Peptides (v.31, #9)

Human cancer cell proliferation inhibition by a pentapeptide isolated and characterized from rice bran by Arvind Kannan; Navam S. Hettiarachchy; Jackson O. Lay; Rohana Liyanage (1629-1634).
Food-derived bioactive peptides promote functional activity against diseases and present as nutraceutical agents. The purpose of our research was to isolate and fully characterize peptide(s) derived from rice bran having anti-cancer properties. Gastrointestinal juices resistant peptide fractions were initially generated from heat stabilized de-fatted rice bran from which <5 kDa fraction was shown to inhibit proliferation of cancer cells. Based on these published findings the <5 kDa peptide fraction was selected for further characterization to obtain single pure peptide(s) with enhanced anti-cancer properties. Purification and characterization from the fraction was done employing chromatography and mass spectrometric techniques. Cancer cell viability was measured using a cell titer assay that uses a tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; (MTS)] and the electron coupling reagent, phenazine methosulfate. Ion-exchange chromatography elutes that showed anti-cancer properties were further purified to liberate pure peptide. The pure peptide at 600–700 μg/mL dose caused 84% inhibition to colon cancer cells (Caco-2, HCT-116) growth, 80% to breast cancer cells (MCF-7, MDA-MB-231) growth and 84% to liver cancer cells (HepG-2) growth. Mass spectrometry analysis and de novo sequencing revealed the sequence of Glu-Gln-Arg-Pro-Arg for the peptide with a molecular mass of 685.378 Da. A novel pentapeptide was isolated from rice bran to possess cancer growth inhibitory properties on colon, breast, lung and liver cancer cells. This peptide could serve as a nutraceutical agent against cancer.
Keywords: Rice bran; Peptide; Anti-cancer; Characterization;

Amaranth lunasin-like peptide internalizes into the cell nucleus and inhibits chemical carcinogen-induced transformation of NIH-3T3 cells by Enrique Maldonado-Cervantes; Hyung Jin Jeong; Fabiola León-Galván; Alberto Barrera-Pacheco; Antonio De León-Rodríguez; Elvira González de Mejia; Ben O. de Lumen; Ana P. Barba de la Rosa (1635-1642).
▶ The cancer-preventive peptide in amaranth has in vitro activities similar to those of soybean lunasin. ▶ The amaranth lunasin-like peptide requires less time than the soybean lunasin to internalize into the nucleus of NIH-3T3 cells. ▶ The open reading frame (ORF) of amaranth lunasin, corresponds to a bifunctional inhibitor/lipid-transfer protein (LTP). ▶ Our results open new intriguing questions about the function of lunasin in plants.Because an unbalanced diet is an important risk factor for several illnesses, interest has increased in finding novel health-promoting foods. Amaranth produces seeds that not only have substantial nutritional properties but that also contain phytochemical compounds as rutin and nicotiflorin and peptides with antihypertensive and anticarcinogenic activities. We report that a cancer-preventive peptide in amaranth has activities similar to those of soybean lunasin. The amaranth lunasin-like peptide, however, requires less time than the soybean lunasin to internalize into the nucleus of NIH-3T3 cells, and inhibits histone acetylation (H3 and H4 in a 70 and 77%, respectively). The amaranth lunasin-like peptide inhibited the transformation of NIH-3T3 cells to cancerous foci. The open reading frame (ORF) of amaranth lunasin corresponds to a bifunctional inhibitor/lipid-transfer protein (LTP). LTPs are a family of proteins that in plants are implicated in different functions, albeit all linked to developmental processes and biotic and abiotic stress resistance. Our results open new intriguing questions about the function of lunasin in plants and support that amaranth is a food alternative containing natural peptides with health-promoting benefits.
Keywords: Amaranth; Histone acetylation; Lipid-transfer proteins; Lunasin; Soybean; Western blot;

The broad-spectrum antitumor action of cyclosporin A is due to its tachykinin receptor antagonist pharmacological profile by Miguel Muñoz; Marisa Rosso; Ana González; Javier Saenz; Rafael Coveñas (1643-1648).
Cyclosporin A (CsA) is an immunosuppressive drug. In human cancer cells substance P (SP) and neurokinin-1 (NK-1) receptor antagonists, respectively, induce cell proliferation and inhibition. CsA is a tachykinin receptor antagonist that showed selectivity for both NK-1 and NK-2 receptors. CsA exerts antitumor action against gastric (AGS) and colon (HT29) carcinoma cell lines. However, the mechanisms involved in this action remain unknown, and it is unknown whether CsA exerts an antitumor action on other human cancer cell lines or not. To demonstrate that CsA exerts a broad-spectrum antitumor action, we carried out an in vitro study of the growth-inhibitory capacity of CsA against seven human cancer cell lines, namely GAMG glioma, SKN-BE(2) neuroblastoma, WERI-Rb-1 retinoblastoma, HEp-2 larynx carcinoma, CAPAN pancreas carcinoma, 23132/87 gastric carcinoma, and SW-403 colon carcinoma. A Coulter counter was used to determine viable cell numbers followed by application of the MTS colorimetric method. Micromolar concentrations of CsA inhibited the growth of these tumor cells, both with and without previous administration of nanomolar concentrations of SP; the inhibition occurred in a dose-dependent manner. Moreover, CsA blocks SP-induced mitogen stimulation of tumor cells, suggesting that the NK-1 receptor is involved in such action. Following administration of CsA apoptosis was observed in the above seven tumor cell lines. These findings suggest that the antitumor action of CsA is at least due to its NK-1 receptor antagonist pharmacological profile, since the involvement of NK-2 receptors in the mentioned action must not be discarded, and that CsA has a broad-spectrum antitumor action.
Keywords: Cyclosporin A; Apoptosis; Glioma; Neuroblastoma; Retinoblastoma; Carcinoma;

▶All antibiotics tested induced the release of LPS from P. gingivalis. ▶ Different antibiotic classes promoted varying degrees of LPS release. ▶ LL-37 was the only AMP able to completely inhibit the LPS activity. ▶ KSL-W and histatin 8 neutralized 50% and 20% of LPS activity, respectively. ▶ Comparatively, minimal amounts (1.4 μM) of LL-37 blocked 100% of all cytokines tested.Bacterial lipopolysaccharide (LPS) release during periodontal infection is a significant component of periodontal disease. We hypothesized that some bacterial LPS release results from bacterial exposure to antibiotics. Therefore, we examined the ability of various classes of antibiotics to induce LPS release from Porphyromonas gingivalis as well as the ability of antimicrobial peptides (AMPs) to inhibit purified LPS. All antibiotics tested against P. gingivalis were able to liberate 1.9–12.9 times more LPS as compared to untreated bacteria. Among the three AMPs tested, LL-37 was found to be the most potent inhibitor of cytokine (tumor necrosis factor-α, interleukin-1β, interleukin-6) production and completely neutralized purified P. ginigivalis LPS activity in the chromogenic limulus amebocyte lysate (LAL) and whole blood cytokine stimulation assays. These observations suggest that therapeutic approaches utilizing AMPs as adjuncts to neutralize released LPS should be considered.
Keywords: Porphyromonas gingivalis; Lipopolysaccharide; Antibiotics; Inflammation; Cytokines;

In vitro bactericidal activity of human β-defensin 2 against nosocomial strains by John G. Routsias; Panagiotis Karagounis; Georgeta Parvulesku; Nikolaos J. Legakis; Athanassios Tsakris (1654-1660).
▶ In this report we studied the bactericidal activity of human β-defensin 2 (hBD-2), a 41-amino acid cationic peptide of the innate immune system that serves as antimicrobial molecule. We determined the bactericidal activity of synthetic hBD-2 against nosocomial strains belonging to eight different bacterial species and exhibiting various antimicrobial resistance phenotypes. Our study describes the requirements for the bactericidal action of hBD-2 (e.g. disulfide connectivity and low salt concentrations). One extrapolation of these findings is that the maximum activity of hBD-2 can be found in sites of the human body with low salt concentration (e.g. airway surface fluid) and if the concentration of NaCl in that is increased (e.g. in cystic fibrosis) the bactericidal of hBD-2 is diminished. ▶ The evaluation of the bactericidal activity of hBD-2 against nosocomial strains. hBD-2 was found to be increased against Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus clinical strains. On the other hand, bactericidal activity of hBD-2 was less pronounced against Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis clinical strains. ▶ The evaluation of the bactericidal activity of hBD-2 against multi-drug resistant (MDR) A. baumannii strains as well as against non-MDR A. baumannii strains. It was found that A. baumannii strains that exhibited multi-drug resistant (MDR) phenotypes were susceptible to lower concentrations of hBD-2 (vLD90  = 3.25–4.5 μg/ml) in comparison with non-MDR (wild-type) A. baumannii strains (vLD90  = 3.90–9.35 μg/ml). Given that MDR Acinetobacter isolates were susceptible only to nephrotoxic antibiotic colistin (polymyxin E), hBD-2 could be used as an altelnative, less toxic, therapeutic agent. ▶ The relation of the bactericidal activity of hBD-2 (against E. coli, K. pneumoniae and P. mirabilis) to antibiotic resistance phenotype of the strains. It was found that the bactericidal activity of hBD-2 was enhanced against strains that exhibited resistance to amoxicillin/clavulanate, aztreonam and all cephalosporins (P  < 0.01). Therefore, hBD-2 has a potential therapeutic role against bacterial pathogens exhibiting resistance to several β-lactam antibiotics.Human β-defensin 2 (hBD-2) is a 41-amino acid cationic peptide of the innate immune system that serves as antimicrobial molecule. We determined the bactericidal activity of synthetic hBD-2 against nosocomial strains belonging to eight different bacterial species and exhibiting various antimicrobial resistance phenotypes. The native disulfide connectivity was found essential for the bactericidal activity of hBD-2, while sodium chloride concentration was reversely associated with its potency. hBD-2 exhibited high bactericidal activity against Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus clinical strains. Characteristically, A. baumannii strains that exhibited multi-drug resistant (MDR) phenotypes were susceptible to lower concentrations of hBD-2 (vLD90  = 3.25–4.5 μg/ml) in comparison with non-MDR (wild-type) A. baumannii strains (vLD90  = 3.90–9.35 μg/ml). Bactericidal activity of hBD-2 was less pronounced against Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis strains but was significantly enhanced against strains of these species that exhibited resistance to several β-lactam antibiotics. These observations give indications that the natural hBD-2 has a potential therapeutic role against bacterial pathogens and particularly against those exhibiting MDR phenotypes.
Keywords: Defensins; hBD-2; Antimicrobial peptides; Nosocomial strains;

Purification and characterization of a novel delta-lysin variant that inhibits Staphylococcus aureus and has limited hemolytic activity by Mohammed Al-Mahrous; Stephanie K. Sandiford; John R. Tagg; Mathew Upton (1661-1668).
Delta-lysins (DL) that are produced by various species of staphylococci are not widely known for their antimicrobial activity. We have purified and characterized a novel DL variant, E229DL and examined its spectrum of inhibitory activity. The biological activity of E229DL, produced by Staphylococcus epidermidis strain E229, shows relatively broad-spectrum activity against Gram-positive pathogens, including representatives of MRSA and epidemic MRSA type 15. E229DL was purified to homogeneity from 95% acidified-methanol extracts of cell cultures by using a series of reversed-phase chromatographic separations. The fully processed form of E229DL is a 25-amino-acid peptide with a predicted mass of 2841.4 Da, but the purified biologically active molecule appears to be N-formylated (mass 2867.33 Da). The DL gene (hld) resembles that of other types of DL, but differs in five codons with hld in Staphylococcus aureus (26 residues) and one codon with the closest homolog, the hld-II in S. warneri (25 residues). The characterization of E229DL showed that its activity is stable in agar exposed to high temperatures (80 °C/45 min). In addition, biological testing of the native and synthetic peptides against a range of human and animal erythrocytes and Vero cells indicated that E229DL is an antibacterial agent with no detectable cytopathic effects at concentrations equivalent to the minimum inhibitory concentration for EMRSA15-A208. Initial investigation of the mode of action of E229DL indicated that it is rapidly lytic for target cells. This is the first description of a native form of DL having only limited cytotoxic activity for eukaryotic cells at concentrations that are inhibitory to staphylococci.
Keywords: Delta-lysin (DL); Antibacterial peptides; MRSA; Coagulase-negative staphylococci;

Selective toxicity of antimicrobial peptide S-thanatin on bacteria by Guoqiu Wu; Hongbin Wu; Xiaobo Fan; Rui Zhao; Xiaofang Li; Shenglan Wang; Yihua Ma; Zilong Shen; Tao Xi (1669-1673).
S-thanatin, an analog of thanatin, was synthesized by substituting the 15th amino acid of threonine with serine, which showed a broad antimicrobial activity against bacteria. We reported earlier that membrane phospholipid was found to be the target for S-thanatin with different mechanism from other antimicrobial peptides. In this study, we have performed its structural characterization by circular dichroism (CD) spectroscopy. The CD analysis showed that S-thanatin retained its overall conformation β-sheet in aqueous buffer, β-turn in 50% trifluoroethanol (TFE) and β-hairpin in 0.4 mM POPC-LUVs. In hemolysis assay, S-thanatin exhibited low hemolytic activity and bacteria selectivity. We investigated the effect of the presence of 33 mol percent cholesterol on the interactions of the antimicrobial peptide S-thanatin with phosphatidylcholine (PC) model membrane systems. The results showed that S-thanatin was more potent at disrupting cholesterol-free bacterial than cholesterol-containing eukaryotic membranes. Thus, in all respects, fluorescence dye leakage experiments indicated that cholesterol inhibited the S-thanatin-induced permeabilization of PC vesicles. Finally, flow cytometry was used to monitor changes in bacterial cell membrane potential and cell membrane integrity, with specific fluorescent dyes DiBAC4(3) and PI. Adding the respiratory poison CCCP seemed to prevent peptide-induced membrane damage, which suggested that S-thanatin acted at the metabolic level on respiratory chain. These findings might explain why S-thanatin was selective toxicity towards bacteria, but low toxicity towards erythrocytes. It might be due to three factors at least: electrostatic interaction (namely anionic phospholipids); cholesterol; respiratory chain.
Keywords: Antimicrobial peptide; S-thanatin; Secondary structure; Selective toxicity; Cholesterol; Respiratory chain;

Novel family of antimicrobial peptides from the skin of Rana shuchinae by Ruiqiang Zheng; Bin Yao; Haining Yu; Hanjin Wang; Jianmin Bian; Feifei Feng (1674-1677).
So far numerous antimicrobial peptides have been characterized from amphibians. In this work, a new family of antimicrobial peptides, named shuchin, was purified and characterized from skin secretions of the frog, Rana shuchinae that lives in freezing mountains. Totally two members of shuchin (shuchin 1 and 2) were identified with the amino acid sequence of NALSMPRNKCNRALMCFG and NALSSPRNKCDRASSCFG, respectively. cDNAs encoding shuchins were cloned from the skin cDNA library of R. shuchinae. The precursors of shuchin are composed of 62 amino acid residues including the conserved signal peptides, acidic propieces, and mature antimicrobial peptides. Synthetic shuchins showed strong and broad antimicrobial activities against Gram-positive bacteria (Staphylococcus aureus, and Bacillus cereus; MICs < 12.5 μg/ml), Gram-negative bacteria (Escherichia coli, Bacillus dysenteriae, Pseudomonas aeruginosa; most MICs from 3.1 to 12.5 μg/ml), and yeast (Candida albicans; MICs of 6.25 μg/ml), but no hemolytic activity under the effective concentration, thereby provide more leading templates for designing novel anti-infection agents.
Keywords: Amphibian; Antimicrobial peptides; Skin; Rana shuchinae;

Divergent M- and O-superfamily peptides from venom of fish-hunting Conus parius by Elsie C. Jimenez; Baldomero M. Olivera (1678-1683).
Six novel peptides from the piscivorous cone snail, Conus parius were purified by reverse-phase HPLC fractionation of crude venom. With the use of matrix-assisted laser desorption ionization mass spectrometry and standard Edman sequencing methods, the peptides were characterized. Two peptides were identified as members of the m-2 and m-4 branches of the M-superfamily and were designated as pr3a and pr3b, while four peptides were identified as members of the O-superfamily and were designated as pr6a, pr6b, pr6c and pr6d. Peptide pr3a differs from the majority of the M-superfamily peptides in the presence of two prolines, which are not modified to 4-trans-hydroxyproline. In peptide pr3b, five amino acids out of the 16 non-cysteine residues are identical with those of μ-GIIIA and μ-PIIIA, suggesting that pr3b may be a divergent μ-conotoxin. Peptide pr6a is notable because of its extreme hydrophobicity. Peptide pr6c has three prolines that are unhydroxylated. Peptides pr6b and pr6d differ from the previously characterized O-superfamily peptides in the presence of an extended N-terminus consisting of six amino acids. Peptides pr3a, pr3b, pr6a and pr6b were demonstrated to be biologically active when injected intraperitoneally in fish. The identification and characterization of these peptides in venom of a fish-hunting species establish the divergence of gene products and their patterns of post-translational modification within superfamilies in a single Conus species.
Keywords: Conus parius peptide; M-superfamily conotoxin; O-superfamily conotoxin; Conopeptide gene superfamily; Post-translational modification; Proline hydroxylation;

Identification of a linear epitope for Fc-binding in the mouse FcγRIII by Jun Xi; Li N. Zhang; Guang P. Hu; Li Wang; Song L. Qiao; Jun Q. Guo; Qi Y. Lu; Gai P. Zhang; Yan Y. Yang (1684-1688).
Fc receptors are transmembrane proteins, found on the surfaces of immune cells, that aid in the removal of foreign pathogens by binding to antibody-coated targets via the Fc regions of the antibodies. To identify sites on mouse FcγRIII (moFcγRIII) α-chain that bind to the Fc region, peptides derived from the proximal extracellular domain (EC2) of moFcγRIII α-chain corresponding to the homologous region of human FcγRIIIB α-chain were synthesized. Binding of mouse IgG to the different peptides was tested by Dot-blot assay. The effective peptide 119SFFHNEKSVRYH130 located in the putative C–C′ loop of the EC2 domain was found to bind mouse IgG specifically with an affinity of approximately 5.58 × 10−5  M and inhibit the binding of mouse IgG to the receptor. Such a functional peptide should be very useful for further understanding the IgG–FcγR interaction and development of FcR-targeting drugs.
Keywords: moFcγRIII α-chain; EC2 domain; Peptides;

Lipopolysaccharide differentially decreases plasma acyl and desacyl ghrelin levels in rats: Potential role of the circulating ghrelin-acylating enzyme GOAT by Andreas Stengel; Miriam Goebel; Lixin Wang; Joseph R. Reeve; Yvette Taché; Nils W.G. Lambrecht (1689-1696).
▶ Plasma acyl ghrelin (AG) decreases more than desacyl ghrelin (DG) at 2 h post-LPS. ▶ Both forms equally decrease at 5 and 7 h and return to pre-injection levels at 24 h. ▶ At 2 h post-LPS, plasma GOAT protein decreases while gastric GOAT protein increases. ▶ This may account for lower plasma acyl ghrelin levels. ▶ Decline in AG and DG is not secondary to increased temperature and blood glucose.Bacterial lipopolysaccharide (LPS) in rodents is an established model for studying innate immune responses to gram-negative bacteria and mimicking symptoms of infections including reduced food intake associated with decreased circulating total ghrelin levels. The ghrelin-acylating enzyme, ghrelin-O-acyltransferase (GOAT) involved in the formation of acyl ghrelin (AG) was recently identified. We investigated changes in circulating AG, desacyl ghrelin (DG) and GOAT induced by intraperitoneal LPS (100 μg/kg) and associated changes in food intake. Plasma AG and total ghrelin were assessed by radioimmunoassay, GOAT protein by Western blot and mRNA by RT-qPCR. DG was derived from total minus AG. Plasma AG and DG were decreased at 2, 5 and 7 h (p  < 0.01) post-injection compared to vehicle and recovered at 24 h. At 2 h there was a significantly greater decrease of AG (−53%) than DG (−28%) resulting in a decreased AG/DG ratio (1:5, p  < 0.01), which thereafter returned to pre-injection values (1:3). This altered ratio was associated with a 38% decrease in plasma GOAT protein compared to vehicle (p  < 0.001), whereas gastric GOAT protein was slightly increased by 10% (p  < 0.05). GOAT mRNA expression was unchanged. Food intake was reduced by 58% measured during the 1.5–2 h period post-LPS injection. Decreased plasma AG and DG preceded the rise in rectal temperature and blood glucose that peaked at 7 h. These data indicate that LPS induces a long-lasting reduction of AG and DG levels that may have a bearing with the decrease in food intake. The faster drop in AG than DG within 2 h is associated with reduced circulating GOAT.
Keywords: Acyl ghrelin; Desacyl ghrelin; GOAT; LPS; RAPID method; Rat;

Metabolic and structural properties of human obestatin {1–23} and two fragment peptides by Anusha P. Subasinghage; Brian D. Green; Peter R. Flatt; Nigel Irwin; Chandralal M. Hewage (1697-1705).
Obestatin is a peptide produced in the oxyntic mucosa of the stomach and co-localizes with ghrelin on the periphery of pancreatic islets. Several studies demonstrate that obestatin reduces food and water intake, decreases body weight gain, inhibits gastrointestinal motility, and modulates glucose-induced insulin secretion. In this study we evaluated the acute metabolic effects of human obestatin {1–23} and fragment peptides {1–10} or {11–23} in high-fat fed mice, and then investigated their solution structure by NMR spectroscopy and molecular modelling. Obestatins {1–23} and {11–23} significantly reduced food intake (86% and 90% respectively) and lowered glucose responses to feeding, whilst leaving insulin responses unchanged. No metabolic changes could be detected following the administration of obestatin {1–10}. In aqueous solution none of the obestatin peptides possessed secondary structural features. However, in a 2,2,2-trifluoroethanol (TFE-d 3)–H2O solvent mixture, the structure of obestatin {1–23} was characterized by an α-helix followed by a single turn helix conformation between residues Pro4 and Gln15 and His19 and Ala22 respectively. Obestatin {1–10} showed no structural components whereas {11–23} contained an α-helix between residues Val14 and Ser20 in a mixed solvent. These studies are the first to elucidate the structure of human obestatin and provide clear evidence that the observed α-helical structures are critical for in vivo activity. Future structure/function studies may facilitate the design of novel therapeutic agents based on the obestatin peptide structure.
Keywords: Obestatin; Obesity; NMR; Molecular modelling;

Regulatory effects of Y4 receptor agonist (BVD-74D) on food intake by Jiang-Bo Li; Akihiro Asakawa; Mutsumi Terashi; KaiChun Cheng; Huhe Chaolu; Takahiro Zoshiki; Miharu Ushikai; Sulaiman Sheriff; Ambikaipakan Balasubramaniam; Akio Inui (1706-1710).
▶ We propose that the novel Y4 receptor agonist BVD-74D has suppressive effects on food intake, water intake, and weight gain in normal mice fed with normal diets and on food intake in normal mice fed with high-fat diets and in FLS-ob/ob mice. ▶ These findings indicate that the Y4 receptor and its agonist would be promising targets for obesity.The objective of this study was to clarify the role of a novel agonist with high selectivity and affinity for Y4 receptors in the regulation of food intake. The Y4 receptor agonist BVD-74D was administered to C57BL/6J mice that were fed with a normal or high-fat diet, and to fatty liver Shionogi (FLS)-ob/ob mice; the food intake, water intake, and body weight gain were measured in these mice. In the mice fed with a normal diet, the cumulative food intake significantly decreased at 20 min and 1 h after the administration of 1 mg/kg of BVD-74D and at 1, 2, 4, 8, and 24 h after the administration of 10 mg/kg of BVD-74D. Moreover, the cumulative water intake and body weight gain significantly decreased in these mice. Among the mice that were fed with a high-fat diet, the cumulative food intake and water intake significantly decreased 1, 2, and 4 h after BVD-74D (10 mg/kg) administration. Furthermore, the cumulative food intake significantly decreased 2 and 4 h after BVD-74D (10 mg/kg) administration in the FLS-ob/ob mice. Thus, we propose that the novel Y4 receptor agonist BVD-74D has suppressive effects on food intake, water intake, and weight gain in normal mice fed with normal diets and on food intake in normal mice fed with high-fat diets and in FLS-ob/ob mice. These findings indicate that the Y4 receptor and its agonist would be promising targets for obesity.
Keywords: Y4 receptor; BVD-74D; Food intake; Body weight; Mouse; Obesity;

▶ Neuronostatin-induced anorexia and adipsia are reversed by Pretreatment with SHU9119. ▶ Pretreatment with OVT does not reverse the anorexigenic and antidipsogenic effects of neuronostatin.Neuronostatin, a recently discovered peptide derived from the somatostatin preprohormone, significantly inhibited both food and water intake when administered centrally in adult male rats. Because neuronostatin is highly produced in the hypothalamus, an area of the brain through which important feeding circuits, including the central melanocortin system, communicate, we sought to determine if the anorexigenic and antidipsogenic effects of neuronostatin would be reversed by pretreatment with the melanocortin 3/4 receptor antagonist, SHU9119. SHU9119 pretreatment reversed the effect of neuronostatin on both food and water intake. We have shown recently that the central oxytocin system is a potential downstream mediator of the anorexignic action of alpha-MSH. We therefore tested whether the effects of neuronostatin also were dependent upon central oxytocin receptors. Neuronostatin-induced anorexia was not reversed by pretreatment with the oxytocin receptor antagonist, OVT, suggesting that neuronostatin acts through a unique subset of POMC neurons that do not signal via central oxytocin receptors.
Keywords: Neuronostatin; Melanocortins; Oxytocin; Appetite;

Adiponectin and adiponectin receptor system in the rat adrenal gland: Ontogenetic and physiologic regulation, and its involvement in regulating adrenocortical growth and steroidogenesis by Lukasz Paschke; Tomasz Zemleduch; Marcin Rucinski; Agnieszka Ziolkowska; Marta Szyszka; Ludwik K. Malendowicz (1715-1724).
▶ The paper is the first one in literature to deeply analyze expression and actions of adiponectin in the rat adrenal gland through different experimental approaches.Adiponectin (ADN) is a regulatory peptide secreted mostly by adipose tissue and acting via two receptors: AdipoR1 and AdipoR2. Our aim was to investigate expression of adiponectin system genes in the rat adrenal gland as well as its ontogenetic and physiological control. Furthermore, we examined the effects of acute and prolonged activation of HPA axis on ADN system in adipose tissue. By means of QPCR, ADN and AdipoR1 expression was demonstrated in rat adrenal cortex both at mRNA and protein levels, while AdipoR2 could only be detected at mRNA levels. ADN expression level was significantly upregulated in a developing and regenerating adrenal cortex. Globular domain of adiponectin at 10−9  M stimulated corticosterone output and BrdU incorporation by cultured rat adrenocortical cells. Moreover, both acute (ACTH and ether stress) and prolonged (ACTH) adrenal stimulation resulted in lowered ADN levels, while expression of AdipoR1 and AdipoR2 was upregulated by the acute treatment. Depending on its site of origin, visceral (VAT) or subcutaneous (SAT) adipose tissue responded differently to alterations in HPA axis. VAT expression of ADN and its receptors remained almost unchanged by experimental manipulations. In SAT, on the other hand, expression of ADN and AdipoR2 was markedly increased by ACTH treatment and stress, while dexamethasone suppressed ADN and AdipoR1 mRNA levels. The results of this study provide new evidence for direct and indirect interactions between adipokines and HPA axis.
Keywords: Adiponectin; Adiponectin receptors; Rat; Cell culture; Steroidogenesis; Adrenal cortex; Adipose tissue; HPA axis;

The present study was aimed to investigate the possible effects of β-casomorphin-7, against hyperglycemia and free radical-mediated oxidative stress in streptozotocin-induced diabetic rats by assaying the blood glucose level and the activity of plasma enzymatic antioxidants, such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px). A significant increase in the levels of both blood glucose and oxidative stress with a predominant decrease in antioxidant status was observed in the diabetic rats when compared to control rats. After 15 days oral administration of β-casomorphin-7 (7.5 × 10−8mol/day), the elevated blood glucose level was reduced. Oral administration of β-CM-7 to diabetic rats showed an increase in the level of plasma insulin, the elevated plasma glucagon level was markedly reduced by the oral administration of β-CM-7. Oral administration of β-CM-7 to the diabetic group of rats also showed a significant elevation in the activity of SOD and catalase. Thus, the results of the present study suggest that β-casomorphin-7 can protect rats from hyperglycemia and free radical-mediated oxidative stress in diabetic rats.
Keywords: Diabetes; Streptozotocin; Oxidative stress; β-Casomorphin-7;

Role of the intra-A-chain disulfide bond of insulin-like peptide 3 in binding and activation of its receptor, RXFP2 by Suode Zhang; Richard A. Hughes; Ross A.D. Bathgate; Fazel Shabanpoor; M. Akhter Hossain; Feng Lin; Bianca van Lierop; Andrea J. Robinson; John D. Wade (1730-1736).
INSL3 is a member of the insulin-IGF-relaxin superfamily and plays a key role in male fetal development and in adult germ cell maturation. It is the cognate ligand for RXFP2, a leucine-rich repeat containing G-protein coupled receptor. To date, and in contrast to our current knowledge of the key structural features that are required for the binding of INSL3 to RXFP2, comparatively little is known about the key residues that are required to elicit receptor activation and downstream cell signaling. Early evidence suggests that these are contained principally within the A-chain. To further explore this hypothesis, we have undertaken an examination of the functional role of the intra-A-chain disulfide bond. Using solid-phase peptide synthesis together with regioselective disulfide bond formation, two analogs of human INSL3 were prepared in which the intra-chain disulfide bond was replaced, one in which the corresponding Cys residues were substituted with the isosteric Ser and the other in which the Cys were removed altogether. Both of these peptides retained nearly full RXFP2 receptor binding but were devoid of cAMP activity (receptor activation), indicating that the intra-A-chain disulfide bond makes a significant contribution to the ability of INSL3 to act as an RXFP2 agonist. Replacement of the disulfide bond with a metabolically stable dicarba bond yielded two isomers of INSL3 that each exhibited bioactivity similar to native INSL3. This study highlights the critical structural role played by the intra-A-chain disulfide bond of INSL3 in mediating agonist actions through the RXFP2 receptor.
Keywords: Insulin-like peptide 3; Dicarba bond; Ring closing metathesis; Microwave irradiation;

cDNAs encoding for preproTRH and preproorexin were cloned in winter flounder, a species that undergoes a period of natural fasting during the winter. For both peptides, the deduced amino acid structure of the hormone precursor shows 30–70% similarities with their homologs in other fish species. RT-PCR studies show that these peptides are present not only in the brain, but also in several peripheral tissues, including gastrointestinal tract and testes. Fasting induced increases in both preproorexin and preproTRH expressions in the hypothalamus, but did not affect their expression levels in the telencephalon/preoptic area. In addition, the mRNA expressions of both preproorexin and preproTRH were higher in the winter than in the summer in both hypothalamus and telencephalon/preoptic area. Our results suggest that orexin and thyrotropin-releasing hormone (TRH) might have a role in the seasonal regulation of food intake in winter flounder.
Keywords: Winter flounder; Orexin; TRH; Feeding; Cloning; Expression; Fasting; Season;

GnRH-induced PACAP and PAC1 receptor expression in pituitary gonadotrophs: A possible role in the regulation of gonadotropin subunit gene expression by Indri N. Purwana; Haruhiko Kanasaki; Aki Oride; Tselmeg Mijiddorj; Norihito Shintani; Hitoshi Hashimoto; Akemichi Baba; Kohji Miyazaki (1748-1755).
We examined the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and the PACAP type 1 receptor (PAC1-R) mRNA following gonadotropin-releasing hormone (GnRH) stimulation using the gonadotroph cell line LβT2. GnRH stimulation increased PACAP and PAC1-R mRNA expression in a static culture. Increase in the cell surface density of the PAC1-R following transfection with PAC1-R expression vectors significantly increased gonadotropin LHβ and FSHβ subunit promoter activities following 100 nM PACAP stimulation. In addition, increasing concentrations of PACAP stimulation augmented the promoter activities for both LHβ and FSHβ in PAC-1R overexpressing cells. In the cells with PAC1-R, the effect of GnRH was further potentiated in the presence of PACAP from 5.31 ± 0.93 to 9.89 ± 0.38-fold for LHβ and for FSHβ subunit, respectively; from 2.58 ± 0.31-fold by GnRH alone to 10.90 ± 2.79-fold with PACAP. The combination treatment with GnRH and PACAP did not augment the ERK phosphorylation induced by GnRH alone. PACAP expectedly increased cAMP accumulation and this effect was significantly attenuated in the presence of GnRH. PACAP gene expression was more prominent following lower frequency GnRH pulses (every 120 min) in a perifused culture. Our results suggest that PACAP and PAC1-R are produced locally within the gonadotrophs following GnRH stimulation. They subsequently affect the gonadotrophs in an autocrine manner and modulate the GnRH pulse-dependent specific regulation of gonadotropin subunits.
Keywords: PACAP; GnRH; Gonadotropin;

Scopolamine- and diazepam-induced amnesia are blocked by systemic and intraseptal administration of substance P and choline chloride by Joseane Carvalho Costa; Kauê Machado Costa; José Luiz Martins do Nascimento (1756-1760).
▶ This study investigates interactions between cholinergic and GABA/benzodiazepine systems with the substance P system on inhibitory avoidance retention in rats. We find that post-trial treatment with substance P or choline chloride blocks the amnesic effects of diazepam and the cholinergic antagonist scopolamine. ▶ The results of this study clearly indicate interactions between the substance P and cholinergic systems with GABAergic system in the modulation of inhibitory avoidance retention. Moreover, considering the pharmacological responses of our set-up, the effects of the treatments described in the paper seems to be mediated, at least in part, by interactions involving the septohippocampal pathway.Systemic (IP) and/or intraseptal (IS) administration of scopolamine (SCP) and diazepam (DZP) induce amnesia, whereas IP injection of the neuropeptide substance P (SP) and choline chloride (ChCl) produce memory facilitation. The septohippocampal cholinergic system has been pointed out as a possible site of SCP and DZP-induced amnesia as well as for the mnemonic effects induced by SP and ChCl. We performed a series of experiments in order to investigate the interactions between cholinergic and GABA/benzodiazepine (GABA/BZD) systems with the SPergic system on inhibitory avoidance retention. Male Wistar rats were trained and tested in a step-down inhibitory avoidance task (1.0 mA footshock). Animals received, pre-training, IP (1.0 mg/kg) or IS (1.0 nM/0.5 μl) injection of DZP, SCP (SCP; 1.0 mg/kg – IP or 0.5 μM/0.5 μl – IS) or vehicle (VEH). Immediately after training they received an IP or IS injections of SP 1-11 (50 μg/kg – IP or 1.0 nM/0.5 μl – IS), SP 1-7 (167 μg/kg – IP or 1.0 nM/0.5 μl – IS), ChCl (20 mg/kg – IP or 0.3 μM/0.5 μl – IS) or VEH. Rats pretreated with SCP and DZP showed amnesia. Post-trial treatments with SP 1-11, SP 1-7 or ChCl blocked the amnesic effects of SCP and DZP. These findings suggest an interaction between SPergic and cholinergic mechanisms with GABAergic systems in the modulation of inhibitory avoidance retention and that the effects of these treatments are mediated, at least in part, by interactions in the septohippocampal pathway.
Keywords: Substance P; Scopolamine; Diazepam; Amnesia; Medial septum; Inhibitory avoidance;

Beta-like hippocampal network activity is differentially affected by amyloid beta peptides by Alvaro Adaya-Villanueva; Benito Ordaz; Hugo Balleza-Tapia; Abraham Márquez-Ramos; Fernando Peña-Ortega (1761-1766).
Alzheimer disease (AD) patients show alterations in both neuronal network oscillations and the cognitive processes associated to them. Related to this clinical observation, it has been found that amyloid beta protein (Aβ) differentially affects some hippocampal network activities, reducing theta and gamma oscillations, without affecting sharp waves and ripples. Beta-like oscillations is another cognitive-related network activity that can be evoked in hippocampal slices by several experimental manipulations, including bath application of kainate and increasing extracellular potassium. Here, we tested whether or not different Aβ peptides differentially affect beta-like oscillatory patterns. We specifically tested the effects of fresh dissolved Aβ25–35 and oligomerized Aβ1–42 and found that kainate-induced oscillatory network activity was affected, in a slightly concentration dependent-manner, by both fresh dissolved (mostly monomeric) Aβ25–35 and oligomeric Aβ1–42. In contrast, potassium-induced oscillatory activity, which is reduced by oligomeric Aβ1–42, is not affected by monomeric Aβ25–35 at any of the concentrations tested. Our results support the idea that different amyloid peptides might alter specific cellular mechanisms related to the generation of specific neuronal network activities, instead of a generalized inhibitory effect of Aβ peptides on neuronal network function.
Keywords: Alzheimer's disease; Network activity; Kainate receptors; Amyloid beta protein;

Subcutaneous injection of endokinin C/D attenuates carrageenan-induced inflammation by Rumi Naono-Nakayama; Natsuki Sunakawa; Tetsuya Ikeda; Osamu Matsushima; Toshikazu Nishimori (1767-1771).
Endokinins, encoded by the human preprotachykinin C (PPT-C)/TAC4 gene, are peptides that consist of endokinin A (EKA), B (EKB), C (EKC) and D (EKD) and belong to the tachykinin family. Intrathecal injection of EKC/D (using the common carboxyl-terminal duodecapeptide in EKC and EKD) markedly attenuated the induction of thermal hyperalgesia and scratching behavior by intrathecal administration of substance P (SP), indicating that EKC/D has an antagonistic effect on the neurokinin 1 receptor (NK1R), SP-preferring receptor, at the spinal level; however, the pharmacological function of EKC/D at the periphery is not yet understood. Therefore, to clarify the effect of EKC/D on the peripheral tissue, the effect of subcutaneous injection of EKC/D on carrageenan-induced inflammation was examined. Subcutaneous injection of EKC/D attenuated an increase in paw volume following carrageenan-induced inflammation in a dose-dependent manner. Indeed, the increased paw volume was significantly decreased 40 min after treatment with 10−4  M (10 nmol) and 10−3  M (100 nmol) EKC/D (100 μl/rat). Similarly, injection of NK1R antagonists such as L-703,606 and Spantide I (10−3  M) attenuated the increased paw volume following inflammation. Furthermore, the reduced withdrawal latency evoked by inflammation following subcutaneous injection of carrageenan was also dose-dependently attenuated by EKC/D administration. These results indicate that subcutaneous injection of EKC/D elicits an anti-inflammatory effect on carrageenan-induced inflammation.
Keywords: Endokinin C/D; Neurokinin 1 receptor; Subcutaneous injection; Inflammation; Thermal hyperalgesia;

Hemodynamic effect of apelin in a canine model of acute pulmonary thromboembolism by Jing-hui Feng; Wei-min Li; Xiu-ping Wu; Xiang-yang Tan; Yan-hui Gao; Chun-liu Han; Shu-qing Li; Hua-ning Xie (1772-1778).
The peptide apelin is expressed in the pulmonary vasculature and is involved in the pathogenesis of many cardiovascular diseases. It has a biphasic role in the regulation of vasomotor tone related to the vascular endothelium. In this study, we induced acute pulmonary embolism (APE) in dogs with autologous blood clots to assess the effect of apelin on pulmonary and systemic circulation in the acute phase of APE. The expression of apelin mRNA was found to be upregulated in the lung tissue in the early several hours after APE induction and decreased at 24 h. The expression of apelin protein in the pulmonary arteries did not change within 24 h after APE, but significantly increased in the bronchial epithelial cells as early as 1 h and decreased at 24 h. In normal anesthetized dogs, intravenous bolus administration of apelin significantly reduced the mean arterial pressure (MAP), but did not significantly affect the mean pulmonary arterial pressure (MPAP). In the dogs with APE, apelin decreased MPAP, whereas its impact on MAP was not significantly different from that in the control group. Taken together, the level of endogenous apelin did not change significantly in the pulmonary arterial wall, whereas its expression in the bronchial epithelium was upregulated in the early stage of APE. The effect of exogenous apelin on vasomotor tone was complicated: it resulted in differential changes in the pulmonary and systemic arterial pressures under different physiological and pathological conditions.
Keywords: Apelin; Acute pulmonary embolism; Lung; Pulmonary arterial pressure; Arterial pressure;

Angiotensin-(1–7) attenuates hyposmolarity-induced ANP secretion via the Na+–K+ pump by Amin Shah; Young-Bin Oh; Gao Shan; Chang Ho Song; Byung-Hyun Park; Suhn Hee Kim (1779-1785).
▶ Perfusion of hyposmolar solution into the atria increased the ANP secretion. ▶ Ang-(1–7) decreased the hyposmolarity-induced ANP secretion via Na+–K+ pump. ▶ In DM atria, the response of ANP secretion to hyposmolar solution was robustly accentuated. ▶ Ang-(1–7) had no effect on hyposmolarity-induced ANP secretion in DM atria partly because of potent inhibition of Na+–K+ pump.The alteration in osmolarity challenges cell volume regulation, a vital element for cell survival. Hyposmolarity causes an increase in cell volume. Recently, it has been reported that the renin–angiotensin system (RAS) plays a role in cell volume regulation. We investigated the effect of angiotensin-(1–7) [Ang-(1–7)] on hyposmolarity-induced atrial natriuretic peptide (ANP) secretion in normal and diabetic (DM) rat atria and modulation of the effect of Ang-(1–7) by the Na+–K+ pump. Using isolated control rat atria, we observed that perfusion of hyposmotic solution into the atria increased ANP secretion. When Ang-(1–7) [0.1 μM or 1 μM] was perfused in a hyposmolar solution, it decreased the hyposmolarity-induced ANP secretion in a dose-dependent manner. This effect of Ang-(1–7) could be mediated by the Na+–K+ pump, since ouabain, an Na+–K+ pump inhibitor, significantly decreased the effect of Ang-(1–7) on hyposmolarity-induced ANP secretion. In contrast, Nω Nitro-l-arginine methyl ester hydrochloride (l-NAME) did not modify the effect of Ang-(1–7) on the hyposmolarity-induced ANP secretion. Interestingly, the ANP secretion was increased robustly by the perfusion of the hyposmolar solution in the DM atria, as compared to the control atria. However, the inhibitory effect of Ang-(1–7) on the hyposmolarity-induced ANP secretion was not observed in the DM atria. In the DM atria, atrial contractility was significantly increased. Taken together, we concluded that Ang-(1–7) attenuated hyposmolarity-induced ANP secretion via the Na+–K+ pump and a lack of Ang-(1–7) response in DM atria may partly relate to change in Na+–K+ pump activity.
Keywords: Angiotensin II; Angiotensin-(1–7); Hyposmolarity; Atrial natriuretic peptide; Na–K ATPase;

The iron-regulatory peptide hepcidin is upregulated in the ischemic and in the remote myocardium after myocardial infarction by Gregor Simonis; Katrin Mueller; Peggy Schwarz; Stephan Wiedemann; Guido Adler; Ruth H. Strasser; Hasan Kulaksiz (1786-1790).
Recent evidence suggests that iron metabolism contributes to the ischemic damage after myocardial infarction. Hepcidin, a recently discovered peptide hormone, regulates iron uptake and metabolism, protecting the body from iron overload. In this study we analyzed the regulation of hepcidin in the heart and blood of rats after myocardial infarction. To induce a myocardial infarction in the rats, left anterior descending coronary artery ligation was performed. After 1–24 h, biopsies from the ischemic and the non-ischemic myocardium were taken. In these biopsies, the mRNA levels and the protein expression of hepcidin were analyzed by quantitative RT-PCR and immunoblot analysis, respectively. In parallel, the serum levels of prohepcidin were measured by ELISA. Six hours after myocardial infarction, the hepcidin mRNA expression was temporally upregulated in the ischemic and in the non-ischemic myocardium. The upregulation was specific for hepcidin, since other iron-related genes (hemojuvelin, IREG-1) remained unchanged. Furthermore, the alteration of the hepcidin protein expression in the ischemic area was connected to the level of hepcidin in the serum of the infarcted rats, where hepcidin also raised up. Angiotensin receptor blockade with candesartan did not influence the mRNA regulation of hepcidin. Together, these data show a particular upregulation of the iron-regulatory peptide hepcidin in the ischemic and the non-ischemic myocardium after myocardial infarction. It is speculated that upregulation of hepcidin may reduce iron toxicity and thus infarct size expansion in an infarcted heart.
Keywords: Iron; Hepcidin; Myocardial infarction; Ischemic damage;

Antimicrobial peptides are predominantly small cationic polypeptides that are classified together on the basis of these molecules to directly kill or inhibit the growth of microorganisms including mycobacteria, and to activate mechanisms of cellular and adaptive immunity. Various families of antimicrobial peptides have been identified, including the cathelicidins. The cathelicidin family is characterised by a conserved N-terminal cathelin domain and a variable C-terminal antimicrobial domain that can be released from the precursor protein after cleavage by proteinases. LL-37 is the C-terminal part of the only human cathelicidin identified to date called human cationic antimicrobial protein (hCAP18), which is mainly expressed by neutrophils and epithelial cells. The cathelicidin hCAP18/LL-37 is a multifunctional molecule that may mediate various host responses, including bactericidal action, chemotaxis, epithelial cell activation, angiogenesis, epithelial wound repair and activation of chemokine secretion. The antimicrobial peptide LL-37 is produced from human cells during infection of mycobacteria and exerts a microbicidal effect. The discussion will (1) describe recent work on the antimicrobial and immunomodulatory functions of the cathelicidin hCAP18/LL-37, (2) highlight the effectiveness of the cathelicidin hCAP18/LL-37 as a potent component in antimycobacterial immune responses and (3) summarise current progress in the understanding of the therapeutic application of hCAP18/LL-37 and its derivates antimicrobial peptides in mycobacterial infection.
Keywords: Antimicrobial peptides; Cathelicidin; LL-37; Innate immunity; Mycobacteria;