Peptides (v.30, #7)

Expression of Jug r 1, the 2S albumin allergen from walnut (Juglans regia), as a correctly folded and functional recombinant protein by Camille Sordet; Raphaël Culerrier; Claude Granier; Fabienne Rancé; Alain Didier; Annick Barre; Pierre Rougé (1213-1221).
Jug r 1, the 2S albumin allergen from walnut, was isolated from ripe nuts as a native allergen and expressed in Escherichia coli using the Gateway® technology as a recombinant allergen. The recombinant Jug r 1 (15 kDa) differs from the native allergen by the absence of cleavage of the polypeptide chain in two covalently associated light (3.5 kDa) and heavy (8 kDa) chains. Recombinant rJug r 1 adopts the canonical α-helical fold of plant 2S albumins as checked on CD spectra. Four IgE-binding epitopic stretches were identified along the amino acid sequence of Jug r 1 and localized on the molecular surface of the modeled allergen. Both native and recombinant allergens exhibit similar IgE-binding activity and similarly trigger the degranulation of a FcɛRI-expressing rat basophilic leukaemia cell line previously treated by IgE-containing sera. Native Jug r 1 resists to heat denaturation and to the proteolytic attack of trypsin and chymotrypsin but is readily hydrolyzed in the presence of pepsin at acidic pH after 1 h of incubation at 37 °C in vitro. Recombinant Jug r 1 could be used for a component-resolved diagnosis of food-allergy.
Keywords: Recombinant allergen; Jug r 1; IgE-binding epitopes; Pepsin cleavage;

Remarkable inter- and intra-species complexity of conotoxins revealed by LC/MS by Jasmine Davis; Alun Jones; Richard J. Lewis (1222-1227).
Cone snails have evolved an assortment of venom peptides as an evolutionary strategy for rapid prey immobilization and defence. Earlier studies estimated ∼100 conopeptides per species. In this study we optimized liquid chromatography and electrospray ionization mass spectrometry for the detection of conopeptides in crude venom to characterize conopeptides present in the venom of individual specimens of Conus textile, C. imperialis and C. marmoreus. Using this approach, we have expanded the predicted number of venom peptides 10-fold to an estimate of 1000–1900 conopeptides per species. Our investigation has also revealed a surprisingly high level of intra-species variation that distinguishes cone snails from other venomous species including spiders and scorpions. Given this inherent diversity and variability, more sensitive bioassays and sequencing techniques will be required to fully explore conotoxin bioactivity.
Keywords: Venom peptide; Venomics; Mass spectrometry; Cone snail; Conotoxin;

While investigating the innate defense of brackish water-living amphibian and its comparison with freshwater-living amphibians, two novel 12-residue antimicrobial peptides were purified from the skin secretions of the crab-eating frog, Fejervarya cancrivora which typically inhabits brackish water of mangrove forests of Southeast Asia. These two antimicrobial peptides, tigerinin-RC1 and -RC2 share significant structural similarity with tigerinins found in the skin of Indian frog, Hoplobatrachus tigerinus. cDNAs encoding tigerinin-RC1 and -RC2 were also cloned from the skin cDNA library of F. cancrivora. Tigerinin-RC precursors are composed of 71 amino acid residues including a signal peptide, acidic spacer peptide, which are very similar to other amphibian antimicrobial peptide precursors and mature tigerinin-RC. The current results confirmed that both amphibians inhabiting freshwater and brackish water share the same antimicrobial peptide family to exert innate defense. Furthermore, the current work was also the first report of precursor and cDNA cloning of the tigerinin antimicrobial peptide family.
Keywords: Amphibian; Antimicrobial peptide; Fejervarya cancrivora; Brackish water; Tigerinin;

Novel gene encoding precursor protein consisting of possible several neuropeptides expressed in brain and frontal ganglion of the silkworm, Bombyx mori by Kanako Mitsumasu; Yoshiaki Tanaka; Teruyuki Niimi; Okitsugu Yamashita; Toshinobu Yaginuma (1233-1240).
A novel gene (BmK5) expressed in the central nervous system of the silkworm, Bombyx mori, was isolated using a cDNA subtraction method. BmK5 was first cloned as a candidate regulator of diapause hormone release from subesophageal ganglion via corpus cardiacum–corpus allatum into the hemolymph; however, subsequent analyses revealed that the gene expression patterns in brain–subesophageal ganglion complexes did not differ between diapause and nondiapause egg producers. The deduced amino acid sequence showed the characteristics of secretory protein precursor or nuclear localization protein. Immunohistochemical experiments with an anti-BmK5 antibody revealed that BmK5 precursor protein exists in the cytoplasm of specific cells of brain and frontal ganglion, but not in the nuclei. In addition, a peptide (GSGTKVGGAGAATKVVTKSGS-NH2) possibly processed from the BmK5 precursor protein was immunohistochemically detected in the axons connecting the anti-BmK5 antibody-positive cells to the neurohemal organ, corpus cardiacum–corpus allatum. These results suggest that BmK5 encodes a precursor of the novel neurosecretory protein and that several mature peptides are released into the hemolymph via the corpus cardiacum–corpus allatum, although the functions of these peptides are yet unclear.
Keywords: Bombyx mori; BmK5 gene; Novel neuropeptide; Frontal ganglion; Brain;

FGLamide Allatostatin genes in Arthropoda: Introns early or late? by Francisco Martínez-Pérez; William G. Bendena; Belinda S.W. Chang; Stephen S. Tobe (1241-1248).
FGLamide allatostatins are invertebrate neuropeptides which inhibit juvenile hormone biosynthesis in Dictyoptera and related orders and also show myomodulatory activity. The FGLamide allatostatin (AST) gene structure in Dictyoptera is intronless within the ORF, whereas in 9 species of Diptera, the FGLamide AST ORF has one intron. To investigate the evolutionary history of AST intron structure, (intron early versus intron late hypothesis), all available Arthropoda FGLamide AST gene sequences were examined from genome databases with reference to intron presence and position/phase. Three types of FGLamide AST ORF organization were found: intronless in I. scapularis and P. humanus corporis; one intron in D. pulex, A. pisum, A. mellifera and five Drosophila sp.; two introns in N. vitripennis, B. mori strains, A. aegypti, A. gambiae and C. quinquefasciatus. The literature suggests that for the majority of genes examined, most introns exist between codons (phase 0) which may reflect an ancient function of introns to separate protein modules. 60% of the FGLamide AST ORFs introns were between the first and second base within a codon (phase 1), 28% were between the second and third nucleotides within a codon (phase two) and 12% were phase 0. As would be required for correct intron splicing consensus sequence, 84% of introns were in codons starting with guanine. The positioning of introns was a maximum of 9 codons from a dibasic cleavage site. Our results suggest that the introns in the analyzed species support the intron late model.
Keywords: Allatostatins; Arthropoda; Gene structure; Introns; Late model;

A potential insect growth regulator: Synthesis and bioactivity of an allatostatin mimic by Zhen-peng Kai; Juan Huang; Stephen S. Tobe; Xin-ling Yang (1249-1253).
Insect growth regulators play an important role in Integrated Pest Management systems. Cockroach-type allatostatins (FGLamides) (ASTs), which are a family of basic peptides first isolated from brains of Diploptera punctata were originally discovered on the basis of their ability to inhibit the production of juvenile hormone by the corpora allata. For this reason, the ASTs can be regarded as possible IGR candidates for pest control although the absence of effect in vivo, rapid degradation and high production costs of the natural peptides preclude their use in pest management. However, we have synthesized a new AST mimic, H17, from the pentapeptide C-terminal active core of the AST. This mimic is able to significantly inhibit the biosynthesis of JH by cockroach CA in vitro (IC50 value: 12 nM) and in vivo following injection (IC50 value: 33 nM). H17 also shows a highly significant inhibition of JH production in topical cuticular assays in vivo. Our results suggest that H17 has potential as an IGR for cockroach control.
Keywords: Allatostatin; IGR; Juvenile hormone; Biosynthesis; Cockroach; Peptidomimetic;

Evaluation of a PK/PBAN analog with an (E)-alkene, trans-Pro isostere identifies the Pro orientation for activity in four diverse PK/PBAN bioassays by Ronald J. Nachman; Xiaodong J. Wang; Felicia A. Etzkorn; Orna Ben Aziz; Michael Davidovitch; Krzysztof Kaczmarek; Janusz Zabrocki; Allison Strey; Nan Pryor; Miriam Altstein (1254-1259).
The pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family plays a multifunctional role in an array of important physiological processes in a variety of insects. An active core analog containing an (E)-alkene, trans-Pro isosteric component was evaluated in four disparate PK/PBAN bioassays in four different insect species. These bioassays include pheromone biosynthesis in the moth Heliothis peltigera, melanization in the larval Spodoptera littoralis, pupariation acceleration in the larval fly Neobellieria bullata, and hindgut contraction in the cockroach Leucophaea maderae. The conformationally constrained analog demonstrated activity equivalent to parent PK/PBAN peptides of equal length in all four PK/PBAN bioassays, and matched and/or approached the activity of peptides of natural length in three of them. In the melanization bioassay, the constrained analog exceeded the efficacy (maximal response) of the natural PBAN1-33 by a factor of 2 (at 1 nmol). The results provide strong evidence for the orientation of Pro and the core conformation adopted by PK/PBAN neuropeptides during interaction with receptors associated with a range of disparate PK/PBAN bioassays. The work further identifies a scaffold with which to design mimetic PK/PBAN analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting PK/PBAN-regulated systems.
Keywords: Pheromonotropic; Pupariation; Melanization; Myotropic; Peptidomimetic; Neuropeptide; PBAN;

Pituitary adenylate cyclase-activating polypeptide induces somatolactin release from cultured goldfish pituitary cells by Morio Azuma; Mio Tanaka; Yumika Nejigaki; Minoru Uchiyama; Akiyoshi Takahashi; Seiji Shioda; Kouhei Matsuda (1260-1266).
In the goldfish pituitary, nerve fibers containing pituitary adenylate cyclase-activating polypeptide (PACAP) are located in close proximity to somatolactin (SL)-producing cells, and PACAP enhances SL release from cultured pituitary cells. However, there is little information about the mechanism of PACAP-induced SL release. In order to elucidate this issue, we used the cell immunoblot method. Treatment with PACAP at 10−8 and 10−7  M, but not with vasoactive intestinal polypeptide (VIP) at the same concentrations, increased the immunoblot area for SL-like immunoreactivity from dispersed pituitary cells, and PACAP-induced SL release was blocked by treatment with the PACAP selective receptor (PAC1R) antagonist, PACAP(6–38), at 10−6  M, but not with the PACAP/VIP receptor antagonist, VIP(6–28). PACAP-induced SL release was also attenuated by treatment with the calmodulin inhibitor, calmidazolium at 10−6  M. This led us to explore the signal transduction mechanism up to SL release, and we examined whether PACAP-induced SL release is mediated by the adenylate cyclase (AC)/cAMP/protein kinase A (PKA)- or the phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3)/protein kinase C (PKC)-signaling pathway. PACAP-induced SL release was attenuated by treatment with the AC inhibitor, MDL-12330A, at 10−5  M or with the PKA inhibitor, H-89, at 10−5  M. PACAP-induced SL release was suppressed by treatment with the PLC inhibitor, U-73122, at 3 × 10−6  M or with the PKC inhibitor, GF109203X, at 10−6  M. These results suggest that PACAP can potentially function as a hypophysiotropic factor mediating SL release via the PAC1R and subsequently through perhaps the AC/cAMP/PKA- and the PLC/IP3/PKC-signaling pathways in goldfish pituitary cells.
Keywords: PACAP; Goldfish; Pituitary; Somatolactin; Cell immunoblot; Hypophysiotropic factor; PAC1R; Signal transduction;

Blockade of PrRP attenuates MPTP-induced toxicity in mice by Binggui Sun; Akikazu Mochiduki; Kimie Nakamura; Kotaro Yokoyama; Sachika Adachi; Ken Fujiwara; Hirokazu Matsumoto; Kinji Inoue (1267-1275).
Prolactin-releasing peptide (PrRP) was isolated as an endogenous ligand of the orphan G-protein coupled receptor hGR3. PrRP has been shown to be involved in the regulation of food intake, stress responses, prolactin secretion and release, blood pressure, and the opioid system. Here we report that PrRP and its receptor, GPR10, were found in the mouse substantia nigra pars compacta (SNpc), the main location of dopaminergic (DA) neurons of the nigrostriatal system. We generated PrRP knockout (KO) mice, and then treated PrRP KO mice and their wild type (WT) littermates with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neuron toxin that selectively damages DA neurons in the SNpc. We found that PrRP KO mice were resistant to MPTP-induced lesions of the nigrostriatal system. These effects were further confirmed by the intracerebroventricular injection of P2L-1C, a monoclonal antibody against PrRP into mice. Taken together, our data established a critical role of PrRP in MPTP intoxication in mice.
Keywords: PrRP; MPTP; Dopaminergic neurons; Substantia nigra pars compacta; Mouse;

Mass spectrometric quantification of MIF-1 in mouse brain by multiple reaction monitoring by Indu Kheterpal; Abba J. Kastin; Sahana Mollah; Chuanhui Yu; Hung Hsuchou; Weihong Pan (1276-1281).
MIF-1 (Pro-Leu-Gly-NH2) has potent therapeutic effects in depression and Parkinson's disease, but its CNS sites of production are not yet clear. In this study, the concentration of MIF-1 in different brain regions was measured by the multiple reaction monitoring technique on a 4000 QTRAP mass spectrometer. The limit of quantification was 300 fg of MIF-1, and limit of detection was 60 fg. The low molecular weight fractions of tissue homogenates from different regions of mouse brain were analyzed. The concentration of MIF-1 ranged from 22 ± 3 fg/μg protein in cerebral cortex to 930 ± 60 fg/μg protein in the hypothalamus. Moderate concentrations were also detected in all other regions tested, including the striatum, thalamus, and hippocampus. By incubation of stable isotope-labeled oxytocin with tissue preparations, it was also confirmed that oxytocin at least partially contributed to the production of MIF-1 in the hypothalamus by action of peptidases. Regional differences were also found. The results are the first to show the ultrasensitive quantification of MIF-1 in different brain regions, and support the neuromodulatory actions of MIF-1 in the striatum.
Keywords: MIF-1; Peptide; Brain; Peptidases; Mass spectrometry; Multiple reaction monitoring (MRM);

Hybrid peptides attenuate cytotoxicity of β-amyloid by inhibiting its oligomerization: Implication from solvent effects by Xun Sun; Wei-hui Wu; Qian Liu; Mei-sha Chen; Ye-ping Yu; Ying Ma; Yu-fen Zhao; Yan-mei Li (1282-1287).
Abnormal assembly of monomeric β-amyloid (Aβ) in Alzheimer's disease leads to the formation of most neurotoxic oligomers in vivo. In this study, we explored a linking strategy to design hybrid peptides, by combining the Aβ recognition motif and the solvent disruptive sequences. We found that in vitro all synthetic peptides with the recognition motif can affect Aβ fibrillization and alter the morphology of Aβ aggregates variously, different from those without the recognition motif. The effects of peptides containing recognition motif on Aβ aggregation correlate with their abilities to change the surface tension of solutions. In addition, compounds with the recognition motif, not those without such motif, can inhibit cytotoxicity of Aβ in cell culture probably by decreasing the amount of toxic Aβ oligomers. These results indicate that recognition domain and solvent effect should be considered as important factors when designing molecules to target Aβ aggregation.
Keywords: Alzheimer's disease; β-Amyloid; Aggregation; Hybrid peptide; Cytotoxicity; Solvent effect;

Improved absorption of salmon calcitonin by ultraflexible liposomes through intranasal delivery by Ming Chen; Xin-Ru Li; Yan-Xia Zhou; Ke-Wei Yang; Xing-Wei Chen; Qiu Deng; Yan Liu; Li-Jun Ren (1288-1295).
The objective of this work was to explore the potential of ultraflexible liposomes as carriers for improving the absorption of salmon calcitonin (sCT) through intranasal administration. The average diameters of positively charged ultraflexible liposomes ranged from about 73 to 99 nm, while those of negatively charged ones were 114 and 157.6 nm, respectively. The content of sodium deoxycholate in liposomes markedly affected the size and encapsulated efficiency of liposomes. The absorption of sCT through intranasal administration was evaluated by hypocalcemic efficacy in rats. The total Ca decrease D% of sCT-loaded ultraflexible liposomes with positive and negative charges were significantly bigger than that of sCT solution, while there was no significant difference in the hypocalcemic efficacy between plain liposome and sCT solution. Unexpectedly, the hypocalcemic efficacy of sCT-loaded ultraflexible liposomes with positive charges was not significantly better than those with negative charges. The decrease rate and extent of the serum calcium level for subcutaneous injection of sCT solution were almost equivalent to those for intranasal administration of negatively and positively charged ultraflexible liposomes within the first 2 h, indicating that the ultraflexible liposomes could quickly enhance the penetration of the drug during their residence in the nasal cavity. The results of the toxicity of sCT-loaded ultraflexible liposomes to nasal mucosa demonstrated that the ultraflexible liposomes exerted slight toxicity on the nasal mucosa. On an overall evaluation, the ultraflexible liposomes may be a useful vehicle for intranasal delivery of sCT.
Keywords: Ultraflexible liposome; Salmon calcitonin; Nasal delivery; Toxicity of nasal cilia; Peptide; Hypocalcemic efficacy;

Chemokine receptor-derived peptides as multi-target drug leads for the treatment of inflammatory diseases by C. Ezerzer; M. Dolgin; J. Skovorodnikova; N. Harris (1296-1305).
The rationale for multi-target drugs has been strengthened both on theoretical and empirical grounds. Serious diseases that are intractable to treatment were found to have multiple pathogenic factors and examples of successful drugs were shown to affect multiple disease targets. The salient features of multiple-target drugs, low target affinity and rapid binding kinetics, have been responsible for their late discovery and slow development. We predicted that peptides from the ligand-binding domains of chemokine (CK) receptors could be used to modulate the activities of disease-related chemokines (CKs) for therapeutic effect. We developed innovative technologies to produce, screen and optimize low affinity, chemokine-binding peptides (CBPs) derived from chemokine receptors (CRs). The peptides were found to have therapeutic activity in animal models of disease, confirming our prediction and validating the related technologies.
Keywords: Chemokine; Peptide; Inflammatory; Disease;

Inhibitory effect of carnosine and N-acetyl carnosine on LPS-induced microglial oxidative stress and inflammation by Sigal Fleisher-Berkovich; Chen Abramovitch-Dahan; Shimon Ben-Shabat; Ron Apte; Elie Beit-Yannai (1306-1312).
Chronic inflammation and oxidative stress have been implicated in the pathogenesis of neurodegenerative diseases. A growing body of research focuses on the role of microglia, the primary immune cells in the brain, in modulating brain inflammation and oxidative stress. One of the most abundant antioxidants in the brain, particularly in glia, is the dipeptide carnosine, β-alanyl-l-histidine. Carnosine is believed to be involved in cellular defense such as free radical detoxification and inhibition of protein cross-linking. The more stable N-acetyl derivative of carnosine has also been identified in the brain. The aim of the present study was to examine the role of carnosine and N-acetyl carnosine in the regulation of lipopolysaccharide (LPS)-induced microglial inflammation and oxidative damage. In this study, BV2 microglial cells were stimulated with bacterial LPS, a potent inflammatory stimulus. The data shows that both carnosine and N-acetyl carnosine significantly attenuated the LPS-induced nitric oxide synthesis and the expression of inducible nitric oxide synthase by 60% and 70%, respectively. By competitive spectrophotometric measurement and electrospray mass spectrometry analysis, we demonstrated a direct interaction of N-acetyl carnosine with nitric oxide. LPS-induced TNFα secretion and carbonyl formation were also significantly attenuated by both compounds. N-acetyl carnosine was more potent than carnosine in inhibiting the release of the inflammatory and oxidative stress mediators. These observations suggest the presence of a novel regulatory pathway through which carnosine and N-acetyl carnosine inhibit the synthesis of microglial inflammatory and oxidative stress mediators, and thus may prove to play a role in brain inflammation.
Keywords: Carnosine; N-acetyl carnosine; LPS; Microglia; BV-2 cells; Nitric oxide;

Central Neuropeptide S inhibits distal colonic transit through activation of central Neuropeptide S receptor in mice by Ren-Wen Han; Min Chang; Ya-Li Peng; Lian-yong Qiao; Xin-Qiang Yin; Wei Li; Rui Wang (1313-1317).
Neuropeptide S (NPS), the endogenous ligand of NPS receptor (NPSR), regulates many biological functions, including arousal, anxiety, locomotion and food intake. NPSR mRNA is expressed in several regions of central autonomic network through which the brain controls visceromotor and other responses essential for survival. However, the role of NPS/NPSR system in regulating gastrointestinal motor is still unknown. Here, we studied the effects of NPS on distal colonic transit in mice. Intracerebroventricular (i.c.v.) injection of NPS (1–1000 pmol) inhibited fecal pellet output and bead expulsion in a dose-dependent manner. However, intraperitoneal injection of NPS (1000 and 10 000 pmol) did not affect fecal pellet output and bead expulsion. In vitro, NPS (0.1–10 μM) also did not modulate distal colonic contractions. Furthermore, i.c.v. co-administration of [D-Val5]NPS, a pure and potent NPSR antagonist, dose-dependently antagonized the inhibitory effects of NPS on fecal pellet output and bead expulsion. In conclusion, our results firstly indicate that central NPS inhibits distal colonic transit through the activation of central NPSR, which implicate that NPS/NPSR system might be a new target to treat function disorder of distal colon.
Keywords: Neuropeptide S (NPS); Neuropeptide S receptor (NPSR); Fecal pellet output; Bead expulsion; Distal colonic transit;

Comparison of independent and combined chronic anti-obese effects of NPY Y2 receptor agonist, PYY(3-36), and NPY Y5 receptor antagonist in diet-induced obese mice by Ryuichi Moriya; Satoshi Mashiko; Akane Ishihara; Toshiyuki Takahashi; Takashi Murai; Junko Ito; Yuko Mitobe; Zenjun Oda; Hisashi Iwaasa; Fukami Takehiro; Akio Kanatani (1318-1322).
Neuropeptide Y (NPY) and its family of peptides are thought to have a major role in the physiological control of energy homeostasis. Among five NPY receptors described, stimulation of the Y2 receptor (Y2R) or inhibition of the Y5 receptor (Y5R) has recently been shown to produce weight-lowering effects in obese rodents. The present study examined and compared the effects of a Y2R agonist, PYY(3-36), and a Y5R antagonist, alone and in combination, on food intake and body weight in diet-induced obese (DIO) mice. Acute intraperitoneal injection of PYY(3-36) dose-dependently reduced spontaneous feeding in lean and DIO mice. In contrast, acute oral administration of the Y5R antagonist had no effect on spontaneous feeding or the anorexigenic effects of PYY(3-36). In a chronic study, subcutaneous infusion of PYY(3-36) (1 mg/kg/day for 14 days) significantly reduced food intake and body weight in DIO mice. The Y5R antagonist (10 mg/kg/day for 14 days, orally) reduced body weight to the same extent as PYY(3-36) without a significant feeding reduction. Combined administration of PYY(3-36) and the Y5R antagonist resulted in a greater body weight reduction than treatment with either agent alone. The combined effects on food intake, body weight, and adiposity are almost the same as a hypothetical sum of the effects of each drug alone. These results illustrate that the combination of a Y2R agonist, PYY(3-36), and a Y5R antagonist resulted in additive effects on body weight and adiposity in DIO mice, suggesting that Y2R stimulation signal and Y5R blockade signal act by distinct pathways.
Keywords: PYY(3-36); NPY Y2 receptor; NPY Y5 receptor;

Effects of obestatin on feeding and body weight after standard or cafeteria diet in the rat by Luigi Brunetti; Sheila Leone; Giustino Orlando; Lucia Recinella; Claudio Ferrante; Annalisa Chiavaroli; Chiara Di Nisio; Pierpaolo Di Michele; Michele Vacca (1323-1327).
Obestatin is a gastric derived 23 amino acid peptide, which has shown anorectic effects in a number of experimental paradigms after both peripheral and central administration. On the other hand, several researchers were not able to confirm these data. Since all previous experiments have been performed in animals fed a standard laboratory diet, we studied obestatin effects in male Wistar rats fed both a standard laboratory chow (STD) diet (3.5% fat, 63% carbohydrate, 14% protein, 19.5% other components without caloric value; 3.20 kcal/g) and a highly palatable cafeteria-style (CAF) diet (30% fat, 56% carbohydrate, 14% protein; 4.20 kcal/g). Vehicle or obestatin (10, 50 or 100 nmol/kg) was injected intraperitoneally daily for 12 days. In STD diet rats, obestatin decreased daily caloric intake and body weight gain compared to vehicle treated rats. The anorectic and weight reducing effects of obestatin treatment were evidenced since day 6 and day 8 of treatment, respectively, and were consistent through the end of treatment. On the other hand, in CAF diet rats, obestatin treatment did not modify either daily caloric intake or body weight gain. In CAF diet rats, the percentage intake from standard food was decreased, balanced by an increase in cafeteria food intake. Obestatin treatment affected neither water consumption nor the intake of any specific food within the cafeteria diet. In conclusion, obestatin decreases caloric intake and body weight gain, but only in rats fed a STD diet.
Keywords: Body weight; Cafeteria; Caloric intake; Feeding; Obestatin;

Orexin-A and ghrelin depolarize the same pedunculopontine tegmental neurons in rats: An in vitro study by Juhyon Kim; Kazuki Nakajima; Yutaka Oomura; Matthew J. Wayner; Kazuo Sasaki (1328-1335).
Orexin (ORX), also called hypocretin, and ghrelin are newly identified peptides in the brain and/or peripheral organs, and they are involved in the regulation of sleep–wakefulness as well as feeding. In our previous studies we have found that ORX and ghrelin each depolarizes more than half of the cholinergic neurons recorded in the pedunculopontine tegmental nucleus (PPT) via a dual ionic mechanism including a decrease of K+ conductance and an increase of nonselective cationic conductance. Thus, the present study was carried out to investigate whether ORX-A and ghrelin both depolarize the same PPT neuron. About 60% of PPT neurons examined was depolarized by both ORX-A and ghrelin, 20% by ORX-A alone, and 10% by ghrelin alone. The remaining 10% did not respond to these peptides. In neurons which were responsive to both ORX-A and ghrelin, the depolarizations induced by ORX-A and ghrelin were additive. In addition, the ORX-A- and ghrelin-induced depolarizations were both blocked by D609, a phosphatidylcholine-specific phospholipase C (PLC) inhibitor. These results suggest that same PPT neurons with receptors for ORX and ghrelin are involved in the cellular process through which ORX and ghrelin participate in the regulation of sleep wakefulness, and that the excitatory effects of ORX and ghrelin on PPT neurons are mediated by PLC.
Keywords: Orexin/hypocretin; Ghrelin; Pedunculopontine tegmental nucleus; Cholinergic neurons; Sleep–wakefulness; Phospholipase C;

Effect of reducing hypothalamic ghrelin receptor gene expression on energy balance by Yogendra B. Shrestha; Kathie Wickwire; Silvia Giraudo (1336-1341).
Central and peripheral injections of fghrelin potently stimulates food intake via its receptor, GHSR1a expressed in the brain. In this study, we explored the role of GHSR1a in the paraventricular nucleus of the hypothalamus (PVN) by reducing their gene expression using the RNA interference (RNAi). pSUPER plasmids inserted with sh (short hairpin)-GHSR1a were injected into the PVN to reduce its expression. The transfected rats were monitored daily for their food intake and body weight throughout the experimental period lasting 8 days. We found that knockdown of GHSR1a did not affect daily food intake but significantly reduced body weight and blood ghrelin levels. This suggests that the central ghrelin system could selectively regulate body weight without affecting energy intake.
Keywords: RNAi; Ghrelin; Paraventricular nucleus; Hypothalamus; GSHR1a;

Ghrelin modulates fatty acid synthase and related transcription factor mRNA levels in a tissue-specific manner in neonatal broiler chicks by Johan Buyse; Sara Janssen; Sofie Geelissen; Quirine Swennen; Hiroyuki Kaiya; Veerle M. Darras; Sami Dridi (1342-1347).
The endogenous ligand for the growth hormone (GH) secretagogue receptor ghrelin is a peptide secreted by the stomach of mammals and stimulates food intake and enhances adiposity. In avian species, ghrelin is mainly produced by the proventriculus but reduces food intake whereas its effect on lipogenesis in different tissues is unknown. We therefore investigated the effects of a single intravenous injection of 2.8 μg (1 nmol per chick) recombinant chicken ghrelin in neonatal broiler chicks. Besides food intake and plasma corticosterone levels, mRNA levels of the key lipogenic enzyme fatty acid synthase (FAS) and its related transcription factors sterol regulatory element binding protein-1 (SREBP-1) and peroxisome proliferator-activated receptor-γ (PPARγ) were determined in diencephalon, liver and quadriceps femoris muscle before, and 15, 30, and 60 min after injection. Chicken ghrelin administration induced a significant short-term (<30 min) reduction in food intake and markedly elevated plasma corticosterone levels. In diencephalon, FAS, SREBP-1 and PPARγ mRNA levels were significantly increased within 15 min after ghrelin injection. These observations suggest that central fatty acid metabolism is involved in the anorectic effects of ghrelin. In contrast, hepatic mRNA levels of FAS and both transcription factors were significantly reduced within 30 min after ghrelin injection. In muscle, FAS and transcription factor gene expression was very low and not affected by ghrelin. Overall, our results indicate that ghrelin has opposite effects on FAS and transcription factor mRNA amounts with increased levels in diencephalon (central anorectic effect) and decreased levels in liver (peripheral anti-lipogenic effect) in chickens.
Keywords: Ghrelin; Chicken; Diencephalon; Liver; Fatty acid synthase; Transcription factors;

Effects and underlying mechanisms of human opiorphin on colonic motility and nociception in mice by Xiao-zhu Tian; Juan Chen; Wei Xiong; Tian He; Qiang Chen (1348-1354).
In the present study, we investigated the effects of human opiorphin on colonic motility and nociception in mice. In in vitro bioassay, opiorphin (10−6 to 10−4  M) caused colonic contraction in a concentration-dependent manner, which was completely blocked by naloxone and partially attenuated by β-funaltrexamine and naltrindole. Moreover, opiorphin (10−4  M) significantly enhanced the contractile response induced by Met-enkephalin. The data suggested that the effect of opiorphin on colonic contraction may be due to the protection of enkephalins. In in vivo bioassay, intracerebroventricular (i.c.v.) administration of opiorphin (1.25–10 μg/kg) dose- and time-dependently induced potent analgesic effect (ED50  = 3.22 μg/kg). This effect was fully blocked by naloxone and significantly inhibited by co-injection (i.c.v.) with β-funaltrexamine or naltrindole, but not by nor-binaltorphimine, indicating the involvement of both μ- and δ-opioid receptors in the analgesic response evoked by opiorphin. In addition, i.c.v. administration of 5 μg/kg opiorphin produced the comparative effect as 10 μg/kg morphine on the analgesia, suggesting that opiorphin displayed more potent analgesic effect than that induced by morphine.
Keywords: Human opiorphin; Antinociception; Opioid receptor; Enkephalins; Neutral endopeptidase; Aminopeptidase N;

Arginine vasopressin antinociception in the rat nucleus raphe magnus is involved in the endogenous opiate peptide and serotonin system by Jun Yang; Huifeng Yuan; Jiegen Chu; Yu Yang; Hongtao Xu; Gen Wang; Wen-Yan Liu; Bao-Cheng Lin (1355-1361).
Arginine vasopressin (AVP) in the nucleus raphe magnus (NRM) has been implicated in antinociception. This communication was designed to investigate which neuropeptide and neurotransmitter are involved in AVP antinociception in the rat NRM. The results showed that (1) in the NRM perfuse liquid, pain stimulation could increase the concentrations of AVP, leucine-enkephalin (L-Ek), methionine-enkephalin (M-Ek), β-endorphin (β-Ep), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), but not change the concentrations of dynorphinA1–13 (DynA1–13), oxytocin, achetylcholine, choline, γ-aminobutyric acid, glutamate, dopamine, 3,4-dihydroxyphenylacetic acid, homovanilic acid, norepinephrine and epinephrine; (2) in the NRM perfuse liquid, AVP increased the concentrations of L-Ek, M-Ek, β-Ep, DynA1–13, 5-HT and 5-HIAA, but did not change the concentrations of oxytocin and the other studied neurotransmitters; (3) AVP antinociception in the NRM was attenuated by cypoheptadine (a 5-HT-receptor antagonist) or naloxone (an opiate receptor antagonist), but was not influenced by the other studied receptor antagonists. The data suggested that AVP antinociception in the NRM might be involved in endogenous opiate peptide and 5-HT system.
Keywords: Arginine vasopressin; Nucleus raphe magnues; Antinociception; Endogenous opiate peptide; Serotonin;

Expression of prosalusin in human neuroblastoma cells by Chisato Nakayama; Masayoshi Shichiri; Kengo Sato; Yukio Hirata (1362-1367).
Salusins, which are derived from the prosalusin precursor molecule, regulate hemodynamics, mitogenesis and atherogenesis. The preprosalusin gene is ubiquitously expressed, while the salusin-β peptide is present in systemic endocrine cells and the neuroendocrine system. However, the regulatory mechanisms for the preprosalusin gene and prosalusin expression remain to be investigated. Real-time quantitative RT-PCR and salusin-α radioimmunoassay revealed that the neuroblastoma cell line, SK-N-SH, exhibited marked upregulation of preprosalusin mRNA and salusin-α-like immunoreactivity (LI) when incubated under 2% serum condition. However, SK-N-SH cells released a limited amount of salusin-α-LI into the culture supernatant. Reverse-phase high performance liquid chromatography coupled with radioimmunoassay after extraction of proteins from the conditioned media using an octyl-silica column did not reveal a component that co-eluted with authentic salusin-α. Western blotting of the nuclear extracts from SK-N-SH showed the expression of prosalusin and its cleaved fragments, but not authentic salusin-α. Addition of Jak-2 inhibitors to growing SK-N-SH cells cultured under 10% serum condition resulted in increased salusin-α-LI expression. Suppression of Jak-2 mRNA using siRNAs upregulated intracellular salusin-α-LI, as detected by immunofluorescence. In summary, the preprosalusin gene and prosalusin protein are expressed in a neuroblastoma cell line and upregulated by reduced serum. The Jak-2 pathway may be involved in the regulation of salusin expression.
Keywords: Salusin-α; Jak-2; Prosalusin; Radioimmunoassay; Neuroblastoma;

Role of TRPC1 and NF-κB in mediating angiotensin II-induced Ca2+ entry and endothelial hyperpermeability by Li-xia Yang; Rui-wei Guo; Bei Liu; Xian-mei Wang; Feng Qi; Chuan-ming Guo; Yan-kun Shi; Hong Wang (1368-1373).
Endothelial dysfunction is associated with cardiovascular diseases. The Ca2+ influx occurring via activation of plasmalemma Ca2+ channels was shown to be critical in signaling the increase in endothelial permeability in response to a variety of permeability-increasing mediators. It has been reported that angiotensin II (AngII) could induce Ca2+ signaling in some cells, and transient receptor potential canonical 1 (TRPC1) had an important role in this process. The objective of this study was to examine the mechanism of AngII-induced Ca2+ entry and vascular endothelial hyperpermeability. Human umbilical vein endothelial cells (HUVECs) exposed to AngII exhibited dose-dependent increase in [Ca2+]i and endothelial permeability. Quantitative real-time RT-PCR and Western blotting showed that the level of TRPC1 expression had increased significantly at 12 h and at 24 h after treatment of HUEVCs with AngII. The expression of p65 was suppressed using an RNAi strategy. The results showed that the NF-κB signaling pathway and type-1 receptor of AngII was involved in AngII-induced TRPC1 upregulation. Moreover, knockdown of TRPC1 and NF-κB expression attenuates AngII-induced [Ca2+]i and endothelial permeability. NF-κB and TRPC1 have critical roles in AngII-induced Ca2+ entry and endothelial permeability.
Keywords: Angiotensin II; NF-κB; TRPC1; Human umbilical vein endothelial cell;

Structural and functional diversity of proopiomelanocortin in fish with special reference to barfin flounder by Akiyoshi Takahashi; Yuki Kobayashi; Masafumi Amano; Takeshi Yamanome (1374-1382).
Proopiomelanocortin (POMC) is a precursor of adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone (MSH), and endorphin (END). We have characterized POMC systems in barfin flounder. The results revealed unique aspects of POMC systems. Notable features in terms of pituitary functions are the occurrence of three functional POMC genes, the mutation of an essential sequence in the β-END in one of the genes, occurrence of α-MSH in addition to ACTH in the pars distalis of the pituitary, and expression of the three genes in a single cell. While MSHs stimulate pigment dispersion, expression of the POMC gene and plasma levels of MSH do not always respond to background color changes between black and white. The functions of MSHs in skin pigmentation are very unique, because acetylation at the N-terminal of α-MSH inhibits its pigment dispersing activity. This is in contrast to results from other teleosts and amphibians, in which acetylation increases the activity. In the skin, the POMC gene is expressed in the non-chromatophoric dermal cells, indicating that MSH produced in the skin de novo has a paracrine function. The detection of MSH peptides in skin extracts seems to show that the control of skin pigmentation by MSHs is twofold—endocrine control by the pituitary, and paracrine control by the skin itself. Thus, fish provide an interesting model to help understand the structural and functional diversity of POMC systems. In this review, we provide an overview of our recent studies on the characterization of molecules and biological significance of POMC systems in barfin flounder.
Keywords: Adrenocorticotropic hormone; Background color; Endorphin; Flounder; Gene expression; Melanocortin receptor; Melanocyte-stimulating hormone; Primary structure; Pituitary; Proopiomelanocortin;

Hypothalamic melanocortin signaling and leptin resistance—Perspective of therapeutic application for obesity-diabetes syndrome by Hiroaki Masuzaki; Tomohiro Tanaka; Ken Ebihara; Kiminori Hosoda; Kazuwa Nakao (1383-1386).
The adipocyte-derived hormone, leptin controls feeding behavior, augments fatty acid β-oxidation in the skeletal muscle, attenuates insulin secretion but enhances whole body insulin sensitivity and glucose disposal, thereby serving as a promising therapeutic candidate for the treatment of insulin resistance and dyslipidemia. Along with other researchers, we demonstrated the clinical efficacy and safety of leptin in the treatment of diabetes and dyslipidemia for patients with generalized lipodystrophy. However, the clinical application of leptin has been hampered by the notion that leptin does not fully exert its metabolic effects in human obesity and diet-induced obese rodents. We found that the activity of skeletal muscle AMP-activated protein kinase (AMPK) parallels hypothalamic leptin sensitivity and metabolic phenotype in transgenic mice overexpressing leptin. Our data indicate that the activation of skeletal muscle AMPK is mediated by the hypothalamic melanocortin pathway. In fact, intracerebroventricular administration of melanocortin agonist, MT-II in mice robustly overcomes high-fat diet-induced leptin resistance and ameliorates fuel dyshomeostasis and hyperphagia, with a concomitant recovery of AMPK activity in skeletal muscle. Conversely, AMPK/ACC phosphorylation by leptin was abrogated by the co-administration of melanocortin antagonist, SHU9119 and in the KKA y mice, which centrally express endogenous melanocortin antagonist. Importantly, high-fat diet-induced attenuation of AMPK/ACC phosphorylation in leptin-overexpressing transgenic mice was not reversed by central leptin per se, but was markedly recovered by MT-II. Our data provide evidence for the critical role of the central melanocortin system in leptin-skeletal muscle AMPK axis, and highlight the system as a therapeutic target for leptin insuffciency in obese humans.
Keywords: Leptin; AMP-activated protein kinase (AMPK); Type 4 melanocortin receptor (MC4R); Hypothalamus; Obesity;