Peptides (v.21, #5)

Detailed characterization of an anti-factor IX monoclonal antibody that neutralizes the prolonged ox brain prothrombin time of hemophilia BM by synthetic peptides by Isao Takahashi; Tetsuhito Kojima; Masayuki Sano; Takeshi Watanabe; Tadashi Kamiya; Hidehiko Saito (603-608).
In a previous study, we prepared a monoclonal antibody (MoAb) to coagulation factor IX (FIX), designated 65–10, which interfered with the activation of FIX by the activated factor XI/Ca2+ and neutralized the prolonged ox brain prothrombin time of hemophilia BM. The location of the epitope on the FIX for 65–10 MoAb is 168 Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr-Arg-Val-Val182. In this paper, we studied in more detail an epitope on FIX using the systematic substitution of different amino acids at each residue of the epitope peptides and the influence of the epitope peptide on the prolonged ox brain prothrombin time of the hemophilia BM plasma of 65–10 MoAb. In the replacement set of amino acids, peptides showing low or no reactivity to 65–10 were 175Phe → Asp, Glu, Gly, Lys, Arg, Thr, Val, 176Asn → Asp, Glu, Phe, Ile, Lys, Leu, Pro, Val, Tyr, 177Asp → Cys, Glu, Phe, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, and 178 Phe → Pro. These results imply that a hydrophobic molecule of 175 Phe, a hydrophilic molecule of 176Asn, and a negative charge molecule of 177Asp were important to the epitope. The 65–10 MoAb antibody neutralized the prolonged ox brain prothrombin time of hemophilia BM Nagoya 2 (180Arg →Trp) and Kashihara (181Val → Phe) as well as BM Kiryu (313Val → Asp) and Niigata (390Ala → Val). This reaction was inhibited by preincubation with a 168 Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr-Arg-Val-Val182 peptide conjugated with bovine serum albumin (BSA). 65–10 MoAb that has been useful in detailing epitopes will be useful for qualitative analysis of hemophilia BM.
Keywords: Blood coagulation factor IX; Mouse monoclonal antibody; Epitope; Synthetic peptides; Hemophilia BM;

Two N-terminally truncated forms of C-type natriuretic peptide from habu snake venom by Gilles H Michel; Nobuhiro Murayama; Toshio Sada; Masatoshi Nozaki; Kenichi Saguchi; Hiroaki Ohi; Yoshiaki Fujita; Hiroyuki Koike; Shigesada Higuchi (609-615).
Two N-terminally truncated forms of the C-type natriuretic peptide (CNP) were isolated from the venom of habu snake, Trimeresurus flavoviridis, and their structures were determined by EMI-MS spectrometry and amino acid sequencing. Tf-CNP(6–22), the shorter peptide retaining the 17-membered ring structure formed by an intra-molecular disulfide bridge, has a vasorelaxant activity in rat aortic strips and a diuretic potency in anesthetized rats. Tf-CNP(3–22), the other 20 amino acid residues peptide, also comprised the 17- membered ring with a short N-terminal extention of 3 amino acid residues. Tf-CNP(6–22), the ring, is the shortest naturally occurring CNP peptide identified so far, and as potent as Tf-CNP(1–22), the supposedly intact CNP of 22 amino acid residues.
Keywords: CNP; Habu snake; Trimeresurus flavoviridis; Ring structure; N-terminally truncated form; Vasorelaxant activity;

To evaluate the release and possible role of N-terminal end of atrial natriuretic factor (ANF) prohormone (proANF-1–30) and C-terminal end of ANF prohormone (aANP-1–28) in patients with soft tissue and bone injuries, 20 patients with soft tissue injuries, 18 bone-fractured patients, and 21 healthy controls were examined. Samples were collected from patients within 24 h after injury. Plasma level of proANF-1–30 and aANP-1–28 were higher in orthopedic patients than the soft tissue injury patients compared to controls (P < 0.005, P < 0.05, respectively). proANF-1–30 was over 15-fold greater than aANP-1–28 in the healthy control samples. These data hypothesized that the concentration of proANF-1–30 may be related to tissue damages in man.
Keywords: Atrial-natriuretic factor prohormone; Atrial natriuretic peptide; Creatine kinase; Myoglobin; Elastase; Enzyme-immunoassay;

Cardioactive peptides isolated from the brain of a Japanese octopus, Octopus minor by Eiko Iwakoshi; Miki Hisada; Hiroyuki Minakata (623-630).
Octopus cardioactive peptides (Ocp-1: Gly-d-Phe-Gly-Asp, and Ocp-3: Gly-Ser-Trp-Asp) were isolated from brain extracts of the octopus, Octopus minor, using the isolated systemic heart as a bioassay. These peptides showed both positive chronotropic and inotropic effects on the heart. The stereoisomers at position 2 were also isolated, but their activities were only 1/103-1/104 those of the corresponding isomers. The presence of the peptides in the systemic heart was confirmed by time-of-flight mass spectrometry (MS) and tandem MS analysis. The results suggested that Ocp-1 and Ocp-3 might be involved in excitatory control of the octopus cardiovascular system as neuropeptides and/or neurohormones.
Keywords: Octopus cardioactive peptides; d-amino acid-containing peptides; Time-of-flight MS analysis;

Synthesis and biological activity of adipokinetic hormone analogues with modifications in the 4–8 region by Anastasia Velentza; Spiridoula Spiliou; Constantine P. Poulos; Graham J. Goldsworthy (631-637).
Several structural characteristics in the molecule of the locust adipokinetic hormone, AKH-I, have been investigated in terms of their importance in determining biologic activity. All modifications tested in this study resulted in analogues with decreased potency in comparison with the parent molecule. However, all analogues that were found to be active gave a full response, although often only at very high doses of peptide. This study has highlighted for the locust receptor(s) the vital role of the side chain of Thr5, and the importance of positions 4 and 8. For example, when Trp8 and Phe4 were exchanged, the resulting analogue (Trp4,Phe8-AKH-I) was one of the least active analogues tested in this study. Although Trp is tolerated quite well as a substitute for Phe4, with only a 10-fold loss of potency, Phe is not favored as a substitute for Trp8 (>300 times decrease in potency). On the other hand, 3-[2-napthyl] alanine (Nal) is a better substitute for Trp8 (only a 100-fold loss in potency). We conclude that position 4 requires a phenyl ring in the side chain, and position 8 an indole ring.
Keywords: AKH-I analogues; Insects; Lipid mobilization; Locusta migratoria; Neuropeptides; Solid phase synthesis; Structure-activity relationships;

A complementary DNA (cDNA) of 928 bp encoding a bombesin (BBS)/gastrin-releasing peptide (GRP) precursor was identified from goldfish brain. Goldfish BBS/GRP messenger RNA (mRNA) encodes a 157 amino acid precursor, which contains a signal peptide sequence, the 22 amino acid putative BBS/GRP-like peptide, and a carboxy-terminal extension peptide. Reverse transcription-polymerase chain reaction (PCR) (RT-PCR) demonstrated that the mRNA for this precursor has a widespread distribution in goldfish brain, and is also present in skin, gastrointestinal tract, gonad, and gill. Phylogenetic analysis of BBS/GRP-like peptide precursors in vertebrates shows that goldfish BBS/GRP is more closely related to the known GRP precursors than to BBS precursors.
Keywords: cDNA; Bombesin; GRP; Cloning; Expression; Brain; Goldfish;

(Tyr0,Bpa4)bombesin is a GRP receptor agonist by M. Casibang; T.W. Moody (649-653).
(Tyr0,Bpa4)bombesin, (YB)BB was synthesized and its biologic activity evaluated using T47D breast cancer cells. (125I-Tyr0,Bpa4)BB bound with high affinity (K d = 5 nM) to T47D cells. Specific (125I-Tyr0,Bpa4)BB binding was inhibited with high affinity by BB, BW2258U89, GRP, GRP14–27 and NMB (IC50 values of 10, 2, 15, 20, and 150 nM)but not GRP1–16 (IC50 value of > 1000 nM). (125I-Tyr0,Bpa4)BB bound to the surface of T47D cells at 4°C but was internalized at 37°C. After binding at 4°C followed by irradiation using ultraviolet light, (125I-Tyr0,Bpa4)BB labeled a 75 kDa protein using T47D cells. (Tyr0,Bpa4)BB, 10 nM, elevated cytosolic calcium using T47D cells within 10 s. Also (Tyr0,Bpa4)BB, 10 nM, elevated c-fos mRNA after 45 min. These results indicate that (Tyr0,Bpa4)BB is an agonist for GRP receptors.
Keywords: Receptor binding; GRP receptor; Cytosolic calcium; c-fos mRNA;

Regional distribution and plasma concentration of peptide YY in sheep by Takenori Onaga; Mayumi Yoshida; Hiroki Inoue; Hiroshi Yokota (655-667).
Peptide YY (PYY)-positive cells are distributed in the mucosa of the ileum, cecum, colon, and rectum of sheep, but not in other layers of these regions. By radioimmunoassay, mucosal content of PYY in the ovine large intestine was much less than that in the rat intestine. The plasma concentration of immunoreactive PYY did not significantly fluctuate over a 48-h period in conscious sheep, even after ingestion of roughage and concentrate. Intraluminal nutrients into the ileum and i.v. CCK8 also did not raise the plasma level of PYY. Therefore, PYY seems unlikely to play a role as “ileal brake” in sheep.
Keywords: Alimentary tract; Distribution; Ileal brake; Immunohistochemistry; Peptide YY; Plasma concentration; Radioimmunoassay; Ruminant; Sheep;

Peripheral administration of CRF and urocortin: effects on food intake and the HPA axis in the marsupial Sminthopsis crassicaudata by Perdita J Hope; Helen Turnbull; Sue Farr; John E Morley; Kenner C Rice; George P Chrousos; David J Torpy; Gary A Wittert (669-677).
The hypothalamic peptides corticotrophin releasing factor (CRF) and urocortin (UCN) decrease food intake and increase energy expenditure when administered either centrally or peripherally to rodents. The effects of CRF and UCN on food intake in other mammals (for example marsupials), however, are not known. Peripherally administered CRF induced cortisol release in the marsupial Sminthopsis crassicaudata via the CRF1 receptor, and central CRF administration potently decreased food intake, as in rodents. When peripherally administered, both CRF and UCN decreased food intake in S. crassicaudata, but UCN was considerably more potent (∼50 fold) in this regard. The anorectic effects of CRF and UCN were not blocked by the CRF1 receptor antagonist antalarmin, suggesting that the peripheral effects of CRF and UCN on food intake are mediated primarily by the CRF2 receptor.
Keywords: Urocortin; Corticotrophin-releasing factor; Antalarmin; Marsupial; Sminthopsis crassicaudata; Cortisol; CRF receptor;

Food deprivation and adrenalectomy are associated with low concentrations of leptin in blood and the absence of obesity. Because leptin is known to cross the blood–brain barrier (BBB) by a saturable transport system, we examined whether fasting and adrenalectomy (ADX) also act at the BBB. Multiple-time regression analysis showed that fasting, but not ADX, significantly decreased the entry of leptin into mouse brain. After 3 days of food deprivation, the influx of leptin became indistinguishable from that of the vascular control (albumin); 5 h of refeeding significantly reversed this reduced rate of influx. Thus, the results indicate that the BBB provides a dynamic site for the regulation of physiological processes involving leptin.
Keywords: Fasting; Adrenalectomy; Leptin; Blood–brain barrier;

The agouti-related protein decapeptide (Yc[CRFFNAFC]Y) possesses agonist activity at the murine melanocortin-1 receptor by Carrie Haskell–Luevano; Eileen K Monck; Y.-P Wan; Anzeela M Schentrup (683-689).
Agouti-related protein (AGRP) is a naturally occurring antagonist of the brain melanocortin receptors (MC3R and MC4R) and is physiologically implicated as participating in feeding behavior and energy homeostasis. The human AGRP decapeptide Yc[CRFFNAFC]Y has been previously reported as binding to the human MC3 and MC4 receptors and antagonizing the MC4 receptor. We have synthesized this decapeptide and pharmacologically characterized it at the murine melanocortin receptors and found it to possess MC4R antagonist activity (pA2 = 6.8) and, unexpectedly, MC1R agonist activity (EC50 = 2.89 μM). This study characterizes the first AGRP-based peptide agonist at the melanocortin receptors.
Keywords: Melanocortin; Agouti-related protein; α-Melanocyte stimulating hormone; Melanotropin; Obesity; MC1R; MC4R;

Condition-dependent presence of β-lipotropin-like peptide in human keratinocytes by Marjolein Wintzen; Susana B Zanello; Michael F Holick; Victor M Wiegant; J.Peter H Burbach; Bert Jan Vermeer (691-697).
This study aimed to characterize the β-endorphin-immunoreactive material (βE-IR) detectable in normal human keratinocytes (NHK). The effects of different culturing conditions and UV-irradiation on production of βE-IR by NHK were assessed by radioimmunoassay and HPLC. All culture systems contained low levels of βE-IR that was increased in conditioned media after UV-irradiation under certain conditions. NHK grown in nutrient-poor medium contained highest levels of βE-IR that exhibited β-lipotropin-like properties after HPLC analysis. The other culturing conditions displayed no authentic βE-related peptides. Our results indicate that under certain culturing conditions NHK can produce POMC peptides like β-lipotropin, which can be induced by UV-radiation.Keywords: β-endorphin; β-lipotropin; POMC; Radioimmunoassay; HPLC; Human keratinocyte; Culture; Ultraviolet-radiation

Interaction between α-MSH and acetylcholinergic system upon striatal cAMP and IP3 levels by Marı́a Cecilia Cremer; Susana Rubiales de Barioglio; Marı́a Ester Celis (699-704).
The interaction between the neuropeptide α-MSH and the acetylcholinergic system as reflected by changes in cAMP and inositol 1–3-5 triphosphate(IP3)production was investigated in an in vitro model of striatal slices. The possible involvement of D1 receptors in cholinergic and α-MSH- stimulated cAMP and IP3 production in slices of rat striatum was also examined, because it has been demonstrated that acetylcholinergic drugs induce endogenous dopamine release in the striatum. α-MSH, pilocarpine(PL) and the selective muscarinic M1 agonist McN-A-343 increased cAMP and IP3 striatal levels, effects blocked by the D1 antagonist SCH-23390, except for the effects of α-MSH on IP3 .The muscarinic M2 antagonist gallamine (GL) brought about an increase in cAMP levels, an effect blocked by SCH-23390. The M1 antagonist pirenzepine (Pz) induced a decrease both in cAMP and IP3 content, and the nicotinic antagonist di-hydro-β-eritroidine(DBE) only diminished cAMP production. When α-MSH and cholinergic agents were simultaneously added, cAMP and IP3 levels were modified with respect to the values reached when these agents were added alone. An interaction between the acetylcholinergic system and α-MSH through M1 and nicotinic receptors was also observed. These results suggest that the intracellular signaling pathways related to cAMP and IP3 production gated by α-MSH and these cholinergic receptors are probably related. α-MSH striatum cAMP IP3 muscarinic and nicotinic receptors an in vitro model.

Sex differences have been observed in antinociception after morphine administered into either the lateral ventricles, rostral ventromedial medulla, or ventrolateral periaqueductal gray such that male rats exhibit significantly greater antinociception than female rats. Adult gonadectomy produced small, but significant changes in morphine antinociception relative to same-sex sham-operated controls. The present study examined whether sex and adult gonadectomy differences were observed in antinociceptive responses after d-Pro2-Endomorphin-2 (1–50 μg) elicited from the ventrolateral periaqueductal gray (vlPAG) on the tail-flick and jump tests in rats, and compared these effects with morphine antinociception. d-Pro2-Endomorphin-2 antinociception in the vlPAG was significantly greater in estrous-phase, sham-operated and ovariectomized female rats relative to sham-operated and castrated male rats on the tail-flick, but not jump test that differed markedly from the greater magnitude of morphine antinociception noted for male rats on both tests. In testing whether d-Pro2-Endomorphin-2’s antinociceptive sex differences were secondary to alterations in activity, similar decreases in the pattern of total activity were observed after d-Pro2-Endomorphin-2 in the vlPAG in male and female rats. In evaluating whether male and female rats differed in their behavioral activation responses after d-Pro2-Endomorphin-2 in the vlPAG, significantly more excessive grooming, seizures, barrel rolls and explosive running behaviors were observed after d-Pro2-Endomorphin-2 in male, but not female rats during the precise periods of time when they were failing to display robust antinociceptive responses on the tail-flick test. Thus, the different patterns of sex differences after d-Pro2-Endomorphin-2 in the vlPAG appear to be attributable to sex-dependent alterations in behavioral activation rather than nociceptive processing per se.
Keywords: Pain; Antinociception; Locomotor activity; Behavioral activation; Sex differences; Adult gonadectomy; d-Pro2-Endomorphin-2; Morphine;

An enzymatically stable kyotorphin analog induces pain in subattomol doses☆ by Hiroshi Ueda; Makoto Inoue; Grazyna Weltrowska; Peter W Schiller (717-722).
Intraplantar injection of the enzymatically stable, N-methylated kyotorphin analog Tyr(NMe)-Arg-OH produced marked and sharp nociceptive flexor responses in a dose-dependent manner. A significant response was observed with this compound at a dose of 0.01 amol (6000 molecules). Tyr(NMe)-Arg-OH-nociception was completely blocked by the kyotorphin antagonist leucyl-arginine and its enzymatically stable, N-methylated analog, as well as by CP-99994, a specific neurokinin 1 antagonist. These findings suggest that the nociceptive effect produced by Tyr(NMe)-Arg-OH in subattomol doses occurs via specific interaction with the kyotorphin receptor and that the extraordinary potency observed may result from amplification through local substance P release.
Keywords: Kyotorphin; Kyotorphin analogs; Kyotorphin antagonists; Nociception; Peripheral nociception assay; Subattomol potency; Substance P;

Dual effect of secretin on nephron filtration and proximal reabsorption depending on the route of administration by Giulio Romano; Pietro Giagu; Grazia Favret; Ettore Bartoli (723-728).
Secretin is a vasoactive peptide capable of acting on transmembrane volume fluxes. We measured nephron filtration (SNGFR) and resorption during secretin microinjection (MIJ) into the tubular lumen or microperfusion (MP) into peritubular capillaries. In 24 rat nephrons, SNGFR, measured by collections from the distal tubule, rose from 25 ± 4 to 61 ± 8 nl/min during MIJ of saline containing secretin 10 9 M into the last convolution of the proximal tubule (LP). Percent and absolute resorptions rose from 70 to 90% and from 20 ± 4 to 56 ± 8 nl/min, respectively. During MIJ of secretin, 3 × 10 9 M into the first convolution of the proximal tubule, SNGFR, measured at LP, rose from 32 ± 4 to 61 ± 8 nl/min, percent and absolute reabsorptions from 52 ± 4 to 78 ± 3% and from 16 ± 2 to 50 ± 7 nl/min, respectively (n = 30). During MP of secretin, 1.5 × 10 9 M, SNGFR fell from 39 ± 6 to 15 ± 4, resorption from 19 ± 4 to 9 ± 2 nl/min, while percent resorption rose from 43 ± 6 to 59 ± 5% (n = 15). While all MIJ and MP changes were significant (P < 0.001), paired pre- versus post-MIJ and MP values were not. Secretin is a powerful vasoconstrictor when perfused into peritubular capillary blood, unlike systemic and intra-arterial injections. When injected into the tubular lumen, it up-regulates SNGFR and increases reabsorption directly.
Keywords: Secretin; SNGFR; Aquaporins; Tubulo-glomerular feedback; Glomerulo-tubular balance; Proximal reabsorption;

Angiotensin-(1–7) decreased mitogen-activated protein (MAP) kinase (Erks) activation in cultured Mardin–Darby bovine kidney (MDBK) epithelial cells. Also, saturable, high-affinity 125I-angiotensin-(1–7) binding was detected in MDBK cell membranes. Together, the data suggested the possible presence of an angiotensin-(1–7) receptor. However, ligand structure-binding studies revealed that angiotensin-(3–7) and AT4 receptor ligands competed with high-affinity for 125I-angiotensin-(1–7) binding. Furthermore, angiotensin-(3–7) and AT4 receptor ligands decreased MAP kinase activation in MDBK cells. These results demonstrate that NH2-terminal-deleted metabolites of angiotensin-(1–7) can bind with high affinity to the AT4 receptor and regulate the MAP kinase/Erk signaling pathway in renal epithelial cells.
Keywords: Angiotensin-(1–7); Ang IV; Angiotensin peptides; AT4 receptor; Mitogen-activated protein kinases; Kidney epithelial cells;

In the migratory locust, the CRF-related diuretic hormone that stimulates fluid secretion by the Malpighian tubules, and the ovary maturing parsin, a neurohormone able to stimulate oogenesis, are produced by the same neuroendocrine cells of the pars intercerebralis in the brain.
Keywords: Diuresis; Reproduction; Insect; Digestion; Neurohormone; CRF;

Peptides and other neuronal markers in transplanted pancreatic islets☆ by Solveig Persson–Sjögren; Sture Forsgren; Inge-Bert Täljedal (741-752).
Functional alterations are developed in transplanted islets over time. Because islets in situ are densely innervated and isolation disconnects the endocrine organ from extrinsic nerves and from ganglia in the exocrine pancreas, it is important to examine the reinnervation of islet grafts. This review describes the patterns of appearances of intrinsic perikarya and reinnervating fibers demonstrating markers for parasympathetic, sympathetic or sensory nerve substances, most notably neuropeptides, in islet transplants. An altered innervation pattern, as compared to normal islets, develops. Presumably the expression of neuronal markers in the grafts is related to factors both in the islets and in the ectopic environment offered by the implantation organ.
Keywords: Acetylcholinesterase; Calcitonin gene-related peptide; Islets of Langerhans; Islet grafts; Neuropeptides; Neuropeptide Y; Neuroinsular complex; Reinnervation; Substance P; Vasoactive intestinal polypeptide; Transplantation; Tyrosine hydroxylase;