Effects of several peptides with pro-arg-leu-NH2 C-terminal sequence on invertebrate muscles

1. Effects of the following PRLamide peptides on some invertebrate muscles were examined: APNFLAYPRLamide (a peptide of the bivalve mollusc Mytilus), AAPLPRLamide (a peptide of the echiuroid Urechis), FTPRLamide (a fragment of a peptide of the insect Pseudaletia), YFSPRLamide (a fragment of a peptide of the insect Heliothis) and pQTSFTPRLamide (a peptide of the insect Leucophaea).2. In the ABRM of Mytilus, the Mytilus and Urechis peptides potentiated phasic contraction by repetitive electrical stimulation. The former was more potent than the latter. The three insect-origin peptides did not show any potentiating effect but showed a weak antagonistic action on the potentiating effect of the Mytilus peptide. Except the Mytilus peptide, the PRLamide peptides showed little or no relaxing effect on catch tension, though they accelerated catch-relaxing response to repetitive electrical stimulation.3. In the inner circular body-wall of muscle of Urechis, the PRLamide peptides showed a potentiating effect on twitch contraction. The Urechis peptide was most potent. In the esophagus of the annelid Perinereis, the PRLamide peptides showed a contractile effect. The Urechis peptide was also most potent.4. In the crop of the cricket Gryllus, the insect-origin peptides markedly potentiated spontaneous rhythmic contractions. The Mytilus peptide showed a weak potentiating effect, but the Urechis peptide showed little or no effect.     

Neural mechanism of the antisecretory effect of peptide YY in the rat colon in vivo

The purpose of this work was to determine the mechanism of the antisecretory effect of peptide YY in the rat colon and whether this effect is physiological. In this prospect, doses of exogenous peptide YY producing physiological and supraphysiological plasma levels were intravenously infused in rats provided with colonic and jejunal ligated loops in vivo, under secretory stimulation by vasoactive intestinal peptide. Peptide YY decreased the secretory effect of VIP in a dose-related fashion. The effect of peptide YY was blocked or strongly decreased by tetrodotoxin, hexamethonium, idazoxan, haloperidol, and the sigma antagonist BMY 14, 802 in both the colon and jejunum. We conclude that peptide YY decreases water and electrolyte secretion in the colonic mucosa by a complex neural mechanism involving at least two neurons connected through a nicotinic synapse, alpha-2 adrenoceptors and sigma receptors, and that this effect can occur with physiological doses of peptide YY.     

The potentiations by insulin-stimulating peptide from bovine serum albumin of the effects of insulin mimickers and insulin in stimulating glucose utilization by rat adipocytes

An insulin-stimulating peptide derived from bovine serum albumin by digestion with trypsin was shown to inhibit insulin degradation. Addition of this peptide (1.2 microM) to the medium of isolated rat adipocytes markedly inhibited the degradation of insulin in the medium, but had a little effect on degradation of cell-associated insulin. Moreover, this peptide did not prevent dissociation of cell-associated insulin, suggesting that it is a bacitracin-type, not a chloroquine-type inhibitor of insulin degradation. The peptide also potentiated the stimulation by insulin mimickers of glucose oxidation by rat adipocytes, strongly indicating that it has some other effects besides inhibition of insulin degradation. Therefore, the effect of the peptide on activation of pyruvate dehydrogenase (PDH), one of the postbinding actions of insulin, was studied. Addition of the peptide (4 microM) to adipocytes was found to activate PDH in the absence or presence of insulin. This stimulatory effect of the peptide on PDH was dose-dependent and was observed in both whole cells and subcellular fractions of rat adipocytes. The peptide also stimulated PDH in a subcellular system of either plasma membranes and mitochondria or mitochondria only. Sodium fluoride, an inhibitor of phosphatase, blocked the action of the peptide almost completely, suggesting that the stimulatory effect of the peptide on PDH activity is at least partly due to its activation of PDH phosphatase. The mechanisms of action of the peptide are discussed. The peptide should be useful in studies on modulation of the action of insulin.     

Stimulation of peptide elongation by thyroxine.

This study suggests that thyroxine stimulates peptide elongation in a cell-free rat liver polyribosome system. The thyroxine effect persists in the presence of sufficient aurintricarboxylic acid to prevent polyuridylic acid-stimulated peptide initiation. In addition, thyroxine stimulates elongation of pre-existing polyphenylalanine chains providing conclusive evidence that the effect does not depend on peptide initiation. Thyroxine does not stimulate release of nascent peptides from ribosomes into the supernatant phase of the reaction mixture. Therefore in this protein-synthesis system the thyroxine effect is expected to occur at one or more of the reactions of peptide chain elongation, which include aminoacyl-tRNA binding, peptide bond synthesis and translocation.     

Pineal peptide with an inhibiting effect on growth of HeLa S3 tumor cells

The aim of the present study was to assess whether a peptide fraction isolated from calf pineal glands has an effect on proliferation and morphology of HeLa S3 tumor cells. Under the experimental conditions adopted, the results showed that the peptide has a marked inhibitory effect on proliferation of HeLa S3 cells and that permeabilization with calcium phosphate of the plasmatic membrane increases this effect. Moreover, the pineal peptide affects the cytoskeletal morphology of HeLa cells by modifying the distribution of actin. The peptide is probably internalized by the cells and irreversibly modifies the cytoskeletal morphology with consequent inhibition of cellular proliferation.     

Characterization of inhibitory mechanism and antifungal activity between group-1 and group-2 phytocystatins from taro (Colocasia esculenta)

Tarocystatin from Colocasia esculenta, a group-2 phytocystatin, is a defense protein against phytopathogenic nematodes and fungi. It is composed of a highly conserved N-terminal region, which is homological to group-1 cystatin, and a repetitive peptide at the C-terminus. The purified recombinant proteins of tarocystatin, such as full-length (FL), N-terminus (Nt) and C-terminus (Ct) peptides, were produced and their inhibitory activities against papain as well as their antifungal effects were investigated. Kinetic analysis revealed that FL peptide exhibited mixed type inhibition (K(ia) = 0.098 microM and K(ib) = 0.252 microM) and Nt peptide showed competitive inhibition (K(i) = 0.057 microM), whereas Ct peptide possessed weak papain activation properties. A shift in the inhibitory pattern from competitive inhibition of Nt peptide alone to mixed type inhibition of FL peptide implied that the Ct peptide has an regulatory effect on the function of FL peptide. Based on the inhibitory kinetics of FL (group-2) and Nt (group-1) peptides on papain activity, an inhibitory mechanism of group-2 phytocystatins and a regulatory mechanism of extended Ct peptide have each been proposed. By contrast, the antifungal activity of Nt peptide appeared to be greater than that of FL peptide, and the Ct peptide showed no effect on antifungal activity, indicating that the antifungal effect is not related to proteinase inhibitory activity. The results are valid for most phytocystatins with respect to the inhibitory mechanism against cysteine proteinase.     

Two conserved residues are important for inducing highly ordered membrane domains by the transmembrane domain of influenza hemagglutinin

The interaction with lipids of a synthetic peptide corresponding to the transmembrane domain of influenza hemagglutinin was investigated by means of electron spin resonance. A detailed analysis of the electron spin resonance spectra from spin-labeled phospholipids revealed that the major effect of the peptide on the dynamic membrane structure is to induce highly ordered membrane domains that are associated with electrostatic interactions between the peptide and negatively charged lipids. Two highly conserved residues in the peptide were identified as being important for the membrane ordering effect. Aggregation of large unilamellar vesicles induced by the peptide was also found to be correlated with the membrane ordering effect of the peptide, indicating that an increase in membrane ordering, i.e., membrane dehydration, is important for vesicle aggregation. The possibility that hydrophobic interaction between the highly ordered membrane domains plays a role in vesicle aggregation and viral fusion is discussed.     

Atrial natriuretic peptide interferes with calcium requirements in vascular tissues of the rat

The inhibitory effect of atrial natriuretic peptide on the myotropic action of phenylephrine on superior mesenteric artery and thoracic aorta rings was studied to test the hypothesis that this peptide interferes with the mobilization of intra- or extra-cellular calcium produced by vasoconstrictor agents. In the absence of calcium in the bathing solution, phenylephrine (10(-6) M) produced a residual effect, which was antagonized in a concentration-dependent manner by the atrial peptide in both mesenteric artery and aorta rings. When calcium (2.5 mM) was added to the bathing solution after the response to phenylephrine in the absence of calcium, a further increase in the tonus of the tissue was observed. This effect was also antagonized by atrial natriuretic peptide in a dose-dependent manner in the two tissues. These results suggest that atrial natriuretic peptide inhibits the effect of vasoconstrictor agents by functionally interfering with the mobilization of intra- and extra-cellular calcium produced by these vasoconstrictors.     

The relationship between micelle formation and biological activity of peptide 562–572 of luteinizing hormone receptor modified with decanoyl radicals

Lipophilic derivatives of peptides corresponding to cytoplasmic regions of G protein-coupled receptors (GPCR) can act as intracellular agonists. Our previous work showed that peptides corresponding to residues 562–572 of luteinizing hormone receptor and modified with palmitate or decanoate at the C-terminus activate adenylate cyclase in rat testes. The stimulating effect of peptide 562–572 modified with decanoates at both the N- and the C-termini (peptide IV) reached its maximum at the peptide concentration of 10^–5 M and diminished with further increase in its concentration. It was supposed that this effect was due to peptide IV ability to form micelles. To verify this hypothesis, the relationship between biological activity, hydrophobicity, and ability to form micelles was investigated for peptide IV and other acylated derivatives of peptide 562–572, including those carrying C-terminal decanoate (peptide III) and palmitate (peptide VI) moieties. It was found that the stimulating effect of peptide IV taken in the concentration of 10^–5 M on adenylate cyclase activity in plasma membranes of rat testes and ovaries was only slightly lower than that of peptide VI and higher than the effect of peptide III. At the concentration of 10^–3 M, the effect of peptide IV was 20–27% lower and amounted to only 50–51 and 87–88% of the effects of peptides VI and III, respectively. In spite of its high hydrophobicity, peptide IV was characterized with an abnormally low retention time when eluted from a Nucleosil C8 column during reverse-phase HPLC: it was even lower than the retention time of nonmodified peptide 562–572. However, the retention time of peptide IV, but not of other peptides, increased significantly when the eluent contained a higher proportion of trifluoroacetic acid, which disrupts micellelike structures (0.5 instead of 0.1%). The surface tension of peptide IV solution in water slightly decreased with increasing peptide concentration, but rapidly dropped and reached a plateau at the concentration of 7 × 10^–6 M, which indicates the beginning of micelle formation. Thus, peptide IV in the concentrations above 10^–5 M forms micelles, which prevents it from interacting with the receptor. The ability of GPCR peptides to aggregate and form micelles should be taken into account in the development of their new membrane-active analogs.     

Limited validity of group additivity for the folding energetics of the peptide group

The principle of group additivity is a standard feature of analyses of the energetics of protein folding, but it is known that it may not always be valid for the polar peptide group. The neighboring residue effect shows that group additivity is not strictly valid for a heteropeptide. We show here that group additivity fails seriously for peptide groups close to either peptide end, even for a homopeptide that has blocked end groups with no formal charges involved. The failure of group additivity is caused by the electrostatic character of the solvation of peptide polar groups and is illustrated with values of the electrostatic solvation free energy (ESF) calculated by DelPhi. Solvation free energies and enthalpies are known experimentally for monoamides and are often used to model the solvation of peptide groups, but ESF results show that monoamide values are very different from those of peptide groups. A main cause of the difference is that peptide solvation depends on the dipole-dipole interactions made between adjacent peptide groups, which vary with peptide conformation. Ligands that interact with the peptide backbone by an electrostatic mechanism could show a similar peptide end effect, and hydrogen exchange results from the literature confirm that exchange rates are position-dependent close to peptide ends.     

Induction of vascular smooth muscle cell growth by selective activation of the thrombin receptor. Effect of heparin.

The synthetic peptide, SFLLRNPNDKYEPF, has been recently described as a peptide mimicking the new amino-terminus created by cleavage of the thrombin receptor, therefore acting as an agonist of the thrombin receptor. This peptide was a potent mitogen for rabbit arterial smooth muscle cells (SMC) and exhibited the same activity as that of native alpha-thrombin. Both compounds stimulated the proliferation of growth-arrested SMCs with half-maximum mitogenic responses at 1 nM. NAPAP, a synthetic inhibitor of the enzymatic activity of thrombin, specifically inhibited thrombin-induced SMC growth (IC50 = 0.35 +/- 0.04 microM) but was without effect on the mitogenic effect of the agonist peptide. These results therefore demonstrate that the mitogenic effect of alpha-thrombin for SMCs is intimately linked to its esterolytic activity. Heparin, which inhibited fetal calf serum-induced SMC growth, was without effect on thrombin-induced SMC growth but strongly reduced the mitogenic effect of the agonist peptide (IC50 = 32 +/- 5 micrograms/ml). This effect was not related to the anti-coagulant activity of heparin but was highly dependent on molecular mass and on the global charge of the molecule and was also observed for other sulphated polysaccharides such as pentosan polysulphate.     

Generation of murine CTL by a hepatitis B virus-specific peptide and evaluation of the adjuvant effect of heat shock protein glycoprotein 96 and its terminal fragments

Previously, we reported that a 7-mer HLA-A11-restricted peptide (YVNTNMG) of hepatitis B virus (HBV) core Ag (HBcAg(88-94)) was associated with heat shock protein (HSP) gp96 in liver tissues of patients with HBV-induced hepatocellular carcinoma (HCC). This peptide is highly homologous to a human HLA-A11-restricted 9-mer peptide (YVNVNMGLK) and to a mouse H-2-K(d)-restricted 9-mer peptide (SYVNTNMGL). To further characterize its immunogenicity, BALB/c mice were vaccinated with the HBV 7-mer peptide. It was found that a specific CTL response was induced by the 7-mer peptide, although the response was approximately 50% of that induced by the mouse H-2-K(d)-restricted 9-mer peptide, as detected by ELISPOT, tetramer, and (51)Cr release assays. To evaluate the adjuvant effect of HSP gp96, mice were coimmunized with gp96 and the 9-mer peptide, and a significant adjuvant effect was observed with gp96. To further determine whether the immune effect of gp96 was dependent on peptide binding, the N- and C-terminal fragments of gp96, which are believed to contain the putative peptide-binding domain, were cloned and expressed in Escherichia coli. CTL assays indicated that only the N-terminal fragment, but not the C-terminal fragment, was able to produce the adjuvant effect. These results clearly demonstrated the potential of using gp96 or its N-terminal fragment as a possible adjuvant to augment CTL response against HBV infection and HCC.     

Further investigation of a potential calcium channel modulator isolated from rat erythrocytes

The contractile effects of a peptide isolated from rat erythrocytes were further studied in rat aortic rings. Previous data showed that preincubation of aortic tissue with the peptide had no effect on resting tension, but significantly enhanced K+ and norepinephrine (NE) induced contraction. The calcium channel antagonist verapamil noncompetitively blocked the effect of the peptide, whereas nifedipine blockage appeared to be competitive. In the present study the peptide enhanced K+, NE, and phenylephrine (PE) induced contraction in a concentration-dependent manner, with a maximum enhancement at peptide concentrations of 10(-7)-10(-6) M. At a concentration as low as 10(-9) M, the peptide significantly enhanced K(+)-induced, but not NE- or PE-induced, contraction. The magnitude of maximal enhancement was greater for K(+)-induced contraction than that for NE- or PE-induced contraction. Preincubation of the tissues with the peptide caused a leftward shift of cumulative concentration-response curves to K+ and NE. The peptide enhancement of contraction increased with increasing K+ and NE concentration. The peptide potentiated the contractile response to Ca2+ in K(+)-depolarizing medium. It also enhanced the contractile response to NE in intracellular Ca2(+)-pool-depleted tissue following the replenishment of extracellular Ca2+, but had no apparent effect on the mobilization of intracellular calcium. Addition of nifedipine caused a rightward shift of both the peptide and Bay K 8644 concentration-response curves.(ABSTRACT TRUNCATED AT 250 WORDS)     

An inhibitory peptide derived from the α-subunit of the epithelial sodium channel (ENaC) shows a helical conformation

Proteolytic activation of the heteromeric epithelial sodium channel (ENaC) is thought to involve the release of inhibitory peptides from the extracellular domains of its α- and γ-subunit. Recently, we demonstrated that an α-13-mer peptide, corresponding to a putative inhibitory region within the extracellular domain of human αENaC, inhibits human αβγENaC. The aim of the present study was to investigate the structural basis of the inhibitory effect of this α-13-mer peptide. Analysis of the peptide by replica exchange molecular dynamics method, circular dichroism spectroscopy, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations suggested that a helical turn at the carboxy-terminus is the preferred conformational state of the α-13-mer peptide. From this we predicted that a specific mutation (leucine 188 to alanine) should have a strong effect on the conformational preferences of the peptide. To functionally test this, we compared the effect of the wild-type α-13-mer with that of a mutant α-L188A-13-mer on ENaC currents in Xenopus laevis oocytes heterologously expressing human αβγENaC. We demonstrated that replacing the leucine 188 by alanine abolished the inhibitory effect of the α-13-mer peptide on ENaC. These findings suggest that a helical conformation in its carboxyterminal part is functionally important to mediate ENaC inhibition by the α-13-mer peptide. However, high resolution structural information on the complex of the inhibitory αENaC peptide and the channel are needed to confirm this conclusion.     

An accessory peptide binding site with allosteric effect on the formation of peptide-MHC-II complexes?

MHC-II molecules bind a single peptide in their groove. Here, the authors summarise evidence that a second peptide could bind transiently to MHC-II molecules outside the groove and have an allosteric effect on peptide-MHC-II complex formation. This effect could modulate, after the antigen processing, the selection of the peptide subset presented by MHC-II molecules to the helper CD4 T cells, which regulate the specific immune response.     

Delta sleep-inducing peptide as a modulator of mediators acting on the heart

Experiments on 51 isolated rabbit hearts have documented, that delta sleep-inducing peptide (6 X 10(-6) M/l) has a modulating effect on the mediators influencing the heart. This peptide enhances negative chronotropic effect of acetylcholine (1 X 10(-6) M/l) and decreases positive chronotropic effect of noradrenaline (1 X 10(-6) M/l). Such effect may be one of mechanisms of changes in the extracardiac regulation on the heart influenced by this peptide.     

Effect of a laminin amphiphatic sequence on DPPC ordered bilayers.

A peptide sequence, stearoyl-GESIKVAVS(NH2), related to a laminin fragment, has been synthesized. Formation of aggregates was controlled by titrating a sodium anilinonaphthalene sulphonate (ANS) solution with peptide and recording fluorescence intensity increases. The results show that this system experiences a sudden increase in fluorescence at peptide concentrations around 2.5 x 10(-4) mol/L. The interaction of this hydrophobic peptide with DPPC vesicles has been studied using fluorescence techniques. Its influence on the microviscosity of bilayers was determined by studying polarization/temperature dependence for ANS and diphenyl hexatriene (DPH) fluorescent probes. With both markers the presence of peptide promotes a clear increase in anisotropy values. This indicates a rigidifying effect. Leakage studies carried out with liposomes loaded with carboxyfluorescein (CF) indicate a stabilizing effect of the peptide on bilayers, in agreement with results obtained with fluorescent probes.     

Structural effect of a recombinant monoclonal antibody on hinge region peptide bond hydrolysis

IgG hinge region peptide bonds are susceptible to degradation by hydrolysis. To study the effect of Fab and Fc on hinge region peptide bond hydrolysis, a recombinant humanized monoclonal IgG1 antibody, its F(ab')2 fragment, and a model peptide with amino acid sequence corresponding to the hinge region were incubated at 40 degrees C in formulation buffer including complete protease inhibitor and EDTA for 0, 2, 4, 6 and 8 weeks. Two major cleavage sites were identified in the hinge region of the intact recombinant humanized monoclonal antibody and its F(ab')2 fragment, but only one major cleavage site of the model peptide was identified. Hinge region peptide bond hydrolysis of the intact antibody and its F(ab')2 fragment degraded at comparable rates, while the model peptide degraded much faster. It was concluded that Fab region of the IgG, but not Fc portion had significant effect on preventing peptide bond cleavage by direct hydrolysis. Hydrolysis of hinge region peptide bonds was accelerated under both acidic and basic conditions.     

Modification of mouse hemopoietic cell proliferation in vivo by a hemoregulatory pentapeptide (HP 5b). Relation to circadian rhythms

The effects on murine hemopoietic cell proliferation of hemoregulatory peptide 5b, a synthetic factor which inhibits granulopoiesis, have been studied in vivo by 3H-thymidine labeling, cell cycle analyses by flow cytometry and total cell counts per femur. Single injections of the peptide seemed to obliterate the normal circadian rhythm of bone marrow cell proliferation. By autoradiography, the main effect was an inhibition of myelopoietic cell proliferation, although erythropoiesis was also affected. This occurred in two ways: a) an immediate effect observed in the first 24 h after a single injection, and b) a delayed effect observed 6-12 days after a week of peptide infusion which could be a result of stem cell inhibition. A secondary accumulation of cells with G2 phase DNA content was seen regularly. Thus the hemoregulatory peptide seems to influence hemopoietic cell proliferation and has its main effect on myelopoiesis. However, inhibition is less marked than observed previously in stem cells.     

Hemolytic activity of a cyclic peptide Ro09-0198 isolated from Streptoverticillium

Ro09-0198, a cyclic peptide isolated from culture filtrates of Streptoverticillium griseoverticillatum, induced lysis of erythrocytes. Preincubation of the peptide with phosphatidylethanolamine reduced the hemolytic activity, whereas other phospholipids present in erythrocytes in nature had no effect. A study of the structural requirements on phosphatidylethanolamine necessary for interaction with the peptide indicates that Ro09-0198 recognizes strictly a particular chemical structure of phosphatidylethanolamine: dialkylphosphoethanolamine as well as 1-acylglycerophosphoethanolamine showed the same inhibitory effect on hemolysis induced by Ro09-0198 as diacylphosphatidylethanolamine, whereas phosphoethanolamine gave no inhibitory effect. Neither phosphatidyl-N-monomethylethanolamine nor alkylphosphopropanolamine had an inhibitory effect. Consequently, the hydrophobic chain is necessary for the interaction and the phosphoethanolamine moiety is exactly recognized by the peptide. Ro-09-0198-induced hemolysis was temperature-dependent and the sensitivity of hemolysis differed greatly among animal species.