Peptides (v.31, #5)

The swaposin-like domain of potato aspartic protease (StAsp-PSI) exerts antimicrobial activity on plant and human pathogens by Fernando F. Muñoz; Julieta R. Mendieta; Mariana R. Pagano; Roberto A. Paggi; Gustavo R. Daleo; María G. Guevara (777-785).
Plant-specific insert domain (PSI) is a region of approximately 100 amino acid residues present in most plant aspartic protease (AP) precursors. PSI is not a true saposin domain; it is the exchange of the N- and C-terminal portions of the saposin like domain. Hence, PSI is called a swaposin domain. Here, we report the cloned, heterologous expression and purification of PSI from StAsp 1 (Solanum tuberosum aspartic protease 1), called StAsp-PSI. Results obtained here show that StAsp-PSI is able to kill spores of two potato pathogens in a dose-dependent manner without any deleterious effect on plant cells. As reported for StAPs (S. tuberosum aspartic proteases), the StAsp-PSI ability to kill microbial pathogens is dependent on the direct interaction of the protein with the microbial cell wall/or membrane, leading to increased permeability and lysis. Additionally, we demonstrated that, like proteins of the SAPLIP family, StAsp-PSI and StAPs are cytotoxic to Gram-negative and Gram-positive bacteria in a dose dependent manner. The amino acid residues conserved in SP_B (pulmonary surfactant protein B) and StAsp-PSI could explain the cytotoxic activity exerted by StAsp-PSI and StAPs against Gram-positive bacteria. These results and data previously reported suggest that the presence of the PSI domain in mature StAPs could be related to their antimicrobial activity.
Keywords: Plant specific domain; SAPLIPs; Antimicrobial proteins;

Purification and identification of three novel antioxidant peptides from Cornu Bubali (water buffalo horn) by Rui Liu; Min Wang; Jin-ao Duan; Jian-ming Guo; Yu-ping Tang (786-793).
Cornu Bubali (water buffalo horn, WBH) has been used in Traditional Chinese Medicine (TCM) for thousands of years. In the present study, three peptides with antioxidant properties were purified from aqueous extract of Cornu Bubali (water buffalo horn, WBH) by consecutive chromatographic methods including gel filtration chromatography, ion-exchange chromatography and high performance liquid chromatography. The sequences of the three peptides were identified to be Gln-Tyr-Asp-Gln-Gly-Val (WBH-1, 708 Da), Tyr-Glu-Asp-Cys-Thr-Asp-Cys-Gly-Asn (WBH-2, 1018 Da) and Ala-Ala-Asp-Asn-Ala-Asn-Glu-Leu-Phe-Pro-Pro-Asn (WBH-3, 1271 Da) by matrix assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-LIFT-TOF/TOF MS). The antioxidant activity of these peptides was tested using 1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay directly. Methylthiazol tetrazolium (MTT) assay and lactate dehydrogenase (LDH) release assay were also used to evaluate the protection of peptides against hydrogen peroxide (H2O2) induced injury. The results showed that these peptides could reduce the DPPH radical and protect rat cerebral microvascular endothelial cells (rCMECs) against H2O2-induced injury, thus demonstrating that these peptides had antioxidant activity. These results suggest that WBH-1, WBH-2 and WBH-3, isolated from the aqueous extract of water buffalo horn are natural antioxidants and may contribute to the efficacy of WBH.
Keywords: Cornu Bubali (water buffalo horn); Aqueous extract; Purified peptides; Sequence identification; Antioxidant activity;

Although several low amphipathic peptides have been known to exhibit antimicrobial activity, their mode of action has not been completely elucidated. In this study, using designed low amphipathic peptides that retain different α-helical content and hydrophobicity, we attempted to investigate the mechanism of these properties. Calorimetric and thermodynamic analyses demonstrated that the peptides induce formation of two lipid domains in an anionic liposome at a high peptide-to-lipid ratio. On the other hand, even at a low peptide-to-lipid ratio, they caused minimal membrane damage, such as flip-flop of membrane lipids or leakage of calcein molecules from liposomes, and never translocated across membranes. Interaction energies between the peptides and anionic liposomes showed good correlation with antimicrobial activity for both Escherichia coli and Bacillus subtilis. We thus propose that the domain formation mechanism in which antimicrobial peptides exhibit activity solely by forming lipid domains without membrane damage is a major determinant of the antimicrobial activity of low amphipathic peptides. These peptides appear to stiffen the membrane such that it is deprived of the fluidity necessary for biological functions. We also showed that to construct the lipid domains, peptides need not form stable and cooperative structures. Rather, it is essential for peptides to only interact tightly with the membrane interface via strong electrostatic interactions, and slight differences in binding strength are invoked by differences in hydrophobicity. The peptides thus designed might pave the way for “clean” antimicrobial reagents that never cause release of membrane elements and efflux of their inner components.
Keywords: Antimicrobial peptide; Low amphipathicity; Domain formation mechanism;

Antimicrobial peptides of an anti-lipopolysaccharide factor, epinecidin-1, and hepcidin reduce the lethality of Riemerella anatipestifer sepsis in ducks by Chieh-Yu Pan; The-Yuan Chow; Chao-Yuan Yu; Chang-You Yu; Jian-Chyi Chen; Jyh-Yih Chen (806-815).
Antimicrobial peptides (AMPs) are effective against a wide range of microbes, but still no research results have reported their use in duck disease therapy. Riemerella anatipestifer (RA) is a Gram-negative bacterium which infects ducks and causes very significant economic losses. The minimum inhibitory concentrations (MICs) of epinecidin-1 for the tested RA strains ranged 6.25–50 μg/ml, those of the SALF55–76 cyclic peptide ranged 12.5–25 μg/ml, those of the SALF55–76 linear peptide ranged 6.25–25 μg/ml, those of hepcidin TH1–5 ranged 25–400 μg/ml, and those of hepcidin TH2–3 ranged 100–400 μg/ml. The antimicrobial activities of these peptides were confirmed by transmission electron microscopy which showed that RA disruption of the outer membrane brought about cell death. In addition, pretreatment, co-treatment, and post-treatment with peptides were all effective in promoting a significant decrease in duck mortality and decreasing the number of infectious bacteria. A quantitative RT-PCR was performed to survey levels of gene expressions of Mn superoxide dismutase in the brain, lipoprotein lipase in the liver, and H5 histone in the spleen induced in response to bacterial infection and an injection of the AMPs in experiments with the duck, Cairina moschata. Our results indicated that the rescue of ducks by the peptides and the behavior of the peptides, which was like an enhancer in immunology, may involve regulation of the expressions of these genes. Collectively, these peptides reduced the mortality in ducks during bacterial challenge, suggesting that AMPs have the potential to serve as therapeutic drugs for use against bacterial infectious diseases in ducks.
Keywords: Antimicrobial peptides; Riemerella anatipestifer; Sepsis; Duck;

Identification of gonadotropin-inhibitory hormone in the zebra finch (Taeniopygia guttata): Peptide isolation, cDNA cloning and brain distribution by Yasuko Tobari; Norio Iijima; Kenta Tsunekawa; Tomohiro Osugi; Kazuo Okanoya; Kazuyoshi Tsutsui; Hitoshi Ozawa (816-826).
Two novel RFamide peptides, kisspeptins and gonadotropin-inhibitory hormone (GnIH) are neuropeptides that appear critical in the regulation of the reproductive neuroendocrine axis. GnIH was first identified in avian brain, however, kisspeptins have not been identified in birds. To determine biochemically the presence of kisspeptins and GnIH in the zebra finch, a study was conducted to isolate these two peptides from zebra finch brain. Peptides were isolated by immunoaffinity purification and only one peptide was characterized by mass spectrometry. This peptide was confirmed to be a 12-amino acid sequence with RFamide at its C-terminus; its sequence is SIKPFSNLPLRFamide (zebra finch GnIH). By this approach, however, identification of kisspeptin from zebra finch brain was not achieved. Cloned zebra finch GnIH precursor cDNA encoded three peptides that possess characteristic LPXRFamide (X = L or Q) motifs at the C-termini. In situ hybridization and immunohistochemical analysis revealed the cellular localization of zebra finch GnIH mRNA and peptide in the paraventricular nucleus and the dorsomedial nucleus of the hypothalamus. Fluorescent immunohistochemistry with confocal microscopy indicated that GnIH-immunoreactive (ir) fibers are very close appositions with gonadotropin-releasing hormone-I (GnRH-I) cells. Furthermore GnIH-ir nerve fibers were widely distributed in the multiple brain regions including the septum, preoptic area, median eminence, optic tectum and median eminence. The prominent fibers were seen in the ventral tegmental area, midbrain central gray and dorsal motor nucleus of the vagus in the medulla. Thus, GnIH may participate in not only neuroendocrine functions but also regulation of motivation for social behavior and autonomic mechanisms.
Keywords: Kisspeptin; Gonadotropin-releasing hormone (GnRH); Gonadotropin-inhibitory hormone (GnIH); Songbird; Neuropeptides; Reproduction;

A palmitoyl conjugate of insect pentapeptide Yamamarin arrests cell proliferation and respiration by Yosinori Sato; Ping Yang; Ying An; Kazushige Matsukawa; Kikukatsu Ito; Shigeo Imanishi; Hirokazu Matsuda; Yusuke Uchiyama; Kunio Imai; Shigeki Ito; Yoji Ishida; Koichi Suzuki (827-833).
A palmitoyl conjugate of an insect pentapeptide that occurs in diapausing insects causes a reversible cell-cycle arrest and suppresses mitochondrial respiration. This peptide compound also causes growth arrest in murine leukemic cell line expressing human gene Bcr/Abl and a farnesoyl peptide induces embryonic diapause in Bombyx mori. These results demonstrate that the insect peptide compounds can lead to the understanding of a common pathway in developmental arrest in animals and may provide a new peptidominetic analog in the development of biopharmaceuticals and pest management.
Keywords: Insect pentapeptide; Yamamarin compound; Cell growth arrest; Mitochondrial respiration arrest, Biopharamaceutical;

A thermally targeted peptide inhibitor of symmetrical dimethylation inhibits cancer-cell proliferation by Gene L. Bidwell; Angela A. Whittom; Emily Thomas; Daniel Lyons; Michael D. Hebert; Drazen Raucher (834-841).
Targeting splicing machinery components is an underdeveloped strategy for cancer therapy. Uridine-rich small nuclear ribonucleoproteins (UsnRNPs) are essential spliceosome components that recognize splice sites in newly transcribed RNA. The major spliceosomal snRNPs are comprised of UsnRNA bound by a ring of Sm proteins. The survival of motor neuron (SMN) complex provides specificity for binding of Sm proteins to UsnRNAs. Three of the seven proteins that comprise the Sm core possess post-translationally modified C-terminal symmetric dimethylarginine (sDMA) residues which promote binding of these proteins to SMN. Here we describe a peptide inhibitor of sDMA that is capable of interfering with SMN/SmB interaction. The inhibitory peptide was attached to elastin-like polypeptide, a thermally responsive macromolecular carrier, in order to increase its stability and allow enhancement of its cellular uptake by thermal targeting. The fusion polypeptide inhibited the interaction of SMN/SmB, inhibited proliferation, and induced apoptosis in HeLa cells.
Keywords: Elastin-like polypeptide; Thermal targeting; Splicing inhibitor; GRG peptide; Symmetrical dimethylation;

Stimulation of N-cadherin-dependent neurite outgrowth by small molecule peptide mimetic agonists of the N-cadherin HAV motif by Susan M. Burden-Gulley; Theresa J. Gates; Sonya E.L. Craig; Mukur Gupta; Susann M. Brady-Kalnay (842-849).
N-cadherin is a cell adhesion molecule that promotes axon outgrowth and synapse formation during the development of the central nervous system. In addition, N-cadherin promotes glial cell adhesion and myelination of axons. Therefore, stimulating N-cadherin function with N-cadherin agonists could be used therapeutically to promote regeneration of the nervous system and remyelination after injury or disease. In the extracellular domain of N-cadherin, the amino acid sequence HAV is required for N-cadherin-mediated adhesion and neurite outgrowth. The ADH-1 cyclic peptide, derived from the N-cadherin HAV site, is an effective antagonist of N-cadherin-mediated neurite outgrowth and is currently being tested in clinical trials for cancer chemotherapy. Of interest, a dimeric version of this cyclic peptide, N-Ac-CHAVDINGHAVDIC-NH2, functions as an N-cadherin agonist. This dimeric peptide agonist and the peptide antagonist ADH-1 both have limitations as drugs due to their metabolic instability and lack of oral delivery. To address this issue Adherex Technologies Inc. generated a small molecule library of peptidomimetics to the HAV region of N-cadherin, which would be more amenable to therapeutic use. We screened the Adherex library for compounds that altered neurite outgrowth and identified eight N-cadherin agonists that stimulated N-cadherin-dependent neurite outgrowth. Five of these agonists also stimulated retinal cell migration. These small molecule agonists may be effective reagents for promoting axon growth and remyelination after injury or disease.
Keywords: N-cadherin; Neurite outgrowth; Cell migration; Peptidomimetic; Small molecules; Agonist;

Anti-tumor effects of a novel chimeric peptide on S180 and H22 xenografts bearing nude mice by Dongdong Wu; Yanfeng Gao; Lixiang Chen; Yuanming Qi; Qiaozhen Kang; Haili Wang; Linyu Zhu; Yong Ye; Mingxia Zhai (850-864).
In recent years, many endogenous peptides have been identified by screening combinatory phage display peptide library, which play important roles in the process of angiogenesis. A heptapeptide, ATWLPPR, binds specifically to NRP-1 and selectively inhibits VEGF165 binding to VEGFR-2. Another heptapeptide, NLLMAAS, blocks both Ang-1 and Ang-2 binding to Tie-2 in a dose-dependent manner. In the present study, we aimed to connect ATWLPPR (V1) with NLLMAAS (V2) via a flexible linker, Ala-Ala, to reconstruct a novel peptide ATWLPPRAANLLMAAS (V3). We firstly investigated the anti-tumor and anti-angiogenic effects of peptide V3 on sarcoma S180 and hepatoma H22 bearing BALB/c nude mice. Mice were continuously subcutaneously administrated with normal saline, V1 (320 μg/kg/d), V2 (320 μg/kg/d), V1  +  V2 (320 μg/kg/d), and V3 (160, 320 and 480 μg/kg/d), for 7 days. Treatment with peptide V3 could significantly reduce the tumor weight and volume. Pathological examination showed that the tumors treated with peptide V3 had a larger region of necrosis than that of peptide V1, V2, and V1  +  V2 at the same dose. A significant decrease of microvessel density (MVD) in a dose-dependent manner was observed in each group of peptide V3. The results of pathological examination on normal tissue, lung, heart, liver, spleen, kidney and white blood cells showed that peptide V3 might have no significant toxicity. In conclusion, our results demonstrated that peptide V3 could be more effective on inhibiting tumor growth and angiogenesis than that of V1, V2, and V1  +  V2. Peptide V3 could be considered as a novel chimeric peptide with potent anti-tumor activity.
Keywords: Peptide; VEGFR-2; Tie-2; Angiogenesis; Tumor;

Adrenomedullin in sinusoidal endothelial cells play protective roles against cold injury of liver by Nobuyoshi Iinuma; Takayuki Sakurai; Akiko Kamiyoshi; Yuka Ichikawa-Shindo; Takuma Arai; Takahiro Yoshizawa; Teruhide Koyama; Ryuichi Uetake; Hisaka Kawate; Shin-ichi Muto; Yoh-ichi Tagawa; Shinichi Miyagawa; Takayuki Shindo (865-871).
Donor organ damage caused by cold preservation is a major problem affecting liver transplantation. Cold preservation most easily damages liver sinusoidal endothelial cells (LSECs), and information about the molecules modulating LSECs function can provide the basis for new therapeutic strategies. Adrenomedullin (AM) is a peptide known to possess anti-apoptotic and anti-inflammatory properties. AM is abundant in vascular endothelial cells, but levels are comparatively low in liver, and little is known about its function there. In this study, we demonstrated both AM and its receptors are expressed in LSECs. AM treatment reduced LSECs loss and apoptosis under cold treatment. AM also downregulated cold-induced expression of TNFα, IL1β, IL6, ICAM1 and VCAM1. AM reduced apoptosis and expression of ICAM1 and VCAM1 in an in vivo liver model subjected to cold storage. Conversely, apoptosis was exacerbated in livers from AM and RAMP2 (AM receptor activity-modifying protein) knockout mice. These results suggest that AM expressed in LSECs exerts a protective effect against cold-organ damage through modulation of apoptosis and inflammation.
Keywords: Adrenomedullin (AM); Receptor activity-modifying protein (RAMP); Liver sinusoidal endothelial cell (LSEC); Vasoactive peptide; Endothelial cell; Liver; Cold injury;

Sepsis is a serious complication for patients with obstructive jaundice. Although administration of adrenomedullin (AM) in combination with its binding protein (AMBP-1) is protective after injury, it remains unknown whether AM/AMBP-1 ameliorates sepsis-induced organ injury and mortality in the setting of biliary obstruction. The aim of this study is, therefore, to test the efficacy of human AM/AMBP-1 in a rat model of obstructive jaundice and polymicrobial sepsis. To study this, obstructive jaundice was induced in male adult rats (275–325 g) by common bile duct ligation (BDL). One week after BDL, the rats were subjected to sepsis by cecal ligation and puncture (CLP). Plasma levels of AM and AMBP-1 were measured at 20 h after CLP. In additional groups of BDL + CLP rats, human AM/AMBP-1 (24/80 μg/kg body weight (BW)) or vehicle (i.e., human albumin) was administered intravenously at 5 h after CLP. Blood and tissue samples were collected at 20 h after CLP for various measurements. To determine the long-term effect of human AM/AMBP-1 after BDL + CLP, the gangrenous cecum was removed at 20 h after CLP and 7-day survival was recorded. Our results showed that plasma levels of AM were significantly increased while AMBP-1 levels were markedly decreased after BDL + CLP (n  = 8, P  < 0.05). Administration of human AM/AMBP-1 attenuated tissue injury and inflammatory responses after BDL + CLP. Moreover, human AM/AMBP-1 significantly increased the survival rate from 21% (n  = 14) to 53% (n  = 15). Thus, human AM/AMBP-1 ameliorates sepsis-induced organ injury and mortality in jaundiced rats. Human AM/AMBP-1 can be further developed as a novel treatment for sepsis in jaundiced patients.
Keywords: Sepsis; Jaundice; Vasoactive peptide; Tissue injury; Survival;

Adrenomedullin 2 attenuates the pressor but not adrenal responses to angiotensin II in conscious sheep by Christopher J. Charles; Miriam T. Rademaker; M. Gary Nicholls; A. Mark Richards (878-882).
Biological actions attributed to the adrenomedullin (AM) peptides, AM and AM2, include reduction of arterial pressure and peripheral resistance. While AM has been shown to reduce aldosterone secretion from the adrenal, little information is available regarding possible actions of AM2 on aldosterone. Evidence suggests that AM may act as a functional antagonist to angiotensin II (Ang II) but such a role has not been investigated for AM2. Accordingly, we have examined hemodynamic and adrenal responses to stepped Ang II infusions with or without co-infusions of AM2 (33 ng/(kg min)) in conscious sheep under controlled conditions of a low sodium intake. The dose-dependent pressor response (5–50 mmHg) of Ang II was both delayed and attenuated (p  < 0.001) by AM2 which also stimulated heart rate (p  < 0.001) and cardiac output (p  < 0.001). AM2 prevented Ang II-induced increases in peripheral resistance (p  < 0.001). In contrast, plasma aldosterone responses to Ang II were not significantly altered by concomitant AM2 infusion. In conclusion, low dose infusion of AM2 administered to conscious sheep on a low salt diet clearly antagonized the vasopressor action of administered Ang II while stimulating cardiac output and heart rate. In contrast to AM, AM2 had no restraining influence on the aldosterone response to Ang II. The data suggest a possible role for AM2 in cardiovascular homeostasis in part through antagonism of the vasopressor action of Ang II.
Keywords: Arterial pressure; Cardiac output; Peripheral resistance; Aldosterone; Adrenal;

Exercise increases the angiotensin II effects in isolated portal vein of trained rats by Agnaldo Bruno Chies; Patrícia de Souza Rossignoli; Elias Fernando Daniel (883-888).
Training in rats adapts the portal vein to respond vigorously to sympathetic stimuli even when the animal is re-exposed to exercise. Moreover, changes in the exercise-induced effects of angiotensin II, a potent venoconstrictor agonist, in venous beds remain to be investigated. Therefore, the present study aimed to assess the effects of angiotensin II in the portal vein and vena cava from sedentary and trained rats at rest or submitted to an exercise session immediately before organ bath experiments. We found that training or exposure of sedentary animals to a single bout of running exercise does not significantly change the responses of the rat portal vein to angiotensin II. However, the exposure of trained animals to a single bout of running exercise enhanced the response of the rat portal vein to angiotensin II. This enhancement appeared to be territory-specific because it was not observed in the vena cava. Moreover, it was not observed in endothelium-disrupted preparations and in preparations treated with N ω-nitro-l-arginine methyl ester hydrochloride, indomethacin, BQ-123 or BQ-788. These data indicate that training causes adaptations in the rat portal vein that respond vigorously to angiotensin II even upon re-exposure to exercise. This increased response to angiotensin II requires an enhancement of the vasocontractile influence of endothelin beyond the influence of nitric oxide and vasodilator prostanoids.
Keywords: Angiotensin II; Endothelin; Exercise; Nitric oxide; Prostanoids;

The renin–angiotensin system (RAS) is an essential regulator of the blood pressure and body fluid balance, but the processing cascade or role of the tissue RAS remains obscure. Proangiotensin-12 (proang-12), a novel angiotensin peptide recently discovered in rat tissues, is assumed to function as a factor of the tissue RAS. To investigate the tissue production of proang-12, we measured the circulating and tissue components of the RAS including proang-12 following low-, normal-, or high-salt feeding in rats. Twelve-week-old male Wistar rats were fed a low-salt 0.3% NaCl or high-salt 8% NaCl diet for 7 days and compared with those fed a normal-salt diet of 0.7% NaCl. Low-salt feeding elevated the plasma renin activity and aldosterone concentration, resulting in significant increases in Ang I and Ang II levels in the plasma or kidney tissue, as compared with the normal- or high-salt group. Despite the increases in plasma renin activity, Ang I, and Ang II, the proang-12 levels in plasma and various tissues including the kidneys, small intestine, cardiac ventricles, and brain remained unchanged following low-salt feeding. These results suggest that peptide levels of proang-12 in rat plasma and tissues are regulated in a manner independent of the circulating RAS.
Keywords: Proangiotensin-12; Renin–angiotensin system; Dietary salt;

Oral and pulmonary delivery of thioether-bridged angiotensin-(1–7) by Louwe de Vries; Christina E. Reitzema-Klein; Anita Meter-Arkema; Annie van Dam; Rick Rink; Gert N. Moll; Marijke Haas Jimoh Akanbi (893-898).
Instability and proteolytic degradation limit the delivery options and in vivo efficacy of many therapeutic peptides. We previously generated a thioether stabilized angiotensin-(1–7) analog, cAng-(1–7), which is resistant against proteolytic degradation in the circulation. We here investigated oral and pulmonary delivery of this compound. In a first step we investigated the in vitro stability of the peptide under conditions that mimic those that will be met after oral administration. We demonstrated that cAng-(1–7) is stable at pH 2.0, a pH value close to that of the stomach, has enhanced resistance to breakdown by proteases from pancreas at pH 7.4, and is resistant to breakdown by proteases from liver at the lysosomal pH 5.0. We subsequently demonstrated that, in the absence of any delivery system or formulation, cAng-(1–7) can be delivered orally and via the lung, with bioavailabilities of 0.28 ± 0.05% and 28 ± 5%, whereas drug uptake was maximal after subcutaneous administration (bioavailability of 98 ± 6%). Therapeutic concentrations could be reached via all three routes of administration. The data prove that introduction of a thioether bridge in peptides opens novel delivery options for medically important peptides.
Keywords: Thioether; Lanthionine; Stability; Cyclic; Angiotensin; Therapeutic peptide;

Regulation of renin release by connexin 43 in As 4.1 cell line by Jeong Hee Han; Kyung-Ah Kim; Amin Shah; Byung Hyun Park; Woo Hyun Park; Suhn Hee Kim (899-902).
Gap junction channels facilitate chemical and electrical communication between adjacent cells. Gap junction protein, connexin (Cx), is expressed in the endothelial cells of vessels, glomerulus, and renin-secreting cells of the kidney. The purpose of this study was to investigate the role of Cx in renin release using Cx-overexpressing As 4.1 cells. The adenovirus-induced Cx overexpression was conducted by using recombinant adenovirus containing the cDNA encoding Cx37, Cx40, Cx43 (Ad-Cx), and β-galactosidase (Ad-β-gal). In 40-overexpressing cells, basal renin release increased in a time-dependent manner but it was significantly lower than that in Ad-β-gal-treated cells. In Cx37- and Cx43-overexpressing cells, basal renin release was increased in a time-dependent manner, which was not different from control cells. 18-β glycyrrhetinic acid (GA), a gap junction blocker, stimulated renin release dose-dependently and increased intracellular Ca2+ in both Cx43-overexpressing cells and control cells. However, no significant differences were observed. An increase in renin release by 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester, a putative antagonist of Ca2+ release from intracellular sequestration sites, was also similar between two groups. These results suggest that Cx43 may unlikely alter the regulation of renin release and intracellular Ca2+ by gap junction blocker in As 4.1 cells.
Keywords: As 4.1 cell line; Ca2+; Gap junction; Hormone; Renin; Connexin;

Atrial natriuretic peptides and urodilatin modulate proximal tubule Na+-ATPase activity through activation of the NPR-A/cGMP/PKG pathway by Diogo Vives; Sílvia Farage; Rafael Motta; Anibal G. Lopes; Celso Caruso-Neves (903-908).
The signaling pathway mediating modulation of Na+-ATPase of proximal tubule cells by atrial natriuretic peptides (ANP) and urodilatin through receptors located in luminal and basolateral membranes (BLM) is investigated. In isolated BLM, 10−11  M ANP or 10−11  M urodilatin inhibited the enzyme activity (50%). Immunodetection revealed the presence of NPR-A in BLM and LLC-PK1 cells. Both compounds increased protein kinase G (PKG) activity (80%) and this effect did not occur with 10−6  M LY83583, a specific inhibitor of guanylyl cyclase. The inhibitory effect of these peptides on Na+-ATPase activity did not occur after addition of 10−6  M KT5823, a specific inhibitor of PKG. LLC-PK1 cells were used to investigate if ANP and urodilatin change the activity of sodium pumps by luminal receptor interaction. ANP and urodilatin inhibited Na+-ATPase activity (50%), with maximal effect at 10−10  M, similar to 10−7  M db-cGMP, and did not occur with 10−7  M LY83583, a guanylyl cyclase inhibitor. ANP and urodilatin specifically inhibit Na+-ATPase activity by activation of the cGMP/PKG pathway through NPR-A located in luminal membrane and BLM, increasing understanding of the mechanism of natriuretic peptides on renal sodium excretion, with proximal tubule Na+-ATPase one possible target.
Keywords: Renal sodium excretion; NPR-A; Na+-ATPase; Second sodium pump; ANP and urodilatin;

Rapakinin, an anti-hypertensive peptide derived from rapeseed protein, dilates mesenteric artery of spontaneously hypertensive rats via the prostaglandin IP receptor followed by CCK1 receptor by Yuko Yamada; Masashi Iwasaki; Hachiro Usui; Kousaku Ohinata; Ewa D. Marczak; Andrzej W. Lipkowski; Masaaki Yoshikawa (909-914).
The anti-hypertensive peptide Arg-Ile-Tyr, which was isolated based on its inhibitory activity (IC50  = 28 μM) for angiotensin I-converting enzyme (ACE) from the subtilisin digest of rapeseed protein, exhibited vasorelaxing activity (EC50  = 5.1 μM) in an endothelium-dependent manner in the mesenteric artery of spontaneously hypertensive rats (SHRs). We named the peptide rapakinin. ACE inhibitors are reported to induce nitric oxide (NO)-dependent vasorelaxation by elevating the endogenous bradykinin level; however, the vasorelaxation induced by 10 μM of rapakinin was blocked only insignificantly by HOE140 or N G-nitro-l-arginine methyl ester (l-NAME), antagonists of bradykinin B2 receptor and an inhibitor of NO synthase, respectively. On the other hand, the vasorelaxation induced by 10 μM rapakinin was significantly blocked by indomethacin and CAY10441, a cyclooxygenase (COX) inhibitor and an antagonist of the IP receptor, respectively. The vasorelaxing activity of rapakinin was also blocked by lorglumide, an antagonist of the cholecystokinin (CCK) CCK1 receptor, although rapakinin has no affinity for the IP and CCK1 receptors. The vasorelaxation induced by 10 μM iloprost, an IP receptor agonist, was also blocked by lorglumide, suggesting that CCK–CCK1 receptor system is activated downstream of the PGI2–IP receptor system. The anti-hypertensive activity of rapakinin after oral administration in SHRs was also blocked by CAY10441 and lorglumide. These results suggest that the anti-hypertensive activity of rapakinin might be mediated mainly by the PGI2–IP receptor, followed by CCK–CCK1 receptor-dependent vasorelaxation.
Keywords: Vasorelaxing activity; Rapakinin; Prostaglandin I2; IP receptor; Cholecystokinin; CCK1 receptor; Spontaneously hypertensive rat;

Further studies on the pharmacological profile of the neuropeptide S receptor antagonist SHA 68 by Chiara Ruzza; Anna Rizzi; Claudio Trapella; Michela Pela’; Valeria Camarda; Valentina Ruggieri; Monica Filaferro; Carlo Cifani; Rainer K. Reinscheid; Giovanni Vitale; Roberto Ciccocioppo; Severo Salvadori; Remo Guerrini; Girolamo Calo’ (915-925).
Neuropeptide S (NPS) regulates various biological functions by selectively activating the NPS receptor (NPSR). Previous studies demonstrated that the non-peptide molecule SHA 68 acts as a selective NPSR antagonist. In the present study the pharmacological profile of SHA 68 has been further investigated in vitro and in vivo. In cells expressing the mouse NPSR SHA 68 was inactive per se up to 10 μM while it antagonized NPS-stimulated calcium mobilization in a competitive manner showing a pA 2 value of 8.06. In the 10–50 mg/kg range of doses, SHA 68 counteracted the stimulant effects elicited by NPS, but not those of caffeine, in mouse locomotor activity experiments. In the mouse righting reflex assay SHA 68 fully prevented the arousal-promoting action of the peptide. The anxiolytic-like effects of NPS were slightly reduced by SHA 68 in the mouse open field, fully prevented in the rat elevated plus maze and partially antagonized in the rat defensive burying paradigm. Finally, SHA 68 was found poorly active in antagonizing the NPS inhibitory effect on palatable food intake in rats. In all assays SHA 68 did not produce any effect per se. In conclusion, the present study demonstrated that SHA 68 behaves as a selective NPSR antagonist that can be used to characterize the in vivo actions of NPS. However the usefulness of this research tool is limited by its poor pharmacokinetic properties.
Keywords: Neuropeptide S; NPS receptor; SHA 68; Calcium assay; In vivo studies;

The newly identified neuropeptide S (NPS) is mainly expressed in a group of neurons located between the locus coeruleus and Barrington's nucleus in the brainstem. Central administration of NPS increases motor activity and wakefulness, and it decreases anxiety-like behavior and feeding. The NPS receptor (NPSR) is widely distributed in various brain regions including the ventral tegmental area (VTA). The mesolimbic dopaminergic system originates in the VTA, and activation of the system produces hypermotor activity. Therefore, we hypothesized that NPS-induced hypermotor activity might be mediated by activation of the mesolimbic dopaminergic pathway via the NPSR expressed in the VTA. Intra-VTA injection of NPS significantly and dose-dependently increased horizontal and vertical motor activity in rats, and the hyperactivity was significantly and dose-dependently inhibited by pre-administration of sulpiride, a DA D2-like receptor antagonist, into the shell of the nucleus accumbens (NAcSh). Intra-VTA injection of NPS also significantly increased extracellular 3,4-dihydroxy-phenyl acetic acid and homovanillic acid levels in the NAcSh of freely moving rats. These results support the idea that NPS activates the mesolimbic dopaminergic system presumably via the NPSR located in the VTA, thereby stimulating motor activity.
Keywords: Neuropeptide S; Shell of the nucleus accumbens; Ventral tegmental area; Mesolimbic dopaminergic system; Hyperactivity; Sulpiride;

Alteration in chromogranin A, obestatin and total ghrelin levels of saliva and serum in epilepsy cases by Ersel Dag; Suleyman Aydin; Yusuf Ozkan; Fazilet Erman; Adile Ferda Dagli; Mehtap Gurger (932-937).
This study was designed to measure the levels of chromogranin A (CgA), ghrelin and obestatin in serum and saliva (including CgA expression in healthy tissue) in epileptic patients to determine any significant differences between these patients and healthy controls. Samples were obtained from a total of 91 subjects: 10 newly-diagnosed primary generalized epilepsy (PGE) patients who had started treatment with valproic acid and phenytoin for seizure control; 18 PGE patients who were previously and currently receiving treatment with valproic acid and phenytoin for seizure control; 37 patients with partial epilepsy (PE) (simple, n  = 17 or complex, n  = 20) who had been and were still being treated with carbazebime for seizures; and 26 healthy controls. CgA immunoreactivity in healthy salivary gland was analyzed by immunohistochemistry and ELISA. The levels of CgA, total ghrelin and obestatin in serum and saliva were measured by ELISA. The results revealed that normal salivary gland produces its own CgA. Before treatment, CgA levels in saliva and serum were significantly greater in patients newly-diagnosed with PGE than controls. Ghrelin and CgA concentrations were also greater in PGE patients previously or currently treated with drugs, and in patients with simple or complex partial epilepsy (PE) previously or currently treated with drugs, than in healthy normal controls. In conclusion, salivary concentrations of CgA, ghrelin and obestatin were similar to their serum levels, so saliva might be a desirable alternative to serum for measuring these hormones because it is easy and painless to collect.
Keywords: Epilepsy; Salivary; Chromogranin A; Ghrelin; Obestain; Seizure;

The present study was designed to determine the dose-dependent effects of endothelin-3 (ET-3) on the secretion of ghrelin and growth hormone (GH) and characterize the receptors involved in these effects. Eight Holstein steers were randomly assigned to receive intravenous bolus injections of vehicle (0.1% bovine serum albumin in saline), bovine ET-3 (0.1, 0.4, 0.7 and 1.0 μg/kg), IRL1620 (selective ETB receptor agonist, 2.0 μg/kg), [d-Lys3]-GHRP-6 (GH secretagogue receptor type 1a [GHS-R1a] antagonist, 20.0 μg/kg) and bovine ET-3 (1.0 μg/kg) combined with [d-Lys3]-GHRP-6 (20.0 μg/kg), respectively. Blood samples were collected at −30, −15, 0, 5, 10, 15, 20, 25, 30, 35, 40, 50 and 60 min relative to injection time. Concentrations of acyl ghrelin, total ghrelin (acyl and des-acyl ghrelin) and GH in plasma were analyzed by a double antibody radioimmunoassay system. Concentrations of acyl and total ghrelin were significantly increased by ET-3 in a dose-dependent manner. Concentrations of GH were markedly elevated by administration of 0.4, 0.7 and 1.0 μg/kg of ET-3, and the effect of 0.7 μg/kg was greater than that of 1.0 μg/kg. The minimum effective dose of ET-3 in the secretion of ghrelin and GH was 0.4 μg/kg. IRL 1620 mimicked the effects of ET-3 on the secretion of ghrelin and GH in plasma. ET-3-induced elevation of plasma GH was blocked by [d-Lys3]-GHRP-6. These results indicate that ET-3 dose-dependently stimulates ghrelin release, and ETB receptors involve in these processes. Moreover, this study shows that endogenous ghrelin response to ET-3 increases GH secretion through GHS-R1a.
Keywords: Ghrelin; Endothelin-3; Growth hormone; ETB receptor; IRL 1620;

Regulation of beta-cell viability and gene expression by distinct agonist fragments of adiponectin by James E.P. Brown; Alex C. Conner; Janet E. Digby; Kenya L. Ward; Manjunath Ramanjaneya; Harpal S. Randeva; Simon J. Dunmore (944-949).
Obesity is an established risk factor for type 2 diabetes. Activation of the adiponectin receptors has a clear role in improving insulin resistance although conflicting evidence exists for its effects on pancreatic beta-cells. Previous reports have identified both adiponectin receptors (ADR-1 and ADR-2) in the beta-cell. Recent evidence has suggested that two distinct regions of the adiponectin molecule, the globular domain and a small N-terminal region, have agonist properties. This study investigates the effects of two agonist regions of adiponectin on insulin secretion, gene expression, cell viability and cell signalling in the rat beta-cell line BRIN-BD11, as well as investigating the expression levels of adiponectin receptors (ADRs) in these cells. Cells were treated with globular adiponectin and adiponectin (15-36) ±leptin to investigate cell viability, expression of key beta-cell genes and ERK1/2 activation. Both globular adiponectin and adiponectin (15-36) caused significant ERK1/2 dependent increases in cell viability. Leptin co-incubation attenuated adiponectin (15-36) but not globular adiponectin induced cell viability. Globular adiponectin, but not adiponectin (15-36), caused a significant 450% increase in PDX-1 expression and a 45% decrease in LPL expression. ADR-1 was expressed at a higher level than ADR-2, and ADR mRNA levels were differentially regulated by non-esterified fatty acids and peroxisome-proliferator-activated receptor agonists. These data provide evidence of roles for two distinct adiponectin agonist domains in the beta-cell and confirm the potentially important role of adiponectin receptor agonism in maintaining beta-cell mass.
Keywords: Adiponectin; Peptide; Beta-cell; Leptin; Cell viability; LPL; PDX-1;

Identification of potent 11mer Glucagon-Like Peptide-1 Receptor agonist peptides with novel C-terminal amino acids: Homohomophenylalanine analogs by Tasir S. Haque; Ving G. Lee; Douglas Riexinger; Ming Lei; Sarah Malmstrom; Li Xin; Songping Han; Claudio Mapelli; Christopher B. Cooper; Ge Zhang; William R. Ewing; John Krupinski (950-955).
We report the identification of potent agonists of the Glucagon-Like Peptide-1 Receptor (GLP-1R). These compounds are short, 11 amino acid peptides containing several unnatural amino acids, including (in particular) analogs of homohomophenylalanine (hhPhe) at the C-terminal position. Typically the functional activity of the more potent peptides in this class is in the low picomolar range in an in vitro cAMP assay, with one example demonstrating excellent in vivo activity in an ob/ob mouse model of diabetes.
Keywords: GLP-1R (Glucagon-Like Peptide-1); Agonist; Homohomophenylalanine;

Previous studies showed a significantly reduced level of hemorphins in the serum of diabetes patients. In order to elucidate the biochemical mechanisms responsible for this anomaly, the influence of hemoglobin glycation on hemorphin generation was studied. The glycation of hemoglobin occurs in the blood of diabetes patients and this could modify its enzymatic digestion and the resulting proteolytic products. Several samples of hemoglobin were obtained from the blood of type 1 diabetes patients (n  = 8) and normal healthy control subjects (n  = 2). The glycated hemoglobin samples were classified on the basis of their HbA1c values expressed as a percentage of total hemoglobin. Four solutions of glycated hemoglobin characterized by HbA1c values of 6%, 9.1%, 10.7% and 12.1% were treated with cathepsin D and the hemorphins obtained following the proteolysis were compared to controls. It was found that hemorphins were produced whatever the level of glycation of hemoglobin and also that the degree of glycation had no effect on the quantity of hemorphins released. Thus the alteration of hemoglobin does not seem to be the essential reason for the decrease in hemorphin concentrations in the sera of diabetic patients.
Keywords: Glycation; Glycated hemoglobin proteolysis; Hemorphins; Diabetes; Cathepsin D;

Structural and pharmacological characteristics of chimeric peptides derived from peptide E and β-endorphin reveal the crucial role of the C-terminal YGGFL and YKKGE motifs in their analgesic properties by Eric Condamine; Karine Courchay; Jean-Claude Do Rego; Jérôme Leprince; Catherine Mayer; Daniel Davoust; Jean Costentin; Hubert Vaudry (962-972).
Peptide E (a 25-amino acid peptide derived from proenkephalin A) and β-endorphin (a 31-amino acid peptide derived from proopiomelanocortin) bind with high affinity to opioid receptors and share structural similarities but induce analgesic effects of very different intensity. Indeed, whereas they possess the same N-terminus Met-enkephalin message sequence linked to a helix by a flexible spacer and a C-terminal part in random coil conformation, in contrast with peptide E, β-endorphin produces a profound analgesia. To determine the key structural elements explaining this very divergent opioid activity, we have compared the structural and pharmacological characteristics of several chimeric peptides derived from peptide E and β-endorphin. Structures were obtained under the same experimental conditions using circular dichroism, computational estimation of helical content and/or nuclear magnetic resonance spectroscopy (NMR) and NMR-restrained molecular modeling. The hot-plate and writhing tests were used in mice to evaluate the antinociceptive effects of the peptides. Our results indicate that neither the length nor the physicochemical profile of the spacer plays a fundamental role in analgesia. On the other hand, while the functional importance of the helix cannot be excluded, the last 5 residues in the C-terminal part seem to be crucial for the expression or absence of the analgesic activity of these peptides. These data raise the question of the true function of peptides E in opioidergic systems.
Keywords: Bovine peptide E; β-Endorphin; Opioid peptides; Chimeric peptides; NMR structure; Molecular modeling; Structure–activity relationships; Analgesic activity;

The immunomodulatory role of endogenous opioid peptides released during stress has been extensively studied in mammals, but least explored in lower vertebrates. The present in vitro study for the first time reports the specific opioid receptor-mediated immunomodulatory role of dynorphin-A(1–17) in ectotherms. Dynorphin-A(1–17) had pleiotropic effects on phagocyte functions, stimulatory on phagocytosis and superoxide production while inhibitory on the nitrite release. However, the effect of dynorphin-A(1–17), whether stimulatory or inhibitory, markedly declined at high (10−5  M) concentration. Dynorphin-A(1–17) seems to mediate its action through opioid receptors since non-selective opioid receptor antagonist, naltrexone, completely blocked the effect of dynorphin-A(1–17) on phagocytosis, superoxide production and nitrite release. Moreover, among specific opioid receptors antagonists, only selective κ (kappa)-opioid receptor antagonist norbinaltorphimine was capable to antagonize the pleiotropic effects on phagocyte functions. The present study provides the direct evidence of immunomodulatory role of dynorphin-A(1–17) via κ-opioid receptor in freshwater teleost Channa punctatus.
Keywords: Dynorphin; Phagocyte; Phagocytosis; Superoxide; Nitrite; Opioid receptors; Fish;

Peptide DV-28 amide: An inhibitor of bradykinin-induced arterial smooth muscle relaxation encoded by Bombina orientalis skin kininogen-2 by Lei Wang; Yong Chen; Mu Yang; Mei Zhou; Tianbao Chen; Da-Yuan Sui; Chris Shaw (979-982).
Skin kininogens from bombinid toads encode an array of bradykinin-related peptides and one such kininogen from Bombina maxima also encodes the potent bradykinin B2-receptor antagonist, kinestatin. In order to determine if the skin secretion of the closely-related toad, Bombina orientalis, contained a bradykinin inhibitory peptide related to kinestatin, we screened reverse phase HPLC fractions of defensive skin secretion using a rat tail artery smooth muscle preparation. A fraction was located that inhibited bradykinin-induced relaxation of the preparation and this contained a peptide of 3198.5 Da as determined by MALDI-TOF MS. Automated Edman degradation of this peptide established the identity of a 28-mer as: DMYEIKGFKSAHGRPRVCPPGEQCPIWV, with a disulfide-bridge between Cys18 and Cys24 and an amidated C-terminal Val residue. Peptide DV-28 was found to correspond to residues 133–160 of skin pre-kininogen-2 of B. orientalis that also encodes two copies of (Thr6)-bradykinin. The C-terminal residue, Gly-161, of the precursor open-reading frame, acts as the C-terminal amide donor of mature DV-28. DV-28 amide thus represents a new class of bradykinin inhibitor peptide from amphibian skin secretion.

Discovering the informational rule(s) underlying structure–function relationships in the protein language is at the core of biology. Current theories have proven inadequate to explain the origins of biological information such as that found in nucleotide and amino acid sequences. Here, we demonstrate that the information content of an amino acid motif correlates with the motif rarity. A structured analysis of the scientific literature supports the theory that rare pentapeptide words have higher significance than more common pentapeptides in biological cell ‘talk’. This study expands on our previous research showing that the immunological information contained in an amino acid sequence is inversely related to the sequence frequency in the host proteome.
Keywords: Peptide modules; Rare amino acid motifs; Information content; Protein information organization;