Peptides (v.23, #10)

Platelet factor 4 fragment induces histamine release from rat peritoneal mast cells by Ryujiro Suzuki; Tomoki Kimura; Kiyoyuki Kitaichi; Yasuaki Tatsumi; Miyoko Matsushima; Ying Lan Zhao; Eiji Shibata; Kenji Baba; Takaaki Hasegawa; Kenzo Takagi (1713-1717).
Platelet factor 4 (PF-4) belongs to a superfamily of low-molecular weight proteins known as chemokines. However, its function has not been fully evaluated. In the present study, we investigated the effect of PF-4 on histamine release from rat peritoneal mast cells by employing its biologically-active carboxyl-terminal fragment, PF-4 (58–70). PF-4 (58–70) stimulated histamine release from mast cells in a dose-dependent manner (10−8 to 10−5  M). Histamine release induced by PF-4 (58–70) occurred rapidly (<30 s) and was inhibited by extracellular Ca2+. These results suggest that PF-4 might play a crucial role at the site of inflammation and/or immune response.
Keywords: Histamine release; Peritoneal mast cells; Platelet factor 4-related peptide (58–70);

Characterization of two peptide epitopes on Mdm2 oncoprotein that affect p53 degradation by M. Balass; E. Kalef; R. Maya; S. Wilder; M. Oren; E. Katchalski-Katzir (1719-1725).
Phosphorylation of Mdm2, in response to DNA damage, resulted in prevention of p53 degradation in the cytoplasm as well as reduction of its binding with monoclonal antibody (mAb) 2A10. Using a 15-mer phage-peptide library, we identified two 2A10-epitopes on human Mdm2 (hdm2): at positions 255–266 (LDSEDYSLSEEG) and 389–400 (QESDDYSQPSTS). Synthetic peptides corresponding to the above sites, inhibit the binding of mAb2A10 to Mdm2 with high (4.5×10−9  M) and moderate affinity (1.1×10−7  M), respectively. Phospho-derivatives of these peptides, and of single human Mdm2 mutations S260D or S395D resulted in a considerable reduction in their binding with mAb2A10. These results provide a molecular explanation for the observation that reactivity of Mdm2 with mAb2A10 is inhibited by phosphorylation.
Keywords: Epitope mapping; Peptide epitopes; Anti-Mdm2 mAb2A10; Phospho-peptides; Phage-peptide library;

Improvement of the enzymatic stability of a cytotoxic T-lymphocyte-epitope model peptide for its oral administration by M.K Marschütz; W Zauner; F Mattner; A Otava; M Buschle; A Bernkop-Schnürch (1727-1733).
Oral administration of peptide antigens, to provide proper mucosal and/or systemic immunity, is largely ineffective. This is mainly due to the very small quantity of antigen that survives degradation in the intestine and that crosses the intestinal absorption membrane. The present study focuses on the improvement of the enzymatic stability of a 13 amino acid long peptide containing a cytotoxic T-lymphocytes (CTL)-epitope. Within this study, it is shown, that simple chemical modification at the N- and C-terminus of the peptide can provide significant stability towards enzymatic attack by intestinal exopeptidases. Around 50% of the modified peptide resisted enzymatic attack on native porcine intestinal mucosa within 3 h of incubation at pH 6.8 and 37 °C, whereas unmodified control peptide was almost completely degraded within the same time period. Additionally, a mucoadhesive drug carrier matrix with specific inhibitory properties towards luminally secreted endopeptidases has been generated. The incorporation of the simply modified peptide in this delivery system should enhance the amount of biologically active antigen being available at the mucosal site for further presentation to immunomodulating systems. This might open the door for a successful oral immunotherapy.
Keywords: CTL-epitope; Cellular immune response; Enzymatic degradation; Mucoadhesion; Oral delivery; Peptide stability;

PACAP enhances the expression of CD11b, CD66b and CD63 in human neutrophils by Johan Kinhult; Arne Egesten; Rolf Uddman; Lars Olaf Cardell (1735-1739).
Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide with strong bronchodilator capacity, present in the human airways. There is recent evidence that PACAP decreases the release of proinflammatory cytokines. We have previously shown that PACAP inhibits neutrophil chemotaxis, but altogether little is known about the effects of PACAP on granulocytes. The present study was designed to investigate if PACAP and the closely related peptide vasoactive intestinal peptide (VIP) could affect the cell surface expression of CD11b, CD63 and CD66b in human neutrophils. Neutrophils isolated from 12 healthy blood donors were incubated with either PACAP or VIP, and the expression of neutrophil cell surface markers was assessed using flowcytometry. Neutrophils incubated with PACAP38 exhibited a marked, concentration-dependent increase in their expression of CD11b, CD63 and CD66b. In contrast, neutrophils incubated with VIP showed no increase of the investigated surface markers. This indicates a role for PACAP in granulocyte activation, mediated via a pathway not shared with VIP. Together with the previously presented data on leukocyte migration it suggests that PACAP acts as a regulator of neutrophil inflammation.
Keywords: PACAP; VIP; Flow cytometry; Neutrophils; CD11b; CD63; CD66b;

A newly developed enzyme-immunoassay for measuring the tissue contents of PACAP in fish by Kouhei Matsuda; Satomi Onoue; Kazuhisa Kashimoto; Aya Hamakawa; Minori Kikuchi; Minoru Uchiyama; Tohru Mochizuki; Akira Arimura (1741-1750).
We have developed a novel and easy enzyme-immunoassay (EIA) for pituitary adenylate cyclase-activating polypeptide (PACAP). We used it to determine immunoreactive PACAP levels in the central nervous system (CNS) and peripheral tissues of two fishes, a teleost (the stargazer) and an elasmobranch (a stingray). An antiserum was raised in a white rabbit immunized with a conjugate of synthetic stargazer PACAP27 plus keyhole limpet hemocyanin. The EIA system used an antiserum/biotin-labeled PACAP/avidin/biotin-conjugated enzyme complex, and a double antibody method was used to precipitate the immune complexes. We call the system the avidin–biotin complex detectable EIA (ABCDEIA) for PACAP. ABCDEIA with biotin-labeled PACAP27 detected only PACAP27, recognizing neither the longer forms of PACAP nor any other peptides. PACAPs with 27, 38, and 44 residues cross-reacted in another ABCDEIA with biotin-labeled PACAP38 or PACAP44. Whole brains of both fishes contained much higher levels of PACAP, 6–30 times as high as the levels in the mammalian brain, but unexpectedly, no immunoreactive PACAP27 was found in any CNS or peripheral tissue in either fish. The gastrointestinal tracts of fish also contained lower, but significant amounts of PACAP.
Keywords: PACAP; Fish; Enzyme-immunoassay;

Proctolin (Arg-Tyr-Leu-Pro-Thr-OH) and crayfish peptide “DF2” (Asp-Arg-Asn-Phe-Leu-Arg-Phe-NH2) enhance spontaneous contractions of isolated crayfish hindguts. Both peptides increase the frequency and amplitude of spontaneous, rapid contractions. Proctolin induces a slow contraction, which gives the appearance of an increase in overall tonus. DF2 has no such effect. To determine whether the peptides affect both longitudinal and circular muscles, hindguts were cut into longitudinal strips and into rings, and contractions were recorded from each. The longitudinal strips generated only rapid contractions, and both peptides increased the frequency and amplitude of such contractions without significantly altering tonus. Rapid contractions were observed in only 1 of 14 preparations of rings. Proctolin induced slow contractions in the rings, and DF2 had no such effect. The results indicate that neuropeptides have different effects on circular and longitudinal muscles of hindgut.
Keywords: Proctolin; FMRFamide; Crustacean; Intestine; Visceral muscle; Procambarus clarkii;

The insect neuropeptides FMRF amide, leucomyosupressin (LMS) and neuropeptide analogues leucosulfakinins (FLSK and LSK II Ser (SO3H)), perisulfakinin (PSK), proleucosulfakinin (PLSK), 14A[φ1]WP-I, 542φ1, and 378A[5b]WP-I were assayed for their effects on the release of amylase and protease from the midgut tissue of larvae of Opisina arenosella. In the bioassay, empty midgut tubes ligated at both ends using hair were incubated with insect saline containing neuropeptides/analogues in a bioassay apparatus at 37 °C for 30 min. After incubation the contents of the midgut preparations were analyzed for amylase and protease activity. In control experiments, the midgut preparations were incubated in insect saline without neuropeptides. The results of the study reveal that for stimulating amylase release from midgut tissue, the peptides require an FXRF amide (X may be methionine or leucine) sequence at the C-terminal. The presence of HMRF amide at C-terminal of peptides may inhibit the release of amylase. Meanwhile, peptides with both FMRF and HMRF amide sequence at the C-terminal are found to be effective in stimulating protease release. The tetrapeptide segment at the C-terminal probably represent the active core of the neuropeptide.
Keywords: Amylase; Coconut pest; Digestive enzyme release; FMRF amide; Leucomyosupressin; Leucosulfakinins; Neuropeptide; Opisina arenosella; Perisulfakinin; Protease;

Functional mapping of NPY/PYY receptors in rat and human gastro-intestinal tract by Laurent Ferrier; Jean-Pierre Segain; Christian Bonnet; Christine Cherbut; Paul-Antoine Lehur; Anne Jarry; Jean-Paul Galmiche; Hervé M Blottiere (1765-1771).
Peptide YY (PYY) is involved in the regulation of several gastro-intestinal functions, including motility. The aims of the present study were (i) to characterize the effects of PYY on smooth muscle strips obtained from the different gastro-intestinal segments in rats and in humans and (ii) to realize a map of the Y receptors expression. Contractions of strips were recorded under isometric conditions, using PYY and acetylcholine as control. We observed that PYY induced a contraction of muscle strips from rat proximal colon, but displayed no effect on other gut segments. Using RT-PCR, mRNA encoding the Y1 and Y4 receptors were detected in muscle strips depending on the segment. In humans, the muscle preparations responded to ACh but not to PYY. Moreover, only Y2 receptor mRNA was found in the ileum and the left colon, but not in other segments. Our study shows the heterogeneity in the expression of Y receptors along the gastro-intestinal tract, and reveals great discrepancies between rats and humans both concerning the expression of Y receptor, and the response of smooth muscle strips to PYY.
Keywords: Gastro-intestinal tract; Motility; PP-fold peptides; Y receptors family;

Peptide fragments released from Phe-caseinomacropeptide in vivo in the rat by S Fosset; G Fromentin; D.W Gietzen; M Dubarry; J.F Huneau; J.M Antoine; V Lang; F Mathieu-Casseron; D Tomé (1773-1781).
The aim of this study was to investigate the pharmacokinetics of bovine Phe-caseinomacropeptide (Phe-CMP) in the rat after oral administration. This polypeptide was monophosphorylated and mainly nonglycosylated: Phe-CMP-1P. During gastrointestinal digestion and absorption, Phe-CMP-1P was degraded. Intact Phe-CMP-1P and CMP-1P were rapidly released from the stomach. In contrast, partial hydrolysis by pancreatic enzymes was observed. In vitro hydrolysis by brush-border membrane vesicles also indicated that the peptide was degraded. In the blood, “CMP-immunoreactive material” appeared rapidly, reaching a maximum level of 5.5 μg/ml at 60 min.
Keywords: Phe-caseinomacropeptide; Caseinomacropeptide; Pharmacokinetics; Rat; Peptide digestion; Absorption;

The peptide substance P (SP) is known to take part in the regulation of the Cl-dependent secretion in the animal and human colonic mucosa. However, no conclusive evidence for the expression of the functional tachykinin NK1 receptor has been found in the human colonic epithelial cells. Using the reverse transcription-polymerase chain reaction (RT-PCR) method we could detect the transcripts of the NK1 receptor in the human colonic epithelial cell line Caco-2. Furthermore, we characterized the mechanism of substance P-induced intracellular signaling in Caco-2 cells. While substance P had no effect on intracellular calcium concentration as measured by fura-2 AM, it induced the activation of the mitogen-activated protein kinases (MAPKs) in a time- and dose-dependent manner. Surprisingly, the peptide NK1 receptor antagonist [d-Pro2, d-Trp7,9]SP stimulated the activity of MAPKs in the same manner as substance P. In contrast, the specific nonpeptide NK1 receptor antagonist CP-96,345 clearly abolished the effect of substance P and [d-Pro2, d-Trp7,9]SP on MAPK activity. CP-96,345 itself did not increase the activity of MAPKs. Thus, we provide the first evidence that a functional NK1 receptor is expressed in the human colonic epithelial cell line Caco-2. The results show that in Caco-2 cells the peptide antagonist [d-Pro2, d-Trp7,9]SP acts as a NK1 receptor agonist in contrast to the nonpeptide antagonist CP-96,345.
Keywords: Caco-2 cells; NK1 receptor; Activation of mitogen-activated protein kinases;

The responsiveness of cultured myenteric neurons to cholecystokinin (CCK-8) was examined using fura-2-based digital microfluorimetric measurement of intracellular calcium ([Ca2+]i). CCK-8 (10−10–10−6  M) evoked concentration-dependent increases in percentage of neurons responding (8–52%) and Δ[Ca2+]i (76–169 nM). Gastrin (1 μM) also induced an increase in [Ca2+]i in 29±6% of neurons (Δ[Ca2+]i: 71±3 nM). L-364,718, an antagonist for the CCK-A receptor, blocked [Ca2+]i response to CCK-8. Removal of extracellular calcium eliminated CCK-induced [Ca2+]i increments, as did the addition of the calcium channel inhibitors nickel (1 mM) and lanthanum (5 mM). Nifedipine (1–50 μM) dose-dependently attenuated CCK-caused [Ca2+]i responses. CCK evokes [Ca2+]i signaling in myenteric neurons by the influx of extracellular calcium, likely through L-type calcium channels.
Keywords: Enteric nervous system; Myenteric plexus; Fura-2 acetoxymethyl ester; Calcium channel; Signal transduction;

Vasoactive intestinal peptide regulation of nerve growth factor in the embryonic mouse by Joanna M Hill; Janice Mehnert; Susan K McCune; Douglas E Brenneman (1803-1808).
Vasoactive intestinal peptide (VIP), a regulator of embryonic growth, increased the concentration of nerve growth factor (NGF)-like immunoreactivity in the conditioned medium of cultured explanted embryonic day (E) 9.5 neural tube preparations compared to control preparations. VIP treatment also induced an increase of NGF-like immunoreactivity (NGF-IR) within the neural tube preparation tissue. A 60 kDa isoform was the primary form of NGF detected. VIP is shown to be a regulator of NGF in the E9.5 embryonic mouse and stimulates the release of a high molecular weight isoform of NGF.
Keywords: Neural tube; Nerve growth factor; Vasoactive intestinal peptide; Pituitary adenylate cyclase activating peptide; Embryogenesis; Neuroepithelium;

Effect of YM471, a nonpeptide AVP receptor antagonist, on human coronary artery smooth muscle cells by Atsuo Tahara; Junko Tsukada; Yuichi Tomura; Koh-ichi Wada; Toshiyuki Kusayama; Noe Ishii; Takeyuki Yatsu; Wataru Uchida; Nobuaki Taniguchi; Akihiro Tanaka (1809-1816).
The antagonistic properties of YM471, a potent nonpeptide vasopressin (AVP) V1A and V2 receptor antagonist, were characterized using human coronary artery smooth muscle cells (CASMC). YM471 potently inhibited specific binding of 3 H -AVP to V1A receptors on human CASMC, exhibiting a K i value of 0.49 nM. Furthermore, YM471 inhibited the AVP-induced increase in intracellular free Ca2+ concentration with an IC50 value of 1.42 nM, but exerted no agonistic activity on CASMC. Additionally, while AVP concentration-dependently induced hyperplasia and hypertrophy in CASMC, YM471 prevented these AVP-induced growth effects, exhibiting IC50 values of 0.93 and 2.64 nM, respectively. These results indicate that YM471 has high affinity for V1A receptors on, and high potency in inhibiting AVP-induced physiologic responses of, human CASMC.
Keywords: Vasopressin; V1A receptor; Coronary artery; Smooth muscle cells; Hyperplasia; Hypertrophy; YM471;

Antisauvagine-30 (aSVG) is the only high-affinity antagonist for the corticotropin-releasing factor (CRF) type 2 (CRF2) receptor. A structure–activity relationship study was performed to pinpoint residues conferring aSVG’s selectivity. The aSVG-analogues being N-terminally extended by one or two residues or containing the Ala22Arg23Ala24 (ARA-motif) of CRF, were synthesized. Additionally, a lactam bridge between positions 29 and 32 was introduced. The modified peptides were analyzed for α-helicity properties, binding affinities and antagonistic potencies at the rat CRF1 and mouse CRF2B receptors. While N-terminal prolongation and replacement of d-Phe11 by Tyr11 increased the affinity for the CRF2 receptor, the introduction of the ARA motif resulted in a loss of CRF2 receptor selectivity. These data show that aSVG10–40 analogues are more potent CRF2 receptor antagonists than aSVG11–40 peptides, while introduction of the ARA-motif or a cyclic constraint between residues 29 and 32 favors binding to the CRF1 receptor.
Keywords: Sauvagine; Antisauvagine; CRF; CRF receptor; Antagonist;

The cloning and characterisation of a procorticotrophin-releasing hormone (proCRH) and the related CRH fragment in the IZD-MB-0503 cell line from the leptidopteran Mamestra brassicae were performed. PCR amplification of the genomic DNA reveals a fragment of 276 bp, while inverse PCR shows the presence of a gene consisting of 1153 bp. The comparison of the insect genomic proCRH gene with proCRH cDNA obtained by RACE shows the presence of three introns. There was a 61% identity with the corresponding coding sequence in Tilapia mossambica, and a 65.2% identity with the human proCRH coding sequence.
Keywords: IZD-MB-0503 cell line; Mamestra brassicae; Lepidoptera; Procorticotrophin-releasing hormone gene; Gene cloning;

Potentiation of morphine analgesia by BQ123, an endothelin antagonist by Shaifali Bhalla; George Matwyshyn; Anil Gulati (1837-1845).
Several neurotransmitter mechanisms have been proposed to play a role in the actions of morphine. The present study is the first to provide evidence that central endothelin (ET) mechanisms are involved in the modulation of pharmacological actions of morphine. The effect of intracerebroventricular (i.c.v.) administration of endothelin-A (ETA) antagonist, BQ123, on morphine-induced analgesia, hyperthermia, and catalepsy was determined in the rat. Morphine produced a significant increase in tail-flick latency as compared to control group. Pretreatment with BQ123 significantly potentiated the effect and duration of morphine (2 and 8 mg/kg, s.c.)-induced analgesia as compared to vehicle-pretreated control rats. The hyperthermic effect of morphine was not only significantly greater in BQ123-pretreated rats but also lasted for more than 6 h. ET antagonist, BQ123, did not affect the pharmacological effect of morphine on cataleptic behavior. These studies demonstrate that BQ123, a specific ETA receptor antagonist, significantly potentiated morphine-induced analgesia and hyperthermia in rats without affecting morphine-induced cataleptic behavior. [ 3 H ]-Naloxone binding was carried out to determine the possibility of BQ123 acting on opiate receptors. It was found that morphine could displace [ 3 H ]-naloxone but BQ123 did not affect [ 3 H ]-naloxone binding even at 1000 nM concentration. Therefore, it can be concluded that BQ123 does not act on opioid receptors. This is the first report suggesting that an ETA antagonist, BQ123, significantly potentiates the analgesic effect of morphine, possibly through a nonopioid mechanism.
Keywords: Morphine; Analgesia; BQ123 [cyclo(–d-Trp–d-Asp–Pro–d-Val–Leu)]; Central nervous system; Rats; Opiates; Endothelin; [ 3 H ]-Naloxone binding;

Aldehyde fixatives are often used to preserve tissue morphology and thereby aid in the identification of cellular structures expressing a target of interest. However, the effect of fixatives on target detection methods is unpredictable and it is currently unknown whether tissue fixation would allow the accurate detection of angiotensin AT4 receptors in the kidney. In vitro receptor autoradiography on tissues fixed with 4% paraformaldehyde and 0.5% glutaraldehyde (±20% sucrose) had differing effects on the density of 125 I -AT4 receptor ligand binding without affecting the tissue distribution of ligand binding in the rat and mouse kidney, whereas an increased expression of specific 125 I -AT4 receptor ligand binding was found in the medulla region of the rabbit kidney. In contrast, such tissue fixation conditions dramatically decreased the renal binding of 125 I -angiotensin II receptor ligands, and altered the distribution of such ligand binding, in all three species. These results suggest that the method of tissue fixation and processing should be used cautiously in angiotensin receptor density measurements but can provide an accurate representation of kidney AT4 receptor distribution only in the rat and mouse.
Keywords: In vitro receptor autoradiography; AT4 receptor; Ang II receptor; Paraformaldehyde; Glutaraldehyde; Kidney;

Isolation and characterization of an angiotensin converting enzyme substrate from vitellogenic ovaries of Neobellieria bullata by Anick Vandingenen; Korneel Hens; Geert Baggerman; Nathalie Macours; Liliane Schoofs; Arnold De Loof; Roger Huybrechts (1853-1863).
Vitellogenic ovaries of the gray fleshfly Neobellieria bullata contain a variety of unidentified substances that interact, either as a substrate or as an inhibitor, with angiotensin converting enzyme (ACE). We here report the isolation and characterization of the first ACE interactive compound hereof. This 1312.7 Da peptide with the sequence NKLKPSQWISL, is substrate to both insect and human ACE. It is a novel peptide that shows high sequence similarity to a sequence at the N-terminal part of dipteran yolk polypeptides (YPs). We propose to call it N. bullata ovary-derived ACE interactive factor or Neb-ODAIF. Both insect and human ACE hydrolyze Neb-ODAIF by sequentially cleaving off two C-terminal dipeptides. K m values of Neb-ODAIF and Neb-ODAIF1-9 (NKLKPSQWI) for human somatic ACE (sACE) are 17 and 81 μM, respectively. Additionally, Neb-ODAIF1–7 (NKLKPSQ) also interacts with sACE (K m/i=90 μM). These affinity-constants are in range with those of the physiological ACE substrates and suggest the importance of Neb-ODAIF and its cleavage products in the elucidation of the physiological role of insect ACE. Alternatively, they can serve as lead compounds in the development of new drugs against ACE-related diseases in humans.
Keywords: ACE inhibitor; Insect; Neobellieria bullata ovary-derived ACE interactive factor (Neb-ODAIF); Pharmacology; Yolk protein;

Elevated immunoreactive-adrenomedullin levels in the aqueous humor of patients with uveitis and vitreoretinal disorders by Tetsuo Udono; Kazuhiro Takahashi; Toshiaki Abe; Shigeki Shibahara; Makoto Tamai (1865-1868).
To clarify possible involvement of adrenomedullin in the pathophysiology of inflammation of eyes, we measured immunoreactive-adrenomedullin concentrations in the aqueous humor and plasma obtained from 14 control subjects and 56 patients with uveitis or vitreoretinal disorders. Immunoreactive-adrenomedullin levels in the aqueous humor were significantly elevated in patients with active uveitis, proliferative vitreoretinopathy and proliferative diabetic retinopathy, as compared with control subjects. The plasma immunoreactive-adrenomedullin levels were not significantly correlated with the aqueous humor levels. These findings suggest that adrenomedullin produced locally in the eyes is involved in the pathophysiology of uveitis and some proliferative vitreoretinal disorders.
Keywords: Adrenomedullin; Eye; Uveitis; Vitreous; Retina; Retinal detachment;

A new synthetic all-d-peptide with high bacterial and low mammalian cytotoxicity by Maxim G Ryadnov; Olga V Degtyareva; Ivan A Kashparov; Yuri V Mitin (1869-1871).
Using the synthetic α-helical peptide ((RLA)2R)2 as a model the effect of net charge, helicity, and epimeric nature of the peptide on bactericidal potency has been examined. Both the nature and the extent of the net charge were shown to be relatively important for antibacterial activity. The loss of the structured character of the peptide resulted in reducing the activity. The all-d-peptide appeared to be a remarkably strong bacteriostatic agent with MIC <1 μM against Escherichia coli. The peptide was neither hemolytic nor cytotoxic, which in conjunction with data on its stability to enzymatic degradation makes this peptide very attractive in terms of designing new bactericidal agents on the basis of d ((RLA)2R)2.
Keywords: Antibacterial peptides; All-d-peptides; Hemolysis; Mammalian cell toxicity;