Peptides (v.21, #6)

Determination of defensin HNP-1, HNP-2, and HNP-3 in human saliva by using LC/MS by C Goebel; L.G Mackay; E.R Vickers; L.E Mather (757-765).
The mass spectral profiling of saliva by liquid chromatography mass spectrometry in relation to particular types of pain is being examined. The aim is to develop a profile that could be useful for the assessment of patients and their treatment programs, as well as identifying unknown compounds observed in saliva. Defensin human neutrophil peptide-1 (HNP-1) and defensin HNP-2 were identified and confirmed, whereas defensin HNP-3 was tentatively identified. Linear calibration range of defensin HNP-1 and HNP-2 was 0.25 to 3 μg/ml with R 2 values of > 0.99 for both. The detection limit for defensin HNP-1 and HNP-2 was estimated at 0.1 μg/ml. The healthy subjects surveyed in this study had readily measurable salivary concentrations of defensin HNP-1 (8.6 ± SD 8.0 μg/ml) and defensin HNP-2 (5.6 ± SD 5.2 μg/ml).
Keywords: Saliva; Defensin; Electrospray; Mass spectrometry;

Expression of functional recombinant scorpion β-neurotoxin Css II in E. coli by Tonny M Johnson; Michael W Quick; Ted T Sakai; N.Rama Krishna (767-772).
The gene for a β-neurotoxin [Centruroides suffusus suffusus toxin II (Css II)] from the scorpion C. suffusus suffusus was synthesized by recursive PCR and cloned into the expression vector, pET15b. This recombinant vector was transformed into a thioredoxin mutant host bacterial cell, AD 494(DE3)pLysS, and expression was induced with isopropyl thiogalactoside (IPTG). Although the level of expression was low, the recombinant toxin was found only in the soluble fraction with no evidence for the formation of inclusion bodies as had been observed previously with other scorpion toxins. The recombinant Css II was purified by successive ion-exchange and hydrophobic interaction chromatography. Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectral measurements indicate that the protein has a native structure with no indication of denatured species. The recombinant neurotoxin inhibits the uptake of [3H]GABA [γ-aminobutyric acid (GABA)] in neuronal cells as effectively as natural β-toxins.
Keywords: Scorpion β-neurotoxin; Recombinant; Escherichia coli;

A novel 1,6-hexanediol diacrylate cross-linked resin was prepared that was subsequently functionalized by using chloromethyl methyl ether to afford a high-capacity resin. The resin exhibited good swelling and its application in the successful synthesis of a 13-residue peptide corresponding to the fragment of seminalplasmin has been illustrated. The resin was chemically inert at peptide synthetic conditions.
Keywords: Solid-phase peptide synthesis; Seminalplasmin; 1,6-Hexanediol diacrylate-cross-linked polystyrene; Polyethylene glycol;

Responses of frog skin Na+ and Cl transport to guanylin by E.P Bernick; D.F Stiffler (779-783).
A possible role for the peptide hormone guanylin was investigated in frog skin (Rana pipiens) epithelium. Sodium and chloride fluxes in response to this peptide were evaluated in Ussing-type chambers. Net and unidirectional Na+ fluxes were measured by using 22Na+ and atomic absorption analysis of total [Na+], whereas net Cl fluxes were measured by using electrometric titration for [Cl]. Mucosal application of guanylin (0.5–2.0 μmol/l) caused marked increases in serosal to mucosal net flux and efflux of Na+. Serosal application of guanylin over the same dose range caused similar large increases in net serosal to mucosal (S→M) Na+ and Cl flux as well as Na+ efflux. Responses of Na+ influx were small and inconsistent. When frog skin was bathed on the serosal side with Cl-free Ringer’s solution mucosal application of guanylin stimulated large efflux and S→M net fluxes of Na+. Serosal treatment yielded large Na+ effluxes and S→M Na+ and Cl net fluxes. When frog skin serosal surfaces were bathed with Na+- free Ringer’s solution mucosal guanylin treatment had no effect but serosal treatment produced large S→M Cl net fluxes.

Uncoupling of proopiomelanocortin (POMC) fragments is related to self-injury by Curt A. Sandman; William Hetrick; Derek Talyor; Sarah Marion; Aleksandra Chicz–DeMet (785-791).
Proopiomelanocortin (POMC) contains several interesting, behaviorally active peptides. Release patterns of these fragments have been related to bizarre episodes of self-injurious behavior (SIB) among autistic individuals. Moreover, elevation in β-endorphin (βE) but not ACTH levels was associated with a positive response to an acutely administered, centrally acting opioid blocker among autistic individuals exhibiting SIB. In the present study, POMC fragments were measured in 12 self-injurious patients before and after long term (3 month) treatment with an opiate blocker naltrexone (NTX). POMC fragments were sampled from blood collected at the beginning of the baseline and placebo-controlled treatment phases of the study. Results indicated that the co-release (coupling) of POMC fragments were stable over time and the profile of POMC fragments in plasma predicted the effectiveness of a CNS acting drug in autistic subjects who self-injure.
Keywords: Self-injury; Autism; β-endorphin; Naltrexone; ACTH; POMC; Opioids;

Hypothalamic neuropeptides play critical roles in the regulation of feeding behavior and body weight (BW). Disruption of signaling in the ventromedial nucleus by microinjection of the neurotoxin, colchicine (COL), produces transient hyperphagia with corresponding BW gain lasting for 4 days. Because the melanocortin system exerts an inhibitory control on food intake, we hypothesized that hyperphagia in COL-treated rats is due to decreased melanocortin-induced restraint on feeding. Melanocortin restraint is exerted through α-melanocortin-stimulating hormone derived from proopiomelanocortin (POMC) and is antagonized by agouti-related peptide produced in neurons located in the arcuate nucleus (ARC). COL (4 μg/0.5 μl saline) or saline was microinjected bilaterally into the ventromedial nucleus of adult male rats. In conjunction with BW gain, blood leptin levels were elevated, whereas POMC mRNA in the ARC was significantly decreased in COL-injected rats. Levels of α-melanocortin-stimulating hormone were also decreased in the micropunched paraventricular nucleus, dorsomedial nucleus, and perifornical hypothalamus, sites implicated in the control of food intake. That diminution in melanocortin signaling underlies hyperphagia was supported by the observation that intracerebroventricular injection of the MC3/MC4 melanocortin receptor agonist, MTII, prevented the hyperphagia and BW gain. Surprisingly, however, mRNA levels of the orexigenic peptide agouti-related peptide in the ARC were decreased perhaps due to the action of elevated leptin. These results show that transient hyperphagia and BW gain induced by disruption of signaling in the ventromedial nucleus results from two neurochemical rearrangements: development of leptin resistance in POMC neurons and diminution in melanocortin signaling as reflected by decreased POMC gene expression in the ARC and decreased availability of α-melanocortin-stimulating hormone for release in feeding relevant sites.
Keywords: Obesity; POMC; AGRP; Leptin; α-MSH; MTII;

Structure-activity studies of α-melanotropin fragments on cAMP production in striatal slices by Marı́a Cecilia Cremer; Mónica Silvina Sanchez; Marı́a Ester Celis (803-806).
In this work, we characterized the active site in the α-melanotropin hormone (α-MSH) sequence responsible for the enhancement of cAMP production in incubated striatal slices by using different α-MSH fragments. We also analyzed the effects of the co-incubation of the SCH23390, a dopaminergic D1 antagonist, with the MSH fragments, to study the involvement of the D1 receptor on this induction. A rise was observed in the levels of cAMP after addition of the 6 μM fragments MSH(1–10), and 0.6 and 6 μM MSH(5–13); however, the values were lower than those induced by 6 μM α-MSH. On the contrary, the addition of MSH(9–13), MSH(7–11), or MSH(6–9) did not affect the cAMP content. The presence of 10 μM SCH23390 blocked the effect of the fragments on cAMP production. We conclude that the biologic activity of α-MSH, as observed through the levels of cAMP, declines when the length of its polypeptide chain is shortened, and that the presence of glutamic acid in the molecule, as well as the core sequence, are of importance for fragments′ activity.
Keywords: α-MSH fragments; Striatal slices; In vitro studies; Dopamine; D1 receptor; SCH23390; cAMP;

Cathepsin B inhibitory peptides derived from β-casein by Hyun Sook Lee; Kye Joon Lee (807-809).
Two cathepsin B inhibitory peptides were isolated from a commercial pancreatic digest of casein. The peptides were identified as the Pro-Phe-Pro-Gly-Pro-Ile and the Gly-Pro-Phe-Pro-Ile corresponding to the sequence 61–66 and 203–207 of bovine β-casein. These peptides showed competitive inhibition for cathepsin B with the K i values of 2.31 and 3.30 mM, respectively. Two related analogues, Tyr-Pro-Phe-Pro-Gly-Pro-Ile and Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile, were synthesized but their cathepsin B inhibitory activity was not detected.
Keywords: β-Casein; Cathepsin B; Inhibitor; Bioactive peptides; K i value;

Vasopressin-containing Long–Evans and vasopressin-deficient Brattleboro rats were maintained in individual cages while telemetered activity (AC) and body temperature (BT) data were collected. Rats were initially exposed to a 12 h/12-h light/dark cycle (photic zeitgeber) and were allowed ad-libitum access to food and water. Daily feeding, care, and handling (nonphotic zeitgebers) occurred at the beginning of the second hour of the dark cycle. After a 14-day habituation period, rats were subjected to continuous light (LL) or dark (DD) and nonphotic cues were presented irregularly. During the habituation period, both strains exhibited clear 24-h circadian rhythms of AC and BT. In LL or DD, photic cues were removed and nonphotic cues were presented irregularly. There was a shift in the rhythm for Long–Evans animals to 26 h for both AC and BT in LL and 24.6 h in DD. Feeding, care, and handling were seen as minor artifact. In Brattleboro rats, although there were robust 26-h and 24.6-h circadian rhythms of AC in the LL and DD, respectively, BT data were inconsistent and showed sporadic fluctuations. In the BT rhythm of Brattleboro animals, strong peaks were associated with feeding, care, and handling times and trough periods were characterized by a dramatic drop in temperature. This experiment demonstrates that AC and BT are controlled by separate oscillators. In addition, the importance of vasopressinergic fibers in the control of circadian rhythms of BT is evidenced by the loss of circadian rhythms in animals lacking these functional fibers when exposed to free-running paradigms where there is no entrainment of photic or nonphotic oscillators.
Keywords: Suprachiasmatic nucleus; Free-running circadian rhythms; Telemetry; Photic zeitgebers; Nonphotic zeitgebers; Brattleboro rat;

The effect of PACAP38 on the LH surge and ovulation was compared with that of PACAP27 and VIP in the same model. The peptides were administered intracerebroventricularly before the critical period of the proestrous stage. PACAP38 was able to inhibit ovulation and to prevent the preovulatory LH surge; however, PACAP27 did not inhibit the ovulation and VIP inhibited the ovulation in 2/11 animals. In those animals of the last two groups in which ovulation occurred, the preovulatory LH surge was higher than in control rats. It is speculated that the opposite effect of PACAP38 and PACAP27 on the preovulatory LH surge and ovulation is possibly mediated through different receptors.
Keywords: PACAP38; PACAP27; VIP; Ovulation; LH; Rat;

Presence of pituitary adenylate cyclase-activating polypeptide (PACAP) and its relaxant activity in the rectum of a teleost, the stargazer, Uranoscopus japonicus by Kouhei Matsuda; Kazuhisa Kashimoto; Terumasa Higuchi; Takayuki Yoshida; Minoru Uchiyama; Seiji Shioda; Akira Arimura; Tadao Okamura (821-827).
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide and a member of the secretin/glucagon superfamily of peptides that include vasoactive intestinal polypeptide. PACAP is not only present in the central nervous system but also in peripheral organs, such as the gastrointestinal tract, gonads and adrenal glands, and plays various roles in mammals. Recently, we isolated and characterized PACAP, which is very similar to PACAP of mammalian origin, from the brain of a teleost, the stargazer, Uranoscopus japonicus. In the present study, the expression of PACAP mRNA was detected in the stargazer rectum using the reverse transcriptase/polymerase chain reaction (RT-PCR) method. The distribution of PACAP-like immunoreactivity in the rectum was also examined immunohistochemically, using an antiserum raised against PACAP 27, and PACAP-like immunoreactive neuronal cell bodies and fibers were found in the myenteric plexuses and the smooth muscle layers of the rectum. The present study also investigated the relaxant activity of synthesized homologous PACAP on rectal contraction. Stargazer PACAP, like that of mammalian origin, inhibited contractions stimulated by acetylcholine or potassium chloride. PACAP-induced inhibition was not affected by preincubation with atropine, propranolol, or phentolamine. These results suggest that PACAP may act directly as an inhibitory neuropeptide in the stargazer rectum.
Keywords: PACAP; Fish; Teleost; Rectum; RT-PCR; Immunohistochemistry; Myenteric plexuses; Relaxant activity;

New bradykinin analogs in contraction of rat uterus,☆ by Henryk I Trzeciak; Wojciech Kozik; Samed Melhem; Aleksandra Kania; Dariusz Dobrowolski; Adam Prahl; Izabela Derdowska; Bernard Lammek (829-834).
In this study, we evaluated 20 of our previously synthesized peptides on isolated rat uterus by Holton’s procedure with minor modifications, and compared their activity with that assessed previously by their ability to inhibit vasodepressor response to exogenous bradykinin (BK) in conscious rats. We used [D-Arg0, Hyp3, Thi5, 8, (D-Phe)7]BK, the B2 antagonist of Vavrek and Stewart as a model when designing our analogs. We observed that, in the case of the rat uterus test, the activity of peptides modified by acylation of the N-terminus with various bulky groups depends substantially on the chemical character of the substituent. We also learned that, contrary to previous examples, acylation of the N-terminus of antagonists, which contain a sterically restricted fragment in the C-terminal part, may not improve their antagonistic potencies. Besides an improved characterization of a series BK analogs, our studies have provided new information on the structure–activity relationship, which in turn may be of value in the design of more potent and selective bradykinin antagonists. The results of our studies appear to support the hypothesis of others about the presence of different subtypes of B2 receptors in rat uterus and blood vessels.
Keywords: Rat uterus assay; Bradykinin analogs; B2 agonists: Antagonists;

Neuropeptide Y is a cotransmitter with norepinephrine in guinea pig inferior mesenteric vein by Lisa Smyth; Janette Bobalova; Sean M. Ward; Violeta N. Mutafova–Yambolieva (835-843).
Neuropeptide Y (NPY) is a cotransmitter with noradrenaline in guinea pig inferior mesenteric vein. Tyrosine hydroxylase-like immunoreactivity and NPY-like immunoreactivity were colocalized in a dense network of fibers within the adventitial layer of guinea-pig inferior mesenteric vein. Vasoconstrictor responses to electrical field stimulation (0.2–64 Hz, 0.1 ms, 12 V, for 10 s) appear to be mediated primarily by norepinephrine at 0.2 to 4 Hz and by NPY at 8 to 64 Hz. NPY Y1 receptors mediate the contractile responses to both endogenous and exogenous NPY. Norepinephrine and NPY are involved in neuromuscular transmission in guinea pig mesenteric vein suggesting that the sympathetic nervous system requires the coordinated action of norepinephrine and NPY to serve capacitance.
Keywords: Neuropeptide Y; Sympathetic cotransmission; Guinea pig mesenteric vein;

Anabolic-androgenic steroids affect the content of substance P and substance P1–7 in the rat brain by Mathias Hallberg; Pia Johansson; Anna M.S. Kindlundh; Fred Nyberg (845-852).
The effects of intramuscular (i.m.) injections of nandrolone decanoate (15 mg/kg/day), an anabolic-androgenic steroid, on the levels of substance P (SP) and on its N-terminal fragment SP1–7 were examined in the male rat brain by radioimmunoassay. The results demonstrated that the SP immunoreactivity in amygdala, hypothalamus, striatum, and periaqueductal gray was significantly enhanced, whereas the concentration of the N-terminal fragment SP1–7 was enhanced in the nucleus accumbens and in periaqueductal gray. In the striatum the steroid induced a decrease in the content of SP1–7. The relevance of these peptides in connection with anabolic-androgenic steroid-induced aggression is discussed.
Keywords: Aggression; Anabolic-androgenic steroids; Brain; Rat; Substance P; N-terminal fragment;

This study reports an improved approach for the determination of neuropeptide levels in human cerebrospinal fluid (CSF). The method is based on sample acidification followed by liquid–liquid extraction (LLE) combined with radioimmunoassay. It was applied to study the recovery and level of some opioid peptides (Met-enkephalin-Arg6-Phe7 and Leu-enkephalin-Arg6), substance P and the substance P1–7 fragment, which are all compounds known to be present in human CSF. The results indicated that the use of LLE highly improved the recovery of these peptides compared to current liquid–solid-phase extraction methods by using silica gel cartridges or mini-columns for ion-exchange chromatography. Peptides added to CSF in concentrations down to 10 fmol/ml were recovered in yields exceeding 80%. The mean recovery of synthetic peptides as recorded by radioimmunoassay in the LLE procedure was significantly improved when HCl was added to the sample. In contrast, when the 125I-labeled analogues of the peptides were added to CSF samples, the mean recovery of the four labeled peptides using the LLE procedure was markedly reduced in acidified samples. We also found that the inclusion of HCl effectively improved the removal of proteins present in the samples. As an application the levels of substance P and Met-enkephalin-Arg6-Phe7 in CSF samples from patients with chronic pain (fibromyalgia syndrome) were measured using the new procedure. It was possible to confirm a significant difference in the CSF levels of both peptides when comparing patients and controls.
Keywords: Analysis; Cerebrospinal fluid; Enkephalins; Opioid peptides; Radioimmunoassay; Substance P;

Binding characteristics of endothelin ETA receptors in normal and post-mortem rat lung by Leonora S Dias; David N Schell; Elizabeth Burcher (861-869).
In control lung homogenates, optimal specific binding of [125I]endothelin-1 and minimal filter binding was achieved using 50 μg/ml bacitracin, 30 μM phenylmethylsulphonyl fluoride (PMSF) and 10 mM EDTA. In post-mortem tissue (8, 16, and 32 h), no significant changes were seen in ETA receptor affinity (K d) or number (B max): control and 32 h K d = 309 ± 75, 225 ± 32 pM and B max = 173 ± 42, 185 ± 17 fmol/mg protein, respectively. Autoradiographic binding sites for [125I]endothelin-1 were densely expressed on bronchiolar smooth muscle and parenchyma with moderate binding on epithelium and blood vessels. Histologic sections of post-mortem lung showed minimal deterioration of structures expressing ETA binding sites. Hence the ETA receptor is stable in the rat lung for up to 32 h post-mortem.
Keywords: Post-mortem; Endothelin-1; Rat lung; Sarafotoxin S6C; BQ-788; Autoradiography; Homogenate binding;

Correlation between apolipoprotein B and endothelin-1-induced vasoconstriction in humans by Silvana C. Romerio; Lilly Linder; Josef Flammer; Walter E. Haefeli (871-874).
Low-density lipoprotein (LDL) concentrations have been linked to altered responses to endogenous vasodilators and vasoconstrictors. We evaluated retrospectively the relationship between LDL and vasoconstrictor (endothelin-1, phenylephrine) responsiveness of the forearm vasculature in 15 elderly healthy volunteers with apolipoprotein B and LDL levels in the normal range. In contrast to phenylephrine, changes in forearm vascular resistance induced by endothelin-1 were correlated with apolipoprotein B, LDL, and total cholesterol concentrations in women but not in men. These findings might suggest that lipids may increase vascular tone through both impaired endothelial vasodilation and increased vasoconstriction to endothelin-1 at least in women.
Keywords: Low-density lipoprotein; Endothelin-1; Phenylephrine; Vasoconstrictors;

Effects of atrial natriuretic peptide in the gut by L.V. González Bosc; M.P. Majowicz; N.A. Vidal (875-887).
The intestinal tract is a target organ for atrial natriuretic peptide (ANP), characterized by various biologic activities, immunoreactivity, as well as specific binding sites for ANP. A review of previous studies reveals that ANP is an important regulator of water and nutrient intake, which acts via multiple signaling pathways including activation of guanylyl cyclase to produce its biologic responses. As a regulator, the peptide locally controls hydrosaline balance and acute systemic effects. Therefore, ANP could also act as a local mediator or paracrine effector of intestinal function.