Peptides (v.30, #6)

IgE-binding epitopic peptide mapping on a three-dimensional model built for the 13S globulin allergen of buckwheat (Fagopyrum esculentum) by Camille Sordet; Raphaël Culerrier; Claude Granier; Alain Didier; Pierre Rougé (1021-1027).
The three-dimensional model built for the 13S globulin allergen of buckwheat (Fagopyrum esculentum) consists of three protomers exhibiting the cupin motif, arranged in a homotrimer around a three-fold symmetry axis. Using the SPOT technique, 11 continuous IgE-binding epitopic peptides were characterized on the molecular surface of the 13S globulin allergen of buckwheat. Except for one of them, they all correspond to well exposed regions containing electropositiveley and/or electronegatively charged residues, which cover up to 40% of the molecular surface of the allergen. Some of these epitopes come in close contact to probably create more extended discontinuous epitopes, especially those located on the edge of the 13S globulin homotrimer. Half of the identified epitope peptides remain unaltered in a core structure protected against hydrolysis by digestive proteases and are thus assumed to promote the allergenicity of the 13S globulin. In addition, a few of these epitopes coincide with sequential IgE-binding epitopes previously characterized in soybean 11S globulins, that could account for the IgE-binding cross-reactions observed between soybean and buckwheat in Western blot experiments.
Keywords: Buckwheat allergen; 13S globulin; IgE-binding epitopic peptide; Three-dimensional model; Conformational analysis; IgE-binding cross-reactivity;

A novel ACE inhibitory peptide isolated from Acaudina molpadioidea hydrolysate by Yuanhui Zhao; Bafang Li; Shiyuan Dong; Zunying Liu; Xue Zhao; Jingfeng Wang; Mingyong Zeng (1028-1033).
Body wall protein from the sea cucumber (Acaudina molpadioidea) was hydrolyzed sequentially with bromelain and alcalase. The hydrolysate was fractionated into two ranges of molecular weight (PH-I, >2 kDa; PH-II, <2 kDa) using an ultrafiltration membrane bioreactor system. The PH-II brought about a high angiotensin I-converting enzyme (ACE) inhibitory activity. An ACE inhibitory peptide was isolated from the PH-II, using the chromatographic methods including gel filtration, ion-exchange chromatography and reversed phase high-performance liquid chromatography. The purified ACE inhibitory peptide was a novel peptide, showing very low similarity to other ACE inhibitory peptide sequences, and was sequenced as MEGAQEAQGD. It was found that the inhibitory activity of the peptide was intensified by 3.5 times from IC50 15.9 to IC50 4.5 μM after incubation with gastrointestinal proteases. The ACE inhibitory peptide from A. molpadioidea showed a clear antihypertensive effect in spontaneously hypertensive rats (SHR), at a dosage of 3 μM/kg.
Keywords: Acaudina molpadioidea; Angiotensin I-converting enzyme inhibitory peptide; Antihypertensive effect; Spontaneously hypertensive rat;

Bioavailability of insect neuropeptides: The PK/PBAN family as a case study by Aliza Hariton; Orna Ben-Aziz; Michael Davidovitch; Ronald J. Nachman; Miriam Altstein (1034-1041).
The ability of unmodified linear peptides to penetrate the insect cuticle and exert bioactivity (e.g., stimulation of sex pheromone biosynthesis) was tested by topical application onto Heliothis peltigera moths of four insect neuropeptides (Nps) of the pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN) family: Helicoverpa zea PBAN (Hez-PBAN), Pseudaletia (Mythimna) separata pheromonotropin (PT), Leucophaea maderae PK (LPK) and Locusta migratoria myotropin (Lom-MT-II). The time kinetic of the peptides applied in double distilled water (DDW) or dimethylsulfoxide (DMSO) was tested and the activities of topically applied and injected peptides were compared. The results clearly indicated that all four peptides were highly potent but with differing activities in the two solvents: PBAN was most active in water, and PT in DMSO. The activity of PBAN in DDW lasted up to 8 h post-application and its activity in this solvent showed a faster onset and a longer persistence than in DMSO. LPK and MT differed less in their kinetics between the two solvents. Topically applied PBAN at 1 nmol exhibited an equivalent or even significantly higher potency than the injected peptide at several different times post-treatment. Similar results were obtained with topically applied and injected LPK. The present results add important information on the bioavailability of unmodified linear peptides in moths, clearly indicate that linear hydrophilic peptides can penetrate the cuticle by contact application in aqueous solutions and in organic solvents very efficiently, reach their target organ and activate it.
Keywords: PBAN; Topical application; Cuticular penetration; Sex pheromone biosynthesis; Bioavailability; Insect neuropeptides;

Expression and characterization of jingzhaotoxin-34, a novel neurotoxin from the venom of the tarantula Chilobrachys jingzhao by Jinjun Chen; Yongqun Zhang; Mingqiang Rong; Liqun Zhao; Liping Jiang; Dongyi Zhang; Meichi Wang; Yucheng Xiao; Songping Liang (1042-1048).
Jingzhaotoxin-34 (JZTX-34) is a 35-residue polypeptide from the venom of Chinese tarantula Chilobrachys jingzhao. Our previous work reported its full-length cDNA sequence encoding a precursor with 87 residues. In this study we report the protein expression and biological function characterization. The toxin was efficiently expressed by the secretary pathway in yeast. Under whole-cell patch-clamp mode, the expressed JZTX-34 was able to inhibit tetrodotoxin-sensitive (TTX-S) sodium currents (IC50  ∼ 85 nM) while having no significant effects on tetrodotoxin-resistant (TTX-R) sodium currents on rat dorsal root ganglion neurons. The inhibition of TTX-S sodium channels was completely reversed by strong depolarization (+120 mV). Toxin treatment altered neither channel activation and inactivation kinetics nor recovery rate from inactivation. However, it is interesting to note that in contrast to huwentoxin-IV, a recently identified receptor site-4 toxin from Ornithoctonus huwena venom, 100 nM JZTX-34 caused a negative shift of steady-state inactivation curve of TTX-S sodium channels by approximately 10 mV. The results indicated that JZTX-34 might inhibit mammalian sensory neuronal sodium channels through a mechanism similar to HWTX-IV by trapping the IIS4 voltage sensor in the resting conformation, but their binding sites should not overlay completely.
Keywords: Toxin; Expression; Tetrodotoxin-sensitive sodium channel; Whole-cell recording;

Synthesis of an iberiotoxin derivative by chemical ligation: A method for improved yields of cysteine-rich scorpion toxin peptides by Jon-Paul Bingham; Joycelyn B. Chun; Margaret R. Ruzicka; Qing X. Li; Zhi-Yong Tan; Yuri A. Kaulin; Darren R. Englebretsen; Edward G. Moczydlowski (1049-1057).
Automated and manual solid phase peptide synthesis techniques were combined with chemical ligation to produce a 37-residue peptide toxin derivative of iberiotoxin which contained: (i) substitution of Val16 to Ala, to facilitate kinetic feasibility of native chemical ligation, and; (ii) substitution of Asp19 to orthogonally protected Cys-4-MeOBzl for chemical conjugate derivatization following peptide folding and oxidation. This peptide ligation approach increased synthetic yields approximately 12-fold compared to standard linear peptide synthesis. In a functional inhibition assay, the ligated scorpion toxin derivative, iberiotoxin V16A/D19-Cys-4-MeOBzl, exhibited ‘native-like’ affinity (K d  = 1.9 nM) and specificity towards the BK Ca2+-activated K+ Channel (KCa1.1). This was characterized by the rapid association and slow dissociation rates (k on  = 4.59 × 105  M−1  s−1; k off  = 8.65 × 10−4 s−1) as determined by inhibition of macroscopic whole-cell currents of cloned human KCa1.1 channel. These results illustrate the successful application of peptide chemical ligation to improve yield of cysteine-rich peptide toxins over traditional solid phase peptide synthesis. Native chemical ligation is a promising method for improving production of biologically active disulfide containing peptide toxins, which have diverse applications in studies of ion-channel function.
Keywords: Peptide synthesis; Chemical ligation; Toxin; Scorpion; Iberiotoxin; BK Ca2+-activated K+ channel; KCa1.1;

The synthetic epinecidin-122–42 peptide was derived from positions 22–42 of Epinephelus coioides epinecidin-1. The synthetic SALF55–76 cyclic peptide (csSALF55–76) and SALF55–76 linear peptide (lsSALF55–76) contained sequences from positions 55 to 76 of the Penaeus monodon anti-lipopolysaccharide factor (ALF), respectively. We studied the in vitro activities of epinecidin-122–42, csSALF55–76, and lsSALF55–76 against Propionibacterium acnes, Candida albicans, and Trichomonas vaginalis. The minimum inhibitory concentrations (MICs) of epinecidin-122–42 for the test pathogen strains ranged 12.5–200 μg/ml, those of csSALF55–76 ranged 100–200 μg/ml, and those of lsSALF55–76 ranged 25–200 μg/ml. epinecidin-122–42 exhibited cytotoxicity towards P. acnes, C. albicans, and T. vaginalis (one strain of which was a metronidazole-resistant strain, while the other strain was not), suggesting that epinecidin-1 functions like a lytic peptide. Similar cytotoxicity was identified against T. vaginalis treated with the csSALF55–76 and lsSALF55–76 peptides. The antimicrobial activities of these peptides were confirmed by scanning electron microscopy (SEM), transmission electron microscopy (TEM), a viable cell count assay, and flow cytometric analysis. TEM and SEM examinations of T. vaginalis treated with these three peptides showed that severe swelling preceded cell death and breakage of the outer membrane, and the intracellular inclusion was found to have effluxed extracellularly. This phenomenon was also found with epinecidin-122–42 treatment of P. acnes and C. albicans. Our results suggest that the epinecidin-122–42, csSALF55–76, and lsSALF55–76 peptides may be good candidates for treating trichomoniasis, and epinecidin-122–42 may have potential as a drug supporting therapy for acne and candidiasis.
Keywords: Antimicrobial peptides; Antibacterial; Antifungal; Antiprotozoan; Scanning electron microscopy; Transmission electron microscopy;

The alyteserins: Two families of antimicrobial peptides from the skin secretions of the midwife toad Alytes obstetricans (Alytidae) by J. Michael Conlon; Anni Demandt; Per. F. Nielsen; Jérôme Leprince; Hubert Vaudry; Douglas C. Woodhams (1069-1073).
Two families of structurally related C-terminally α-amidated antimicrobial peptides have been identified in norepinephrine-stimulated skin secretions of the midwife toad Alytes obstetricans (Alytidae). The alyteserin-1 peptides (Gly-Leu-Lys-(Asp/Glu)-Ile-Phe-Lys-Ala-Gly-Leu-Gly-Ser-Leu-Val-Lys-(Gly/Asn)-Ile-Ala-Ala-His-Val-Ala-(Asn/Ser).NH2) show limited structural similarity to the ascaphins from the skins of frogs of the family Leiopelmatidae. Alyteserin-2a (Ile-Leu-Gly-Lys-Leu-Leu-Ser-Thr-Ala-Ala-Gly-Leu-Leu-Ser-Asn-Leu.NH2) and alyteserin-2b and -2c (Ile-Leu-Gly-Ala-Ile-Leu-Pro-Leu-Val-Ser-Gly-Leu-Leu-Ser-(Asn/Ser)-Lys-Leu.NH2) show limited sequence identity with bombinin H6, present in the skins of frogs of the family Bombinatoridae. The alyteserin-1 peptides show selective growth inhibitory activity against the Gram-negative bacteria Escherichia coli (MIC = 25 μM) whereas alyteserin-2a is more potent against the Gram-positive bacteria Staphylococcus aureus (MIC = 50 μM). The hemolytic activity against human erythrocytes of all peptides tested is relatively weak (LC50  > 100 μM). The data demonstrate that the frogs belonging to the family Alytidae are among those producing dermal antimicrobial peptides that may represent a component of the animal's system of innate immunity.
Keywords: Antimicrobial peptide; Frog skin; Alytidae; Alyteserin; Bombinin; Ascaphin;

(−)-Ternatin inhibits adipogenesis and lipid metabolism in 3T3-L1 cells by Masahiko Ito; Junko Ito; Hidefumi Kitazawa; Ken Shimamura; Takehiro Fukami; Shigeru Tokita; Kenichiro Shimokawa; Kaoru Yamada; Akio Kanatani; Daisuke Uemura (1074-1081).
(−)-Ternatin, a highly N-methylated cyclic peptide, inhibits fat accumulation in 3T3-L1 cells and reduces fat mass in mice. However, the mechanism for its anti-adipogenic effect has remained unknown. To examine the mechanism used by (−)-ternatin to inhibit adipocyte differentiation, we examined the effects of (−)-ternatin and [l-Ala4]ternatin, an inactive analog of (−)-ternatin, on the expression of adipocyte markers and lipogenic enzymes. We found that (−)-ternatin potently reduced mRNA expression of several adipocyte markers in a dose-dependent manner, whereas [l-Ala4]ternatin showed no effects. At the immediate early phase, (−)-ternatin, but not [l-Ala4]ternatin, reduced the expression of Srebp1c, Fas, Acc2 and C/EBP-α while showing no effects on C/EBP-β and C/EBP-δ. These results suggest that (−)-ternatin affects the mid-to late differentiation stages of adipocytes. Consistent with the decreased expression of lipogenic enzymes, (−)-ternatin potently inhibited triglyceride synthesis. Intriguingly, (−)-ternatin also inhibited triglyceride synthesis in rat primary hepatocytes, suggesting that the potential action sites for (−)-ternatin are shared by adipocytes and liver. Although the target molecule of (−)-ternatin remains unknown, our data suggest that (−)-ternatin and its potential target might provide a new therapeutic approach to metabolic disorders.
Keywords: (−)-Ternatin; Adipogenesis; 3T3-L1 cells; Lipogenesis;

Zonulin is not increased in the cardiac and esophageal mucosa of patients with gastroesophageal reflux disease by Thomas Wex; Klaus Mönkemüller; Doerthe Kuester; Lucia Fry; Arne Kandulski; Peter Malfertheiner (1082-1087).
Human Zonulin, related to the Zonula occludens toxin of Vibrio cholerae, regulates intestinal permeability and is induced in inflammatory disorders of the lower GI tract. Gastroesophageal reflux disease (GERD) is associated with an impairment of epithelial barrier function. Here, we studied expression of zonulin in the gastroesophageal mucosa of 58 patients with typical reflux symptoms and 27 asymptomatic controls. During endoscopy, multiple biopsies from gastroesophageal mucosa were obtained for routine histopathology (Helicobacter pylori-status, inflammation) and gene expression analysis (immunohistochemistry, ELISA). Patients with GERD presented with typical histopathological alterations like elongation of papillae (P  = 0.015), basal cell hyperplasia (P  < 0.001) and dilatation of intercellular spaces (P  = 0.002). Zonulin was found to be expressed ubiquitously in gastroesophageal mucosa. Mucosal levels in controls ranged between 2.2 and 3.7 ng/μg total protein. Mean values were significantly higher in antrum (3.3 ± 1.7 ng/μg) than cardia (2.7 ± 1.2 ng/μg) and esophagus (2.2 ± 1.3 ng/μg) (P  < 0.001), but did not differ between GERD and controls for cardia and esophageal mucosa. Immunohistochemistry revealed that predominantly epithelial cells but not stromal cells contribute to the zonulin expression in gastroesophageal mucosa. In conclusion, despite its established role for intestinal permeability, Zonulin seems not to be involved in the regulation of epithelial barrier function in relation to GERD.
Keywords: Gastroesophageal reflux disease; GERD; Zonulin; Esophageal mucosa;

Ability of GHTD-amide and analogs to enhance insulin activity through zinc chelation and dispersal of insulin oligomers by Sarah G. Paule; Biljana Nikolovski; Justin Ludeman; Robyn E. Gray; Leone Spiccia; Paul Z. Zimmet; Mark A. Myers (1088-1097).
GHTD-amide is a tetrapeptide originally isolated from human urine that has hypoglycemic activity. Insulin occurs in secretory granules of beta cells as zinc-stabilized hexamers and must disperse to monomeric form in order to bind to its receptor. The aim of this study was to identify whether GHTD-amide and an analog called ISF402 (VHTD-amide) reduce blood glucose through enhancement of insulin activity by dispersing oligomers of insulin. Peptides containing the HTD-amide sequence and a free α-amino group were optimal at binding Zn2+ and adopting secondary structure in the presence of Zn2+. Binding was concentration dependent and resulted in a 1:1 Zn:peptide complex. In vitro the tetrapeptides dispersed hexameric insulin to dimers and monomers. GHTD-amide and ISF402 potentiated the activity of hexameric insulin when co-injected into insulin resistant Zucker rats. Injection of peptides with insulin caused reductions in blood glucose and C-peptide significantly larger than achieved with insulin alone, and serum insulin time profiles were also altered consistent with a reduced clearance or enhanced dispersal of the injected insulin. Insulin potentiation by ISF402 was reduced when lispro insulin, which does not form zinc-stabilized hexamers, was used in place of hexameric zinc insulin. In conclusion, GHTD-amide and ISF402 are zinc binding peptides that disperse hexameric insulin in vitro, and potentiate the activity of hexameric insulin more so than monomeric lispro insulin. These results suggest that dispersal of hexameric insulin through chelation of Zn2+ contributes to the hypoglycemic activity of these tetrapeptides.
Keywords: Zinc; Chelation; Peptide; Insulin; Type 2 diabetes; C-peptide; Zucker rat; Insulin hexamers;

Melanocortin-4 receptor activation inhibits c-Jun N-terminal kinase activity and promotes insulin signaling by Biaoxin Chai; Ji-Yao Li; Weizhen Zhang; Hui Wang; Michael W. Mulholland (1098-1104).
The melanocortin system is crucial to regulation of energy homeostasis. The melanocortin receptor type 4 (MC4R) modulates insulin signaling via effects on c-Jun N-terminal kinase (JNK). The melanocortin agonist NDP-MSH dose-dependently inhibited JNK activity in HEK293 cells stably expressing the human MC4R; effects were reversed by melanocortin receptor antagonist. NDP-MSH time- and dose-dependently inhibited IRS-1ser307 phosphorylation, effects also reversed by a specific melanocortin receptor antagonist. NDP-MSH augmented insulin-stimulated AKT phosphorylation in vitro. The melanocortin agonist melanotan II increased insulin-stimulated AKT phosphorylation in the rat hypothalamus in vivo. NDP-MSH increased insulin-stimulated glucose uptake in hypothalamic GT1-1 cells. The current study shows that the melanocortinergic system interacts with insulin signaling via novel effects on JNK activity.
Keywords: c-Jun N-terminal kinase (JNK); Melanocortin receptor; AKT; Insulin; Insulin receptor substrate 1 (IRS-1); MT II; NDP-MSH; Hypothalamus;

Orally administered novokinin, an angiotensin AT2 receptor agonist, suppresses food intake via prostaglandin E2-dependent mechanism in mice by Kousaku Ohinata; Yoko Fujiwata; Fukumoto Shingo; Iwai Masaru; Horiuchi Masatsugu; Masaaki Yoshikawa (1105-1108).
Novokinin (Arg-Pro-Leu-Lys-Pro-Trp), having affinity for the AT2 receptor, is a potent vasorelaxing and hypotensive peptide designed based on the structure of ovokinin(2–7), a bioactive peptide derived from ovalbumin. Here we show that intracerebroventricularly (i.c.v.) administered novokinin dose-dependently suppresses food intake at a dose of 30–100 nmol/mouse in fasted conscious mice. Orally administered novokinin (30–100 mg/kg) also suppressed food intake. Novokinin suppressed food intake in wild-type and AT1 receptor-knockout mice but not in AT2 receptor-knockout mice after i.c.v. or oral administration. Novokinin-induced anorexigenic activity after i.c.v. administration was blocked by indomethacin, a cyclooxygenase inhibitor, or ONO-AE3-208, an antagonist for EP4 receptor for PGE2. Taken together, novokinin may suppress food intake via activation of PGE2–EP4, downstream of the AT2 receptor.
Keywords: Novokinin; RPLKPW; Food intake; Angiotensin AT2 receptor; EP4 receptor;

Inhibition of endoplasm reticulum stress by ghrelin protects against ischemia/reperfusion injury in rat heart by Gai-Gai Zhang; Xu Teng; Yue Liu; Yan Cai; Ye-Bo Zhou; Xiao-Hui Duan; Jun-Qiu Song; Yi Shi; Chao-Shu Tang; Xin-Hua Yin; Yong-Fen Qi (1109-1116).
Ghrelin is a multi-functional polypeptide with cardiovascular protective effects. We aimed to explore whether the cardioprotective effect of ghrelin is mediated by inhibiting myocardial endoplasmic reticulum stress (ERS). A Langendorff model of isolated rat heart was used with ischemia/reperfusion (I/R; 40/120 min). Cardiac function was monitored, and histomorphologic features, degree of myocardial injury, level of ERS markers, and number of apoptotic cardiomyocytes were determined. Compared with control group, the I/R group showed significantly decreased cardiac function, seriously damaged myocardial tissue, increased number of apoptotic cells, and overexpression of mRNA and protein of ERS markers. However, preadministration of ghrelin in vivo (10−8  mol/kg, intraperitoneal injection, every 12 h, twice in all) greatly ameliorated the damaged heart function, attenuated myocardial injury and apoptosis, and decreased the expression of ERS markers: it decreased the mRNA and protein levels of glucose-regulated protein78 (GRP78) and C/EBP homologous protein (CHOP), with reduced caspase-12 protein expression. Furthermore, in vitro, ghrelin directly inhibited the myocardial ERS response induced by tunicamycin or dithiothreitol in rat cardiac tissue. Ghrelin could protect the heart against I/R injury, at least in part, through inhibiting myocardial ERS.
Keywords: Ghrelin; Ischemia/reperfusion injury; Endoplasmic reticulum stress; Apoptosis; Unfolded protein response;

Urocortin inhibits mesenteric arterial remodeling in spontaneously hypertensive rats by Jiandong Chen; Jin Tao; Rongjian Zhang; Youhua Xu; TuckWah Soong; Shengnan Li (1117-1123).
Urocortin (UCN), a newly isolated corticotrophin-releasing factor (CRF) related peptide, has been found to have potent cardiovascular protective effects. This study aimed to investigate the long-term effects of UCN on arterial remodeling and related functional alterations. UCN (7 μg/kg/d) was administered to spontaneously hypertensive rats (SHR) for 8 weeks. Systolic blood pressure (SBP) was measured weekly. Functional studies were performed on isolated mesenteric arterial segments. Also, by light microscope and electron microscope, the morphology of mesenteric arteries was examined. Our results showed that mean SBP in UCN-treated SHRs was about 40 mmHg lower than that of the control SHR group, and was similar to that of the enalapril-treated group. In the mesenteric arterial segments pre-contracted with norepinephrine (0.001–10 μM), the maximal relaxation rate induced by acetylcholine (10 μM) in UCN-treated group (about 93.3%) was higher than that in SHR control group (about 40.0%) (n  = 6, P  < 0.01). Furthermore examination under light microscope showed that UCN (3.5 μg/kg/d) treatment significantly reduced media thickness, media/lumen ratio, resulting in larger lumen diameter while analysis of transmission electron microscopic findings revealed that chromatin, internal elastic lamina and densely packed mitochondria displayed a close-to-normal distribution after UCN treatment. These results suggested that long-term UCN treatment not only had hypotensive effects but may also inhibited development of vascular remodeling in mesenteric arteries in SHR.
Keywords: Urocortin; Arterial remodeling; Spontaneously hypertensive rats;

Increased expression of urotensin II, urotensin II-related peptide and urotensin II receptor mRNAs in the cardiovascular organs of hypertensive rats: Comparison with endothelin-1 by Takuo Hirose; Kazuhiro Takahashi; Nobuyoshi Mori; Takashi Nakayama; Masahiro Kikuya; Takayoshi Ohkubo; Masahiro Kohzuki; Kazuhito Totsune; Yutaka Imai (1124-1129).
Urotensin II (UII) and urotensin II-related peptide (URP) are novel vasoactive peptides that share urotensin II receptor (UT). We have recently reported that expressions of URP and UT were up-regulated in kidneys of rats with renal failure or hypertension. To clarify possible changes of the UII system expression in cardiovascular organs with hypertension, we examined the gene expression of UII, URP and UT in hearts and aortae of hypertensive rats. Furthermore, the expression was compared with that of endothelin-1 (ET-1). Quantitative reverse transcription polymerase chain reaction analysis showed that expression levels of UII mRNA and UT mRNA were significantly elevated in the atrium of 11–12-week-old spontaneously hypertensive rats (SHR) compared with age-matched Wistar–Kyoto rats (WKY). Moreover, UT mRNA expression was elevated in the ventricle of 11–12-week-old SHR. In the aorta, expression levels of URP mRNA and UT mRNA were significantly elevated in 11–12-week-old SHR compared with age-matched WKY, similarly to those in the kidney. In contrast, expression levels of ET-1 were significantly decreased in both the heart and the kidney of 11–12-week-old SHR compared with age-matched WKY. Immunohistochemistry showed that URP and UT were immunostained in cardiomyocytes, with weaker immunostaining in vascular endothelial and smooth muscle cells, in both SHR and WKY. These findings indicate that the gene expression of the UII system components (UII, URP and UT) and ET-1 is differently regulated in hypertension, and that the UII system in the heart and aortae may have certain pathophysiological roles in hypertension.
Keywords: Urotensin II; Urotensin II-related peptide; Endothelin-1; Polymerase chain reaction; Immunohistochemistry;

Structure–activity relationship study on Tyr9 of urotensin-II(4–11): Identification of a partial agonist of the UT receptor by Madura Batuwangala; Valeria Camarda; John McDonald; Erika Marzola; David G. Lambert; Leong L. Ng; Girolamo Calo’; Domenico Regoli; Claudio Trapella; Remo Guerrini; Severo Salvadori (1130-1136).
Urotensin-II (U-II) activates the U-II receptor (UT) to modulate a range of biological responses at both central and peripheral sites. Previous studies have demonstrated that the sequence Trp7-Lys8-Tyr9 of the cyclic portion of the peptide is crucial for biological activity. Here, we describe a focused structure–activity study of Tyr9 which has been replaced with a series of non-coded amino acids in the U-II(4–11) template. Thirteen analogs were synthesized and pharmacologically tested for intracellular calcium mobilization in HEK293 cells stably expressing the rat UT receptor. The results of this study demonstrated the following Tyr9 structure–activity features: (i) the position of the OH group of the side chain is not important for biological activity, (ii) the distance of the phenol moiety from the peptide backbone and its conformational freedom are crucial for UT receptor recognition, (iii) this position is important not only for receptor occupation but also for its activation since the 3,5-diiodoTyr9 chemical modification generated a potent partial agonist. This pharmacological activity of [3,5-diiodoTyr9]U-II(4–11) was confirmed in bioassay experiments performed using the rat thoracic aorta as a U-II sensitive preparation.
Keywords: Urotensin-II; UT receptor; Structure–activity study; Peptide synthesis; Ca2+ mobilization; Rat aorta;

Osmoregulation of natriuretic peptide receptors in bromoethylamine-treated rat kidney by Kuichang Yuan; Xuanshun Jin; Shan Gao; Amin Shah; Sun Young Kim; Sung Zoo Kim; Suhn Hee Kim (1137-1143).
Extracellular osmolarity is known as an important factor for the regulation of natriuretic peptide receptors (NPRs). We investigated the intra-renal osmoregulation of NPRs using renal medullectomized rats with bromoethylamine hydrobromide (BEA, 200 mg/kg). The administration of BEA caused the decreased food intake and body weight. Water intake was decreased on the first day and then increased from the second day. Urine volume was persistently increased from the first day and free water clearance was also increased from the second day. Urinary excretions of sodium and potassium were decreased on the second day and then recovered to control level. Plasma levels of atrial natriuretic peptide (ANP) and Dendroaspis natriuretic peptide (DNP) in BEA-treated rats were not different from control rats. The inactive renin was increased. The maximum binding capacities of 125I-ANP as well as 125I-DNP decreased in glomeruli and medulla of BEA-treated rat kidneys but the binding affinity was not changed. In renal cortex, the gene expressions of ANP, NPR-A, and NPR-B were not changed but that of NPR-C decreased. In renal medulla, the gene expressions of NPR-A, -B, and -C decreased without change in ANP mRNA. Both renal medullary osmolarity and sodium concentration by BEA treatment were lower than those in control kidney. The cGMP concentrations in renal medulla and urine in BEA-treated rats were higher than those in control rats. These results suggest that the increased cGMP production may be partly involved in the decrease in NPRs mRNA expression and their binding capacities by BEA-induced medullectomy.
Keywords: Medullectomy; Osmolarity; Bromoethylamine; Natriuretic peptide receptor; Natriuretic peptide; Renal function; cGMP;

Apelin protects heart against ischemia/reperfusion injury in rat by Xiang Jun Zeng; Li Ke Zhang; Hong Xia Wang; Ling Qiao Lu; Li Quan Ma; Chao Shu Tang (1144-1152).
Apelin, the endogenous ligand of the G protein-coupled APJ receptor, is a peptide mediator with emerging regulatory actions in the heart. We aimed to determine whether the endogenous apelin/APJ system is an intrinsic protective pathway in ischemic/reperfusion injury. A Langendorff model of perfused isolated rat hearts and primary cultured myocardial cells from neonatal rats were used. Cardiac function was monitored and apelin/APJ expression was determined by real-time PCR and Western blot analysis. In rats under I/R, cardiac function was significantly decreased as compared with controls, and APJ was over-expressed at both the mRNA and protein levels (by 7-fold and 35%, respectively, both p  < 0.01). However, pre-administration of apelin (30 pmol/L) greatly ameliorated the reduced heart function. To gain mechanistic insight into the cardio-protective effects of apelin/APJ, cultured cardiomyocytes were treated with apelin (30 pmol/L), and those under hypoxia/re-oxygenation showed H/R-induced apoptosis and up-regulated apelin/APJ mRNA expression by 6-fold and 7-fold, respectively (both p  < 0.01). And lactate dehydrogenase leakage was greatly increased as well. Meanwhile, apoptosis, the generation of reactive oxygen species and malonaldehyde content as well as lactate dehydrogenase leakage were inhibited by apelin. Furthermore, apelin enhanced superoxide dismutase activity and phosphorylation of extracellular signal-regulated kinase 1/2 and Akt after hypoxia/re-oxygenation. In conclusion, apelin/APJ has protective effects in ischemic heart disease and might constitute an important therapy target.
Keywords: Apelin; APJ; Ischemia/reperfusion injury; Apoptosis;

The effect of apelin-13 on pain modulation at the supraspinal level was investigated in mice using the tail immersion test. Intracerebroventricular (i.c.v.) administration of apelin-13 (0.3, 0.5, 0.8 and 3 μg/mouse) produced a dose- and time-related antinociceptive effect. This effect was significantly antagonized by the APJ receptor antagonist apelin-13(F13A), indicating an APJ receptor-mediated mechanism. Furthermore, naloxone, β-funaltrexamine and naloxonazine, could reverse the analgesic effect. However, naltrindole or nor-binaltorphimine could not reverse the effect, suggesting that μ opioid receptor (primarily μ1 opioid receptor subtype) is involved in the analgesic response evoked by apelin-13. Moreover, i.c.v. administration of apelin-13 potentiated the analgesic effect induced by morphine (i.c.v., 5 μg/kg) and this potentiated effect can be also reversed by naloxone.
Keywords: Apelin-13; Apelin-13(F13A); Opioid receptor; Antinociception;

The immunoregulatory role of methionine-enkephalin (Met-enk) is well studied in mammals, but has not been explored in ectotherms despite the fact that this peptide is highly conserved in vertebrates. The present study demonstrates the diverse effects of Met-enk depending on its concentration and specific function of splenic phagocytes in the freshwater fish Channa punctatus. Although Met-enk increased both phagocytic as well as respiratory burst activity, the concentration-related response was opposite to each other. It had the maximum stimulatory effect on phagocytosis at 10−9  M, while the same concentration was least effective in increasing superoxide production. Similarly, Met-enk at concentrations lower or higher than 10−9  M was either ineffective or less effective in case of phagocytosis, while highly effective in stimulating superoxide production. On the other hand, concentration-independent inhibitory effect of Met-enk was observed in case of nitrite production. Nonetheless, Met-enk regulated all the functions of phagocyte through opioid receptors since non-specific opioid receptor antagonist naltrexone completely blocked the effect of Met-enk on phagocytosis, superoxide and nitrite production by splenic phagocytes of C. punctatus. Among selective opioid receptor antagonists, δ-opioid receptor antagonist naltrindole completely antagonized the effect of Met-enk on phagocytosis, superoxide and nitrite production, while μ- and κ-opioid receptor antagonist, CTAP and norbinaltorphimine, respectively, were ineffective in influencing any of the functions. This suggests that Met-enk modulates splenic phagocyte functions in the fish C. punctatus via δ-opioid receptor. This is further substantiated by using highly selective δ-opioid receptor agonist, SNC80.
Keywords: Methionine-enkephalin; Phagocyte; Phagocytosis; Superoxide; Nitrite; Opioid receptors; Fish;

Much evidence indicates that endogenous opioid peptides are involved in effects caused by ethanol. The aim of the present study was to determine whether dansyl-PQR amide, a putative antagonist of receptors for an anti-opioid peptide—neuropeptide FF (NPFF) could affect anxiety-like behavior measured during withdrawal from acute-, and chronic ethanol administration in the elevated plus maze test in rats. Our study indicated that intracerebroventricular (i.c.v.) administration of dansyl-PQRamide (2.4 and 4.8 nmol) reversed anxiety-like behavior measured as a percent time spent in the open arms, and a percent open arm entries onto the open arms in the elevated plus-maze test in rats. These effects were inhibited by NPFF (10 and/or 20 nmol, i.c.v.) in the experiments performed during withdrawal from acute- and chronic ethanol administration. During withdrawal from acute ethanol, naloxone (1 mg/kg, i.p.), a nonselective opioid receptor antagonist, attenuated only an increased percent time spent in the open arms induced by dansyl-PQR amide (4.8 nmol). Dansyl-PQR amide, NPFF and naloxone given alone to naive rats did not have influence on spontaneous locomotor activity of animals. Furthermore, NPFF potentiated anxiety-like behavior during withdrawal from chronic, but not acute, ethanol administration in rats. Our data suggest that NPFF system is involved in regulation of affective symptoms of ethanol withdrawal. It seems that involvement of the NPFF system in ethanol withdrawal anxiety-like behavior is associated with regulation of the opioid system activity.
Keywords: Ethanol withdrawal; Anxiety; Elevated plus maze; Dansyl-PQRamide; NPFF; Naloxone;

A novel human parathyroid hormone (1-34) analog for the treatment of osteoporosis by Jiao Feng; Yanhua Liu; Yun Xing; Huaqian Wang; Taiming Li; Jingjing Liu; Hao Fan; Rongyue Cao (1173-1180).
The more effective bioactive peptides which can be efficiently prepared with low cost and high yield are in great demand for the development of therapeutic peptides. In this study, Pro-Pro-[Arg11]hPTH(1-34)-Pro-Pro-Asp (hPTH′), an analog of human parathyroid hormone (1-34) [hPTH(1-34)], was prepared by an efficiently cost-effective preparation strategy. It was repeated eight times on the gene level and expressed in the form of inclusion bodies in Escherichia coli. Following some primary purifications, the inclusion bodies of octapeptide repeats were hydrolyzed into hPTH′ monomers by hydrochloric acid. After further purified by a CM52 chromatographic column, hPTH′ reached a yield of 16.9% and output of 61 mg/l medium. Furthermore, we compared anabolic effects between hPTH′ and hPTH(1-34) on ovariectomized rats. The results indicated that hPTH′ led to more significant increments in bone material density (BMD), trabecular width and bone formation marker as well as less loss in marrow space area than what hPTH(1-34) did. In addition, the active form of hPTH′ was introduced by the excision with dipeptidyl peptidase IV in vivo. These results suggest that hPTH′ is more effective than hPTH(1-34) in stimulating bone formation and improving skeletal microarchitecture. We can conclude that hPTH′ is potentially a more effective therapeutical agent on osteoporosis.
Keywords: Parathyroid hormone; Bone material density; Bone formation; Tandem expression; Acid hydrolysis; Dipeptidyl peptidase IV;

Restrain of bone growth by Estrogen-Mimetic Peptide-1 (EMP-1): A micro-computed tomographic study by Roni Kasher; Alon Bajayo; Yankel Gabet; Nava Nevo; Mati Fridkin; Ephraim Katchalski-Katzir; Fortune Kohen; Itai Bab (1181-1186).
Estrogen has a key role in the regulation of skeletal growth and maintenance of bone mass. Recently, we developed peptides having estrogen-like activity as potential estrogen-based new drugs. The aim of the present study was to evaluate the influence of long-term administration of the most efficacious of these peptides, the hexapeptide EMP-1 (VSWFFE), on bone mass and development. EMP-1 was injected daily to ovariectomized (OVX) and intact young, sexually mature female mice for 10 weeks. Whole femora, including the cartilaginous growth plates were analyzed by micro-computed tomography (μCT). We found that peptide EMP-1 restrains bone growth in OVX mice: it inhibited dramatically bone longitudinal growth (40%), and decreased femoral diaphyseal diameter. Peptide EMP-1 had no effect on bone growth in normal mice, and did not influence the OVX-induced bone loss. We then developed a new μCT methodology to evaluate uncalcified and calcified growth plate parameters. In the OVX mice, peptide EMP-1 reduced volume and thickness of the uncalcified growth plate, a possible cause for the inhibition of bone longitudinal growth. Peptide EMP-1 may be used as a lead compound for the development of drugs to treat acromegalic patients.
Keywords: Estrogen-Mimetic Peptide; Bone growth; Micro-computed tomography; Estrogenic activity;

Ghrelin is a powerful orexigenic peptide predominantly secreted by the stomach. Blood concentration of ghrelin increases before meals and fall postprandial. Its regulation appears to be influenced by the type of macronutrient ingested, the vagus nerve stimulation and by other post-meal stimulated hormonal factors. However, the direct role of nutrients (amino acids or lipids), neuronal (vagal neurotransmitter acetylcholine) and satiety-inducing factor such as CCK are not known. To study this we applied amino acids, lipids, acetylcholine and CCK via vascular perfusion to the isolated stomachs and found that amino acids significantly reduced ghrelin release from the isolated stomach by approximately ∼30% vs. the control while lipids (10% intralipid) had no affect. Acetylcholine (1 μM) increased ghrelin release from the stomach by ∼37% whereas insulin (10 nM) decreased it by ∼30% vs. the control. Interestingly, CCK (100 nM) potently increased ghrelin release by ∼200% vs. the control. Therefore it appears that ghrelin secretion from the stomach is under direct influence of amino acids, neurotransmitter acetylcholine and hormones such as insulin and CCK.
Keywords: Ghrelin; Glucose; Amino acid; Lipid; Acetylcholine; CCK; Insulin;

Phosphoinositide 3-kinase is required for bombesin-induced enhancement of fear memory consolidation in the hippocampus by Rafael Roesler; Samira S. Valvassori; Adalberto A. Castro; Tatiana Luft; Gilberto Schwartsmann; João Quevedo (1192-1196).
Increasing evidence indicates that the neuronal gastrin-releasing peptide-preferring bombesin receptor (GRPR) is a key molecular regulator of fear memory formation. However, the downstream signaling events remain poorly understood. The protooncogene product phosphoinositide 3-kinase (PI3K) has been implicated in regulating memory formation, as well as in mediating cellular responses to GRPR activation in glioma and neuroblastoma cells. We show here that GRPR modulation of fear memory consolidation in the rat hippocampus requires PI3K activation. Male Wistar rats received bilateral infusions of the GRPR agonist bombesin (BB) or the PI3K inhibitor LY294002 into the CA1 region of the dorsal hippocampus immediately after inhibitory avoidance (IA) conditioning. BB enhanced, whereas LY294002 impaired, IA memory retention. The BB-induced memory enhancement was blocked by coinfusion of either a GRPR antagonist or LY294002. These findings provide the first evidence suggesting that PI3K signaling is required for GRPR regulation of CNS function.
Keywords: Bombesin; Gastrin-releasing peptide receptor; Phosphoinositide 3-kinase; Hippocampus; Synaptic plasticity; Fear memory;

Protective effect of S14G-humanin against beta-amyloid induced LTP inhibition in mouse hippocampal slices by Wei Zhang; Jianting Miao; Jian Hao; Zhen Li; Jiang Xu; Rui Liu; Fale Cao; Ruirui Wang; Jun Chen; Zhuyi Li (1197-1202).
Synaptic dysfunction induced by amyloid-beta protein (Aβ) has been shown to play a critical role in cognitive deficits of Alzheimer's disease (AD). Currently, however there is no clinical causative therapy for the disease. S14G-humanin (HNG) is best known for its strong neuroprotective ability against AD-related insults in vitro, and several in vivo studies have shown its effectiveness in ameliorating the cognitive impairment, but the precise mechanism of HNG on neuroprotection still remains to be elucidated. The present study examined the effects of HNG on Aβ-induced inhibition of hippocampal long-term potentiation (LTP) in mouse hippocampal slices. The results disclosed that soluble Aβ25–35 significantly inhibited the induction of early-phase LTP (E-LTP) and late-phase LTP (L-LTP) in the hippocampal CA1 region without affecting the basal synaptic transmission, while HNG significantly ameliorated such inhibition of E-LTP and L-LTP in a dose-dependent manner. In addition, the reduction of phosphorylated CREB trigged by Aβ25–35 was restored by HNG during L-LTP induction, possibly attributing to the improvement of the L-LTP inhibition. Collectively, our findings add to the evidence that soluble Aβ-induced LTP inhibition may represent an early pathological event of AD, and demonstrate for the first time that HNG may improve LTP inhibition by subneurotoxic concentration of soluble Aβ, suggesting that HNG may have therapeutic potential for Aβ-induced synaptic dysfunction closely associated with cognitive deficits in the early stage of AD.

Ghrelin and its biological effects on pigs by Xiao-Ying Dong; Jian Xu; Sheng-Qiu Tang; Hai-Yun Li; Qing-Yan Jiang; Xiao-Ting Zou (1203-1211).
Ghrelin is a 28 amino acid peptide, which produces its marked effects through binding to the endogenous ligand of the growth hormone secretagogue receptor (GHS-R). Based on the contemporary literatures, it was shown that ghrelin was involved in a series of biological functions including regulation of food intake, body weight, gastrointestinal (GI) motility, hormone secretion, glucose release, cardiovascular functions, enzyme release, cell proliferation and reproduction in pigs through binding to GHS-R 1a or unidentified receptors. It was also observed that ghrelin induced adipocyte and hepatocyte proliferation of primary cultured piglet. In this paper, recent research on ghrelin structure, distribution, GHS-R receptor, biological functions and its regulatory mechanisms for pigs are presented.
Keywords: Ghrelin; Peptide; GHS-R; Pig; Function;