The anti-infective peptide, innate defense-regulator peptide, stimulates neutrophil chemotaxis via a formyl peptide receptor

The anti-infective peptide, innate defense-regulator peptide (IDR-1), has been selectively reported to modulate the innate immune response. We found that IDR-1 stimulates the chemotactic migration in human neutrophils. Moreover, IDR-1-induced neutrophil chemotaxis was completely blocked by pertussis toxin, suggesting the importance of the G_i protein in this process. The mechanism governing the IDR-1-induced neutrophil chemotaxis was found to be completely inhibited by the formyl peptide receptor (FPR) antagonist; cyclosporin H. IDR-1 was also found to induce chemotactic migration in FPR but not in vector-expressing HCT116 cells. Meanwhile, IDR-1 failed to stimulate superoxide anion generation and intracellular calcium increase in human neutrophils. Furthermore, IDR-1 was found to inhibit fMLF (an FPR agonist)-induced superoxide generation and calcium signaling in human neutrophils and FPR-expressing HCT116 cells. Taken together, the results demonstrate that IDR-1 is a partial agonist for FPR and further, stimulates neutrophil chemotaxis without inducing calcium signaling and superoxide generation.     

血管钠肽、 C型钠尿肽和心房钠尿肽舒血管作用的对比

本实验采用离体血管灌流方法,观察和比较血管钠肽（ＶＮＰ）,Ｃ型钠尿肽（ＣＮＰ）和心房钠尿肽（ＡＮＰ）对大鼠肺动脉,腹主动脉和腹腔静脉的舒张作用。．结果表明,ＶＮＰ,ＣＮＰ和ＡＮＰ对离体大鼠的保留内皮与去内皮的肺动脉,腹主动脉和腹腔静脉均有浓度依赖性舒张作用。     

Immunocytochemical localization of atrial natriuretic peptide, vessel dilator, long-acting natriuretic peptide, and kaliuretic peptide in human pancreatic adenocarcinomas.

We recently found that four peptide hormones synthesized by the same gene completely inhibit the growth of human pancreatic adenocarcinomas in athymic mice. The present immunocytochemical investigation was designed to determine where in the adenocarcinomas these peptide hormones localize. Atrial natriuretic peptide, vessel dilator, long-acting natriuretic peptide, and kaliuretic peptide localized to the cytoplasm and nucleus of the human pancreatic adenocarcinomas, which is consistent with their ability to decrease DNA synthesis in the nucleus of this cancer. In this first investigation of where these peptide hormones with anticancer effects localize in any cancer, these peptide hormones also localized to the endothelium of capillaries and fibroblasts within these cancers. This is the first demonstration of growth-inhibiting peptide hormones localizing to the nucleus, where they inhibit DNA synthesis and may interact with growth-promoting hormones that localize there as the etiology of their ability to inhibit the growth of adenocarcinomas both in vitro and in vivo.     

Evidence for the phosphorylation of a proenkephalin-derived peptide, peptide B

The potential phosphorylation of a proenkephalin-derived peptide, Peptide B, was investigated in primary cultures of bovine adrenal chromaffin cells and fresh adrenal medullary tissue. Cultures were labeled with [32P]phosphate for 24 h and extracts subjected to immunoprecipitation using affinity-purified anti-serum directed against the carboxyl terminus of Peptide B. A 4.6-kDa-labeled peptide was observed in autoradiograms of immunoprecipitates separated by sodium dodecyl sulfate-polyacrylamide electrophoresis; this peptide was not observed when excess antigen was present during the immunoprecipitation. Radioimmunoassay of extracts prepared from adrenal medullary tissue and separated by isoelectric focusing revealed the presence of four isoelectric forms of Peptide B-immunoreactive peptides; these peptides also exhibited Met-enkephalin-Arg-Phe immunoreactivity. The isoelectric points of these peptides (4.5, 4.3, 4.1, and 3.9) were consistent with the predicted pI values for phosphorylated derivatives of Peptide B. Treatment of samples with alkaline phosphatase prior to isoelectric focusing resulted in the conversion of the more acidic forms to the least acidic form. The presence of phosphate in the more acidic peaks was additionally verified by isoelectric focusing of 32P-labeled immunoprecipitates; the pI values of the radioactive peptides corresponded precisely to the peaks of immunoreactivity. In adrenal medullary tissue, the relative contributions of the various phosphorylated species to the total Peptide B immunoreactivity were as follows: unphosphorylated form, 13%; singly phosphorylated, 31%; doubly phosphorylated, 37%; and triply phosphorylated, 17%. Thus more than 85% of the Peptide B molecules present in the bovine adrenal medulla are phosphorylated.     

Amidation of joining peptide, a major pro-ACTH/endorphin-derived product peptide

Based on sequence data, rat and mouse pro-adrenocorticotropin (ACTH)/endorphin could give rise to joining peptide, a short acidic peptide that could terminate with a glutamic acid alpha-amide. Rat and mouse pituitary cells were found to cleave the pro-ACTH/endorphin precursor at an -Arg-Arg- site to produce primarily joining peptide-sized material. The amounts of joining peptide were approximately equimolar to the other major pro-ACTH/endorphin-derived products. Using antisera specific for the COOH-terminal modifications of joining peptide and three analytical approaches which separate amidated from glycine-extended forms of joining peptide, it was found that most of the joining peptide in murine anterior and intermediate pituitary was amidated. Identification of the amidated and glycine-extended forms of joining peptide was confirmed by amino acid analysis of the purified molecules. When anterior pituitary corticotrope tumor cells were grown in culture medium lacking ascorbate, there was no detectable ascorbate in the cells; nevertheless, a significant fraction of the joining peptide produced was alpha-amidated, indicating that production of alpha-amidated product was not totally dependent on ascorbate. The amidation state of the joining peptide produced by mouse corticotrope tumor cells was responsive to added ascorbate. Cells grown in medium containing ascorbic acid at the levels found in plasma concentrated the ascorbate to the levels normally found in pituitary tissue, and nearly all of the joining peptide produced was alpha-amidated. The amidation state of secreted joining peptide mirrored the amidation state of the joining peptide in the cells.     

Threading a peptide through a peptide: protein loops, rotaxanes, and knots

Proteins adopt complex folds in nature that typically avoid conformations that are knotted or “threaded” through closed loops. Is this the result of fundamental barriers to folding, or have proteins simply evolved to avoid threaded conformations? Organic synthesis has been used in supramolecular chemistry to install topological links in small molecules. By following these principles, we now show that it is possible to assemble a topologically linked protein complex by threading a linear protein through a cyclic protein to form a [2]pseudo‐rotaxane. Subsequent ring closure using native chemical ligation cyclizes the linear protein, forming a [2]heterocatenane. Although the kinetics of protein threading are slower than the folding kinetics of the native protein, threading appears to be a highly efficient process.     

Gastrointestinal satiety signals III. Glucagon-like peptide 1, oxyntomodulin, peptide YY, and pancreatic polypeptide

Many peptides are synthesized and released from the gastrointestinal tract and pancreas, including pancreatic polypeptide (PP) and the products of the gastrointestinal L cells, glucagon-like peptide 1 (GLP-1), oxyntomodulin, and peptide YY (PYY). Whereas their roles in regulation of gastrointestinal function have been known for some time, it is now evident that they also influence eating behavior. This review considers the anorectic peptides PYY, PP, GLP-1, and oxyntomodulin, which decrease appetite and promote satiety in both animal models and humans.     

Vasonatrin peptide stimulates both of the natriuretic peptide receptors,NPRA and NPRB

Vasonatrin peptide (VNP) is an active cardiovascular factor and a novel synthetic natriuretic peptide with unknown natriuretic peptide receptor (NPR) binding properties. We set out to design binding models of NPRA/VNP and NPRB/VNP, and then assessed their recognition and binding affinities using molecular dynamics. Molecular dynamics analysis indicated decreases in the values of Van der Waals, electrostatic energy and potential energy of NPRB/VNP compared to NPRA/VNP. There was a 25% increase in H-bond formation between VNP and NPRB. The cGMP stimulated by VNP in NPRB-transfected HEK-293 cells was 11-fold higher than that of NPRA. We therefore demonstrated that VNP binds with both NPRA and NPRB, but with a preference for NPRB.     

Isolation, characterization, and pharmacological actions of peptide histidine valine 42, a novel prepro-vasoactive intestinal peptide-derived peptide

The primary structure and biological activity of a novel prepro-vasoactive intestinal peptide (prepro-VIP)-derived peptide has been determined from an adrenal pheochromocytoma. The peptide was purified sufficiently for characterization by fast atom bombardment mapping after cation-exchange and reverse-phase fast protein liquid chromatography. The sequence of this novel peptide corresponds exactly to prepro-VIP-81-122 and has been designated peptide histidine valine 42 (PHV-42). Synthetic PHV-42 reduced both the force and frequency of spontaneous contractions of isolated rat uterus and was at least 12 times more potent than peptide histidine methionine (prepro-VIP-81-107), and over a hundred times more potent than noradrenaline. PHV-42 was also more potent than peptide histidine methionine in relaxing smooth muscle preparations of rat stomach and guinea pig trachea, but was approximately 4-fold less potent in reducing blood pressure than VIP. PHV-42 thus forms a separate subsystem in the VIP family of peptides and may be the most biologically active product of prepro-VIP in certain tissues such as the uterus and trachea.     

Antifungal activity of synthetic peptide derived from halocidin, antimicrobial peptide from the tunicate, Halocynthia aurantium

Halocidin is an antimicrobial peptide isolated from the hemocytes of the tunicate. Among the several known synthetic halocidin analogues, di-K19Hc has been previously confirmed to have the most profound antibacterial activity against antibiotic-resistant bacteria. This peptide has been considered to be an effective candidate for the development of a new type of antibiotic. In this study, we have assessed the antifungal activity of di-K19Hc, against a panel of fungi including several strains of Aspergillus and Candida. As a result, we determined that the MICs of di-K19Hc against six Candida albicans and two Aspergillus species were below 4 and 16 microg/ml, respectively, thereby indicating that di-K19Hc may be appropriate for the treatment of several fungal diseases. We also conducted an investigation into di-K19Hc's mode of action against Candida albicans. Our colony count assay showed that di-K19Hc killed C. albicans within 30s. Di-K19Hc bound to the surface of C. albicans via a specific interaction with beta-1,3-glucan, which is one of fungal cell wall components. Di-K19Hc also induced the formation of ion channels within the membrane of C. albicans, and eventually observed cell death, which was confirmed via measurements of the K+ released from C. albicans cells which had been treated with di-K19Hc, as well as by monitoring of the uptake of propidium iodide into the C. albicans cells. This membrane-attacking quality of di-K19Hc was also visualized via confocal laser and scanning electron microscopy.     

Mycoplasma pneumoniae infection induces a neutrophil-derived antimicrobial peptide, cathelin-related antimicrobial peptide

In innate immunity, cationic antimicrobial peptides including cathelin-related antimicrobial peptide (CRAMP) are known to play critical roles in protecting the host from infection by invasive microbes, including Gram-positive and -negative bacteria. However, little is known about the interactions between CRAMP and mycoplasmas. In the present study, the antimicrobial activity of CRAMP against M. pneumoniae and the expression of CRAMP in bronchoalveolar lavage fluid (BALF) of M. pneumoniae-infected mice was examined. CRAMP at 10-20 μg/mL reduced the growth of two strains of M. pneumoniae by 100 to 1000-fold. The amount of CRAMP in the BALF of M. pneumoniae-infected mice was 20～25 ng/mL by ELISA. The presence of mature CRAMP in BALF was observed by Western blotting. Neutrophils in BALF showed a fair amount of CRAMP in their cytoplasm by immunofluorescence. Furthermore, the addition of M. pneumoniae resulted in the release of a large amount of CRAMP from neutrophils induced by thioglycolate. These results suggest that CRAMP from neutrophils may play an important role in protection against M. pneumoniae infection.     

Vessel dilator, long acting natriuretic peptide, and kaliuretic peptide increase circulating prostaglandin E2

Prostaglandin E2 (PGE2) increases in the circulation of persons with congestive heart failure (CHF), but the cause of this increase is unknown. Prostaglandins are not stored, therefore, they cannot be released in response to congestive heart failure itself but rather need to have their synthesis stimulated by a hormone or some other substance. Prostaglandin E2's biologic properties are nearly identical to four peptide hormones originating from amino acids 1-30 [long acting natriuretic peptide], 31-67 [vessel dilator], 79-98 [kaliuretic peptide] and 99-126 [atrial natriuretic peptide, ANP] of the 126 amino acid ANP prohormone. ANP previously has been found to have no effect on circulating PGE2 concentrations in persons with CHF. The present investigation was designed to determine if one or more of the other three atrial natriuretic peptides might increase PGE2 when infused at their respective 100 ng/kg body weight/minute concentrations for 60 minutes in persons with congestive heart failure. Vessel dilator increased PGE2 8-fold (P<0.001) in the first 20 minutes of its infusion with PGE2 remaining 2-3 fold increased (P<0.05) for 60 minutes after stopping its infusion. Long acting natriuretic peptide did not increase PGE2 until 40 minutes of its infusion but it caused the maximal increase (27-fold; P<0.001) of PGE2 of the three peptide hormones tested. Kaliuretic peptide's stimulated increase of PGE2 also began in a delayed fashion but its effects lasted the longest, with PGE2 being increased (P<0.05) for two hours after the cessation of kaliuretic peptide's infusion. This investigation demonstrates that 1) three endogenous peptide hormones increase PGE2 in the circulation and 2) suggests that the known increase in PGE2 in CHF may be in part secondary to these peptides.     

F2L, a peptide derived from heme-binding protein, inhibits formyl peptide receptor-mediated signaling

F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein. Very recently, F2L was identified as an endogenous chemoattractant peptide acting specifically through formyl peptide receptor-like (FPRL)2. In the present study, we report that F2L stimulates chemotactic migration in human neutrophils. However, F2L inhibits formyl peptide receptor (FPR) and FPRL1 activities, resulting in the complete inhibition of intracellular calcium increases, and superoxide generation induced by N-formyl-Met-Leu-Phe, MMK-1, or Trp-Lys-Tyr-Met-Val-d-Met (WKYMVm) in human neutrophils. In terms of the inhibitory role of F2L on FPR- and FPRL-mediated signaling, we found that F2L competitively inhibits the binding of (125)I-WKYMVm to its specific receptors, FPR and FPRL1. F2L is the first endogenous molecule that inhibits FPR- and FPRL1-mediated signaling, and is expected to be useful in the study of FPR and FPRL1 signaling and in the development of drugs to treat diseases involving the FPR family of receptors.     

Lactoferricin, a new antimicrobial peptide

Lactoferricin B (LF-B) is a peptide derived from acid-pepsin digestion of bovine lactoferrin, which has antimicrobial properties. In order to assess the antimicrobial spectrum of LF-B and its possible in vivo uses, the minimum inhibitory and microbicidal concentrations of pure lactoferricin B were determined for a range of bacterial species and under varying conditions of growth including growth phase and size of the inoculum, pH and ionic strength of the medium. Lactoferricin B was bactericidal against a wide range of bacteria and Candida albicans. Proteus spp., Pseudomonas cepacia and Serratia spp. were resistant. The bactericidal activity of LF-B was inhibited by increasing ionic strength and bacterial inoculum and at acid pH. The activity of lactoferricin B was completely inhibited by the addition of 5% whole cow's milk and was reduced in the presence of increasing concentrations of mucin. These results indicate the potential of LF-B to reduce the numbers of organisms in a simple medium, but raise doubts about its role in vivo because of its sensitivity to changes in physical variables. It may be that lactoferricin exerts a transient antimicrobial effect at mucosal surfaces.     

Siderophore peptide, a new type of post-translationally modified antibacterial peptide with potent activity

Microcin E492 (MccE492, 7886 Da), the 84-amino acid antimicrobial peptide from Klebsiella pneumoniae, was purified in a post-translationally modified form, MccE492m (8717 Da), from culture supernatants of either the recombinant Escherichia coli VCS257 strain harboring the pJAM229 plasmid or the K. pneumoniae RYC492 strain. Chymotrypsin digestion of MccE492m led to the MccE492m-(74-84) C-terminal fragment that carries the modification and that was analyzed by mass spectrometry and nuclear magnetic resonance at natural abundance. The 831-Da post-translational modification consists of a trimer of N-(2,3-dihydroxybenzoyl)-l-serine linked via a C-glycosidic linkage to a beta-d-glucose moiety, itself linked to the MccE492m Ser-84-carboxyl through an O-glycosidic bond. This modification, which mimics a catechol-type siderophore, was shown to bind ferric ions by analysis of the collision-induced dissociation pattern obtained for MccE492m-(74-84) by electrospray ion trap mass spectrometry experiments in the presence of FeCl(3). By using a series of wild-type and mutant isogenic strains, the three catechol-type siderophore receptors Fiu, Cir, and FepA were shown to be responsible for the recognition of MccE492m at the outer membrane of sensitive bacteria. Because MccE492m shows a broader spectrum of antibacterial activity and is more potent than MccE492, we propose that by increasing the microcin/receptor affinity, the modification leads to a better recognition and subsequently to a higher antimicrobial activity of the microcin. Therefore, MccE492m is the first member of a new class of antimicrobial peptides carrying a siderophore-like post-translational modification and showing potent activity, which we term siderophore-peptides.     

Tissue transglutaminase-mediated glutamine deamidation of beta-amyloid peptide increases peptide solubility, whereas enzymatic cross-linking and peptide fragmentation may serve as molecular triggers for rapid peptide aggregation

Tissue transglutaminase (TGase) has been implicated in a number of cellular processes and disease states, where the enzymatic actions of TGase may serve in both, cell survival and apoptosis. To date, the precise functional properties of TGase in cell survival or cell death mechanisms still remain elusive. TGase-mediated cross-linking has been reported to account for the formation of insoluble lesions in conformational diseases. We report here that TGase induces intramolecular cross-linking of β-amyloid peptide (Aβ), resulting in structural changes of monomeric Aβ. Using high resolution mass spectrometry (MS) of cross-linked Aβ peptides, we observed a shift in mass, which is, presumably associated with the loss of NH3 due to enzymatic transamidation activity and hence intramolecular peptide cross-linking. We have observed that a large population of Aβ monomers contained an 0.984 Da increase in mass at a glutamine residue, indicating that glutamine 15 serves as an indispensable substrate in TGase-mediated deamidation to glutamate 15. We provide strong analytical evidence on TGase-mediated Aβ peptide dimerization, through covalent intermolecular cross-linking and hence the formation of Aβ1-40 dimers. Our in depth analyses indicate that TGase-induced post-translational modifications of Aβ peptide may serve as an important seed for aggregation.     

Atrial natriuretic peptide, B-type natriuretic peptide, and serum collagen markers after acute myocardial infarction.

Experimental data suggest that atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) act locally as antifibrotic factors in heart. We investigated the interrelationships of natriuretic peptides and collagen markers in 93 patients receiving thrombolytic treatment for their first acute myocardial infarction (AMI). Collagen formation following AMI, evaluated as serum levels of amino terminal propeptide of type III procollagen, correlated with NH(2)-terminal proANP (r = 0.45, P < 0.001), BNP (r = 0.55, P < 0.001) and NH(2)-terminal proBNP (r = 0.50, P < 0.01) on day 4 after thrombolysis. Levels of intact amino terminal propeptide of type I procollagen decreased by 34% (P < 0.001), and levels of carboxy terminal cross-linked telopeptide of type I collagen (ICTP) increased by 65% (P < 0.001). ICTP levels correlated with NH(2)-terminal proBNP (r = 0.25, P < 0.05) and BNP (r = 0.28, P < 0.05) on day 4. Our results suggest that ANP and BNP may act as regulators of collagen scar formation and left ventricular remodeling after AMI in humans. Furthermore, degradation of type I collagen is increased after AMI and may be regulated by BNP.     

Cellular localization, half-life, and secretion of peptide YY

Tissue and plasma concentration of peptide YY (PYY) were measured by means of a radioimmunoassay (RIA) developed in our laboratory, using a specific PYY antiserum generated in New Zealand white rabbits against synthetic PYY, and dextran-coated charcoal to terminate the assay. Cellular localization of PYY was studied immunohistochemically using the peroxidase-antiperoxidase (PAP) technique. The highest tissue concentration of PYY was found in the mucosa of the terminal ileum and colon. PYY-containing secretory granules were primarily found in the basal pole of open-type endocrine cells. Basal plasma concentration of PYY was 70 +/- 9 pg/ml and rose to 357 +/- 30 pg/ml during the IV administration of PYY at 400 pmol/kg-h. A significant correlation was found (r = 0.94, p less than 0.05) between dose of PYY (12.5, 25, 50, 100, 200, 400 pmol/kg-h, IV) and plasma concentration of PYY. The calculated half-life of PYY in plasma was 8.3 +/- 1.9 minutes. Plasma concentration of PYY during the intraduodenal administration of sodium oleate (150 +/- 20 pg/ml) or long-chain triglyceride (187 +/- 37 pg/ml) was similar to plasma concentration of PYY obtained during the IV administration of PYY at 100 pmol/kg-h. Plasma concentration of PYY raised (126 +/- 10 pg/ml) after the administration of bombesin (400 pmol/kg-h, IV). Bile enhanced release of PYY. The present study suggests a hormonal role for PYY.