Cytokine-inducing activity of a proline-rich polypeptide complex (PRP) from ovine colostrum and its active nonapeptide fragment analogs.

A complex of proline-rich polypeptides (PRP) was isolated from ovine colostrum in our laboratory and was shown to possess immunomodulatory properties and psychotropic activity, including beneficial effects in the treatment of Alzheimer's disease. A nonapeptide fragment (NP): Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro, isolated from the chymotryptic digestion products of PRP, and its C-terminal fragment, a hexapeptide (HP): Tyr-Val-Pro-Leu-Phe-Pro also exhibited immunoregulatory activity. Although NP and HP expressed activity similar to that of PRP in studies on humoral and cellular immune responses, in other immune processes, e.g. induction of cytokines, they showed markedly lower activity than PRP. In the search for more active peptides, in the present study, we compared the cytokine-inducing ability of PRP, NP, HP, and linear oligomers of NP or HP. For this purpose, the induction of IFN, TNF-alpha, IL-6, and IL-10 in human whole blood cell cultures was measured. NP, HP, and their oligomers showed differential effects in the induction of cytokines, generally lower than that of PRP. Only the PRP complex showed a bell-shaped dose-response dependence suggesting regulatory properties. There were no distinct differences between monomeric forms of NP (NP1) or HP (HP1) and their oligomers in the induction of IFN and TNF-alpha (Th1 cytokines) but such differences were found in the induction of IL-6 and IL-10 (Th2 cytokines). Dimer (NP2) was less active than the monomeric NP1 nonapeptide in the induction of IL-6 and IL-10. On the other hand, oligomers: HP3 and HP4, showed a significantly higher ability to induce Th2 cytokines compared to HP1, HP2 or NP peptides. This was especially evident in the case of IL-10 induction, where the activity of HP4 surpassed the activity of PRP and approached the activity of LPS-PHA. The results obtained showed that some of the peptides studied, when used at higher concentrations (100 microg/ml) may replace the PRP complex as cytokine inducers. Our data also suggest the possibility of using certain oligomers for selective induction of particular cytokines.

Chromogenic substrates of bovine beta-trypsin: the influence of an amino acid residue in P1 position on their interaction with the enzyme.

The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design and synthesize a series of trypsin chromogenic substrates modified in position P1: Ac-Ala-Val-Abu-Pro-X-pNA, where X = Orn, Lys, Arg, Har, Arg(NO(2)), Cit, Hci, Phe(p-CN), Phe(p-NH(2)); pNA = p-nitroanilide. The most active compounds (as determined by specificity constant k(cat)/K(m)) were peptides with the Arg and Lys residues in the position discussed. Changes in the length and the decrease of the positive charge of the amino acid residue side chain in position P(1) resulted in the decrease or loss of the affinity towards bovine beta-trypsin. Among peptides containing amino acid residues with uncharged side chains in position P1, only one with p-cyano-l-Phe revealed activity. These results correspond well with trypsin inhibitory activity of CMTI-III analogues modified in the equivalent position, indicating the same type of interaction between position P1 of the substrate or inhibitor and S1 site specificity of trypsin.

Specific induction of Z-DNA conformation by a nuclear localization signal peptide of lupin glutaminyl tRNA synthetase.

Recently we have sequenced cDNA of plant glutaminyl-tRNA synthetase (GlnRS) from Lupinus luteus. At the N terminal part the protein contains a lysine rich polypeptide (KPKKKKEK), which is identical to a nuclear localization signal (NLS). In this paper we showed that two synthetic peptides (20 and 8 amino acids long), which were derived from lupin GlnRS containing the NLS sequence interact with DNA, but one of them (8aa long) changing its conformation from the B to the Z form. This observation clearly suggests that the presence of the NLS polypeptide in a leader sequence of GlnRS is required not only for protein transport into nucleus but also for regulation of a gene expression. This is the first report suggesting a role of the NLS signal peptide in structural changes of DNA.

Design, chemical synthesis and kinetic studies of trypsin chromogenic substrates based on the proteinase binding loop of Cucurbita maxima trypsin inhibitor (CMTI-III).

A series of trypsin chromogenic substrates with formula: Y-Ala-X-Abu-Pro-Lys-pNA, where X = Gly, Ala, Abu, Val, Leu, Phe, Ser, Glu and Y = Ac, H; pNA = p-nitroanilide was synthesized. The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design the trypsin substrates. To evaluate the influence of position P(4) on the substrate-enzyme interaction, kinetic parameters of newly synthesized substrates with bovine beta-trypsin were determined. The increasing hydrophobicity of the amino acid residue (Gly, Ala, Abu, Val) introduced in position P(4) significantly enhanced the substrate specificity (k(cat)/K(m)) which was over 8 times higher for the last residue than that for the first one. The introduction of residues with more hydrophilic side chain (Glu, Ser) in this position reduced the value of this parameter. These results correspond well with those obtained using molecular dynamics of bovine beta-trypsin with monosubstituted CMTI-I analogues, indicating that in both trypsin substrate and inhibitor position 4 plays an important role in the interaction with the enzyme.

The effect of statherin and its shortened analogues on anaerobic bacteria isolated from the oral cavity.

The susceptibility (MIC) of 44 strains of anaerobic bacteria isolated from the oral cavity and 3 standard strains to statherin and its C-terminal fragments with sequences QYQQYTF, YQQYTF, QQYTF, QYTF and YTF was determined by means of plate dilution technique in Brucella agar with 5% content of defibrinated sheep's blood, menadione and hemin. The culture was anaerobic. As shown, at concentrations from 12.5 to 100 microg/ml statherin and its C-terminal fragments inhibited the growth of anaerobic bacteria isolated from the oral cavity. Peptostreptococcus strains were the most susceptible to statherin and YTF (MIC < or = 12.5 mg/ml), whereas the most susceptible to the peptides investigated were Fusobacterium necrogenes and Fusobacterium necrophorum strains: QYQQYTF, YQQYTF, QQYTF, QYTF (MIC < or = 12.5 microg/ml). Prevotella oralis, Bacteroides forsythus and Bacteroides ureolyticus strains exhibited the lowest susceptibility (MIC > 100 microg/ml). When analysing the bacteriostatic activity of statherin it should be pointed out that the concentrations of this peptide used in microbiological investigations are within the range of physiological concentrations determined for whole saliva when at rest and stimulated in healthy donors of 19-25 years of age. Since the anaerobes investigated may be involved in the diseases of periodontum, the results presented seem to have also a practical aspect, i.e. a possibility to apply the C-terminal fragments of statherin as a novel therapeutic agent, affecting favourably the oral cavity.

Studies on binding of HIV-1 p24gag peptide to HLA-Cw3+ cells.

Human major histocompatibility complex class I antigens, HLA-C, are expressed on the cell surface at approximately a tenfold lower level than HLA-A and -B. We hypothesized that the expression of HLA-C is limited by the quantity of high affinity peptides which bind to these molecules, thus allowing only a small fraction of HLA-C molecules to be transported and/or to remain stable on the cell surface. If this assumption is correct, then the addition of exogenous peptide should increase cell surface HLA-C expression. To verify the hypothesis, we pulsed lymphoblastoid cell line PAJ (HLA-Cw3+) with synthetic HIV-1 p24gag 145-152 peptide, known to be presented to T-lymphocytes by HLA-Cw3 molecule. PAJ (HLA-Cw3+) cells bound approximately two times more of the peptide than HAJ (HLA-Cw3-), and four times more than 500/C9 (HLA-Cw3-) cells. Accordingly, overnight pulsing of PAJ cells with the p24gag 145-152 peptide caused an increase in class I HLA expression detected on the cell surface by flow cytofluorimetric analysis with anti-HLA-B,C monoclonal antibodies but not by anti-HLA-A antibody. In contrast, HLA-Cw3- cells treated in the same manner did not show any increase of HLA class I expression. Our data suggest that low concentration of high affinity peptides within the cell may be one of the factors limiting cell surface expression of HLA-C molecules.

Modifications outside the proteinase binding loop in Cucurbita maxima trypsin inhibitor III (CMTI-III) analogues change the binding energy with bovine beta-trypsin.

Five 26-peptide analogues of the trypsin inhibitor [Pro18]CMTI-III containing Leu or Tyr in position 7 and Val or Tyr in position 27: 1 (Leu7, Tyr27), 2 (Tyr7, Val27), 3 (Tyr7, Tyr27), 4 (Leu7, Val27) and 5 (Leu7, Ala18, Tyr27) were synthesized by the solid-phase method. Analogues 1-4 displayed Ka with bovine beta-trypsin of the same order of magnitude as the wild CMTI-III inhibitor, whereas for analogue 5, this value was lower by about 3 orders of magnitude. This indicated that for the analogues with Pro (but not with Ala) in position 18, the side-chain interactions between positions 7 and 27 did not play a critical role for the stabilization of the active structure. In addition, these results also suggest that Tyr7 is involved in an additional aromatic interaction with position 41 of the enzyme.

Antiserum against connexin32 fragment reacts with a 32-kD protein localised in gap junctions of mouse and rat liver, endometrium and in the fish heart.

The expression of various connexins so far identified is metabolically and developmentally regulated. Examples include uterine endometrium where the expression of gap junction protein, connexin32 (Cx32) is regulated by steroid hormones. In this study we attempted to synthesise a short peptide which matches the portion of the amino acid sequence of the Cx32. Cx32 has primary structure predicted from the nucleotide sequence of cDNA clone. A fragment of Cx32 molecule was synthesised to produce anti-peptide antibody for detecting gap junction protein in mouse and rat liver and endometrium. The 12-peptide, plus Abu residue that corresponds to residues 108-119 (LEGHGDPLHLEE-Abu) of the rat Cx32 (283-mer) was synthesised by the solid phase method. Antibodies against this peptide were raised in rabbits, screened for reactivity and specificity using dot blot assays [15] and used for immunocytochemical staining at the light and at electron microscope levels. The antibodies also reacted with fish heart myocardium.

Effect of colostrinin, an immunomodulatory proline-rich polypeptide from ovine colostrum, on sialidase and beta-galactosidase activities in murine thymocytes.

Colostrinin: a proline-rich polypeptide (PRP) from ovine colostrum and its nonapeptide active fragment (NP) induce maturation and differentiation of murine thymocytes, formation of helper cells from PNAhigh thymocytes and cytotoxic T cells from PNAlow thymocytes. These processes are accompanied by changes in expression of receptors for peanut agglutinin (PNA), PNAhigh thymocytes were transformed into PNAlow cells, and vice versa. It was shown, in various laboratories, that sialyltransferases are involved in the transformation of PNAhigh thymocytes into PNAlow cells. To find out whether the expression of receptors for PNA on murine thymocytes might also be influenced by other enzymes, we decided to study the effect of PRP and NP on sialidase and beta-galactosidase activities in these cells. The results obtained showed that the most of sialidase activity of murine thymocytes is present in the plasma membrane compartments. Both thymocyte subpopulations PNAhigh and PNAlow, showed similar sialidase activity, which was not affected either by PRP or NP. In contrast to sialidases, most of beta-galactosidase activity was present in the cytosol. PNAhigh, thymocytes showed a higher beta-galactosidase activity than PNAlow cells. Incubation of immature, PNAhigh, thymocytes with PRP or NP enhanced the beta-galactosidase activity in these cells. The presented results suggest that sialidases seem not to be involved in modulation of surface sialic acid content during murine thymocyte maturation. On the other hand, stimulation of activity of beta-galactosidase in PNAhigh, immature thymocytes by PRP and NP suggests that beta-galactosidase in murine thymocytes might be involved in transformation of PNAhigh into PNAlow cells.

Distance between the basic group of the amino acid residue's side chain in position P1 of trypsin inhibitor CMTI-III and Asp189 in the substrate pocket of trypsin has an essential influence on the inhibitory activity.

Three new analogues of trypsin inhibitor CMTI-III were synthesized by the solid-phase method: [Lys5]-CMTI-III, [Orn5]CMTI-III and [Dab5]CMTI-III. Only one analogue with L-lysine residue in position P1 showed inhibitory activity of the same order of magnitude as did wild CMTI-III. Two remaining analogues were completely inactive. A conclusion was drawn that the distance between the basic group of the amino acid residue's side chain in position P1 of the trypsin inhibitor CMTI-III and Asp189 in the substrate pocket of trypsin plays an essential role for the trypsin-inhibitor interaction.

Influence of L-naphthylalanine in position 3 of AVP and its analogues on their pharmacological properties.

We describe the synthesis and pharmacological properties of two series of analogues: one which consists of three peptides having L-1-naphthylalanine in position 3 and the second composed of analogues substituted in position 3 with L-2-naphthylalanine. All peptides were tested in bioassays for pressor and antidiuretic activities. We also checked the uterotonic activity in vitro. We observed that the activity of counterparts in both series is, in two cases, strikingly different. One of the new analogues, [(L-2-Nal)3,(D-Arg)8]VP is among the most potent antagonists of the vasopressor response to AVP. Moreover, it is the first potent V1 antagonist devoid of antiuterotonic activity. This analogue was designed without modification of position 1, which was previously thought to be essential for substantial pressor antagonism. Two other peptides, [Mpa1;(L-2-Nal)3;(D-Arg)8]VP and [Mpa1,(L-1-Nal)3,D-Arg)8]VP, are highly potent V2 agonists. The second analogue is highly selective. With the exception of [(L-2-Nal)3]AVP, which showed weak antioxytocic activity, (L-Nal)3 modification resulted in the almost complete removal of interaction of our analogues with oxytocic receptors in vitro. Our results suggest that position 3 in AVP and its analogues is important not only for binding and recognition as previously though, but also for pressor, antidiuretic and uterotonic activities. We also assume that the hindering effect caused by bulky naphthyl moiety has a significant impact on the bioactive conformations of molecules which contain Nal residue, and can thus influence their interaction with V1, V2 and oxytocic receptors.

Interaction of HIV Tat model peptides with tRNA and 5S rRNA.

New data are presented on the interaction of model synthetic peptides containing an arginine-rich region of human immunodeficiency virus (HIV-Tat), with native RNA molecules: tRNA(Phe) of Saccharomyces cerevisiae and 5S rRNA from Lupinus luteus. Both RNA species form complexes with the Tat1 (GRKKRRQRRRA) and Tat2 (GRKKRRQRRRAPQDSQTHQASLSKQPA) peptides, as shown by electrophoretic gel shift and RNase footprint assays, and CD measurements. The nucleotide sequence UGGG located in the dihydrouridine loop of tRNAPhe as well as in the loop D of 5S rRNA is specifically protected against RNases. Our data indicate direct interactions of guanine of RNA moieties with arginine residues. These interactions seem similar to those observed in DNA-protein complexes, but different from those previously observed in the TAR RNA-Tat complexes.

Specific interactions of Cu2+ ions with fragments of envelope protein of hepatitis B virus.

Potentionmetric and spectroscopic (EPR, CD and absorption spectra) data have shown that a fragment of envelope proteins of the hepatitis B virus could be very specific bind molecules for Cu2+ ions using arginine lateral NH2 donor sites. The presence of Pro and Asp residues makes Arg binding not only very specific, but also very efficient.

Surprising pharmacological activity of analogues designed by substitution of position 3 in arginine vasopressin (AVP) and 8-D-arginine vasopressin with L-2-napthylalanine.

In an attempt to develop more active and selective analogues of arginine vasopressin (AVP), two peptides have been designed, synthesized and tested for vasopressor (V1-receptors) and antidiuretic (V2-receptors) activities. We also estimated the uterotonic and anti-uterotonic activities of these compounds in-vitro. The first peptide, [(L-2-Nal)3] AVP is a highly active V2-agonist. The second analogue, [(L-2-Nal)3, (D-Arg)R]VP is among the most potent antagonists of the vasopressor response to AVP. Moreover, it is the first V1-antagonist devoid of anti-uterotonic activity. High antipressor potency of the second peptide was achieved without modification of position 1.

Fluorescence and Monte Carlo conformational studies of the (1-15) galanin amide fragment.

Galanin (GAL) is a 29 amino acid C-terminally aminated linear neuropeptide showing diverse biological activities. The N-terminal (1-15)GAL-NH2 fragment was shown to have a very high affinity to the galanin receptor. In this work we describe the results of a combined fluorescence and Monte Carlo studies, the latter carried out using the ECEPP/3 force field with and without including hydration, on the (1-15)GAL-NH2 fragment. Using the time-domain technique we measured fluorescence decay times of the tyrosine residue in position 9. Based on the Forster energy transfer theory we calculated the distance and distance distribution between the Trp2 (acceptor) and Tyr9 (donor) aromatic side chains. The distance obtained was about 10.5 angstrom and half-width, hw, of the distance distribution was 5.6 angstrom. This results were found to be in good agreement with the chromophore distances calculated for the low-energy solution confirmations obtained in Monte Carlo simulations. All the low-energy conformations obtained in the absence of water were almost all-helical with the exception of a few C-terminal residues. In contrast, none of the low-energy solution conformations contained any significant amount of secondary structure. These findings are in agreement with the results of earlier CD and NMR conformational studies of galanin in water and non-aqueous solvents. On the other hand, the conformations obtained in the presence of water turned out to be largely compact in the N-terminal hydrophobic part. This explains the relatively short distance between chromophores and narrow distance distribution obtained in fluorescence measurements.

Mechanism of the activation of proteinase inhibitor synthesis by systemin involves beta-sheet structure, a specific DNA-binding protein domain.

We analyzed a tertiary structure of systemin, the first identified polypeptide plant hormone, using two-dimensional NMR spectroscopy. From these data and molecular dynamics calculations we concluded that the peptide can adopt a Z-like-beta-sheet structure, which has previously been found in many specific DNA-binding proteins. Using DNA-cellulose affinity chromatography, we showed that systemin binds strongly to DNA. We suggest that the specific systemin-DNA interaction, particularly in a promoter region of the proteinase inhibitors, could effect gene expression and thus explain the biological activity of systemin.

Thermoregulatory activity of sodium nitroprusside and arginine vasopressin.

1. Thermal responses to sodium nitroprusside (SNP, 3 mg/kg/hr) and arginine vasopressin (AVP, 3 micrograms/kg) were investigated in normothermic and febrile rabbits (LPS, 1 microgram/kg) at ambient temperature of 20.0 +/- 1.0 degrees C. Furthermore, blood pressure after these drugs was tested on a separate group of animals. 2. I.v. infusion of SNP produced hypothermia and attenuated pyrogen fever. On the other hand, AVP increased body temperature and intensified the febrile response. 3. Both drugs affected in an opposite way blood pressure, i.e. SNP produced falls and AVP increases in this parameter. 4. The relationship between the activity of the vascular and thermoregulatory systems in normothermic or febrile state is discussed.

Cu(II) binding by angiotensin II fragments: Asp-Arg-Val-Tyr-Ile-His and Arg-Val-Tyr-Ile-His. Competition between amino group and imidazole nitrogens in anchoring of metal ions.

Potentiometric and spectroscopic (absorption, circular dichroism and electron paramagnetic resonance) study on the coordination of two angiotensin II fragments (Asp-Arg-Val-Tyr-Ile-His and Arg-Val-Tyr-Ile-His) to Cu(II) ions has shown that competition between amino and imidazole nitrogens to anchor metal ions is a complicated process and may lead to formation of macrochelate rings. The important factor that influences this competition is the distance between competing His and N-terminal residues (number of spacer residues in a peptide sequence).

Intracerebroventricular galanin and N-terminal galanin fragment enhance the morphine-induced analgesia in the rat.

Behavioral effect of galanin and its fragments, galanin1-15 and galanin16-29 (200 ng, 1 and 5 micrograms), after intracerebroventricular (i.c.v.) administration was studied in rats. The number of crossings and pippings and the time of locomotion (an open field test) showed a similar sedative action of galanin and galanin16-29, with no significant effect of galanin1-15. Galanin and its fragments, injected in doses of 200 ng, 1 and 5 micrograms, did not affect nociception, as measured by a tail-flick and paw pressure test. Galanin and galanin1-15, but not galanin16-29 (5 micrograms i.c.v.), injected together with morphine (2.5 micrograms i.c.v.), significantly potentiated the analgetic effect of morphine assessed by a paw pressure test; a similar tendency was also observed in a tail-flick test. Galanin and its two fragments injected in doses of 200 ng, 1 and 5 micrograms, did not change the effect of morphine given in a dose of 1 microgram. These data suggest that galanin, having no effect when given alone, potentiate the analgetic effect of morphine. The fact that the N-terminal fragment of galanin acts like a natural peptide suggests a receptor mediated action. In conclusion, the analgesic effect of morphine was potentiated by galanin and its N-terminal fragment galanin1-15. On the other hand, behavioral study showed a similar sedative action of galanin and C-terminal fragment galanin16-29. This suggests that the N- and C-terminal fragments of galanin are differentially involved in behavioral effects of the peptide.