of these proteins were investigated. Protein phosphorylation was found to be modulated by Ca2+ in the physiological concentration range (10(-8) to 10(-6)M free Ca2+). Incorporation of 32P from [gamma-32P]ATP into the 17kDa protein was dramatically increased within the 10(-7) to 10(-6)M free Ca2+ range, whereas an opposite effect was observed for the 3.5kDa polypeptide. Strong de-phosphorylation of the 3.5kDa polypeptide and enhanced 32P-incorporation into the 17 and 23kDa proteins were found with supra-threshold Ca2+ loads and these effects were eliminated or reduced in the presence of cyclosporin A, an inhibitor of Permeability Transition Pore (PTP) opening. In the presence of calmidazolium (Cmz), a calmodulin antagonist, enhanced levels of phosphorylation of the 17 and 3.5kDa polypeptides were observed and the 17kDa protein phosphorylation was suppressed by H-8, a protein kinase A inhibitor. It is concluded that Ca2+ in physiological concentrations, as a second messenger, can control phosphorylation of the low molecular mass phospoproteins in RBM, in addition to well known regulation of some Krebs cycle dehydrogenases by Ca2+. The protein phosphorylation was strongly dependent on the Ca2+-induced PTP opening.     

Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor

Keratinocytes and fibroblasts isolated from human neonatal foreskin can be plated and grown through multiple rounds of division in vitro under defined serum-free conditions. We utilized these growth conditions to examine the mitogenic potential of acidic and basic fibroblast growth factor (aFGF and bFGF) on these cells. Our results demonstrate that both aFGF and bFGF can stimulate the proliferation of keratinocytes and fibroblasts. aFGF is a more potent mitogen than bFGF for keratinocytes. In contrast, bFGF appears to be more potent than aFGF in stimulating the growth of fibroblast cultures. Heparin sulfate (10 micrograms/ml) dramatically inhibited the ability of bFGF to stimulate the proliferation of keratinocytes. In comparison, heparin slightly inhibited the stimulatory effect of aFGF and had no effect on epidermal growth factor (EGF) stimulation in keratinocyte cultures. In fibroblast cultures the addition of heparin enhanced the mitogenic effect of aFGF, had a minimal stimulatory effect on the mitogenic activity of bFGF, and had no effect on EGF-stimulated growth. Our results demonstrate that the proliferation in vitro of two normal cell types found in the skin can be influenced by aFGF and bFGF and demonstrate cell-type specific differences in the responsiveness of fibroblasts and keratinocytes to these growth factors and heparin.     

A study of the uterine protein variations during the estrous cycle in the cow: molecular weights determination

This study was carried out to investigate molecular weights (MW) of the uterine protein content as the first step in their identification, and their variations during the estrous cycle as a part of the uterine adjustments for provision of an appropriate environment for physiological events in the bovine. Of the 24 pro-estrous, 21 estrous, 24 met-estrous and 46 diestrous uterine fluid samples prepared for our previous work, five samples in each phase were selected randomly and subjected to SDS-PAGE analysis in parallel with standard marker proteins to separate their protein content into fractions. By comparing these fractions with those of the standards, MWs of <14.4, 20, 30, 38, 40, 46, 67, 75, 85, 90, 160, 200, 210, 270 and 330 kiloDaltons (kDa) were calculated, respectively. Proteins with MWs of less than 14.4kDa [5.3 mg/ml (7.5% of total protein)] were observed only in met-estrus. Proteins with the MW of 20 kDa [14.0 mg/ml (19.4%)] were found only in diestrus. Proteins with MW of 30 kDa in met-estrus [20.6 mg/ml (28.9%)] and diestrus [4.5 mg/ml (6.3%)], and 38 kDa proteins were observed in a considerable amount in pro-estrus [41.4 mg/ml (44.5%)] and estrus [40.0 mg/ml (44.2%)]. Forty kiloDaltons proteins were observed only in diestrus [5.5 mg/ml (7.6%)], and 46 kDa proteins were observed only in met-estrus [5.3mg/ml (7.6%)]. Sixty seven, 75 and 330 kDa proteins were observed in all the phases of the cycle with different densities, the higher values recorded in diestrus, pro- and met-estrus, respectively. Proteins with MW of 85 kDa were observed in all the phases but diestrus [9.8 mg/ml (10.5%), 8.6 mg/ml (9.5%) and 7.3 mg/ml (10.2%) for pro-estrus, estrus and met-estrus, respectively], and proteins with MW of 90 kDa were observed in estrus [20.0mg/ml (22.1%)] and diestrus [3.3mg/ml (4.6%)]. Proteins of 160 kDa fraction were observed in pro-estrus [9.8 mg/ml (10.5%)] and met-estrus [10.7 mg/ml (15%)], 200 kDa proteins were observed only in diestrus [6.3mg/ml (8.7%)]; finally, 210 and 270 kDa proteins were observed only in estrus [3.4 mg/ml (3.8%) and 2.0 mg/ml (2.2%), respectively]. It was concluded that protein profile of the bovine uterine lumen changes during the estrous cycle, both in quality and quantity, which may be a part of uterine adaptation for the physiological events. Further identification of these proteins would give us a better insight into their role in the bovine reproduction.     

Protein and peptide factors from Bacillus sp.739 inhibit the growth of phytopathogenic fungi

Thermolabile peptides inhibiting the growth of Helminthosporium sativum, a facultative phytopathogen, have been isolated from the low-molecular-weight fraction of extracellular metabolites of the strain Bacillus sp. 739. Paper chromatography of the fraction, followed by bioautography, revealed the presence of three components exhibiting antifungal activity. These components were separated by gel chromatography on Toyopearl HW-40. SDS-PAGE (the Laemmli procedure) demonstrated that only one component was a protein (MW, approximately 14 kDa). The other two substances were polypeptides with molecular weights less than 6 kDa each. The protein factor inhibited the growth of H. sativum with a minimum effective concentration of 0.1 to 0.2 mg/ml.     

Val-Gly-Val-Ala-Pro-Gly, a repeating peptide in elastin, is chemotactic for fibroblasts and monocytes

Recent studies have demonstrated that tropoelastin and elastin-derived peptides are chemotactic for fibroblasts and monocytes. To identify the chemotactic sites on elastin, we examined the chemotactic activity of Val-Gly-Val-Ala-Pro-Gly (VGVAPG), a repeating peptide in tropoelastin. We observed that VGVAPG was chemotactic for fibroblasts and monocytes, with optimal activity at approximately 10(-8) M, and that the chemotactic activity of VGVAPG was substantial (half or greater) relative to the maximum responses to other chemotactic factors such as platelet-derived growth factor for fibroblasts and formyl-methionyl-leucyl-phenylalanine for monocytes. The possibility that at least part of the chemotactic activity in tropoelastin and elastin peptides is contained in VGVAPG sequences was supported by the following: (a) polyclonal antibody to bovine elastin selectively blocked the fibroblast and monocyte chemotactic activity of both elastin-derived peptides and VGVAPG; (b) monocyte chemotaxis to VGVAPG was selectively blocked by preexposing the cells to elastin peptides; and (c) undifferentiated (nonelastin producing) bovine ligament fibroblasts, capable of chemotaxis to platelet-derived growth factor, did not show chemotactic responsiveness to either VGVAPG or elastin peptides until after matrix-induced differentiation and the onset of elastin synthesis. These studies suggest that small synthetic peptides may be able to reproduce the chemotactic activity associated with elastin-derived peptides and tropoelastin.