Peptides (v.32, #4)

Minimal antimicrobial peptidic sequence from hemoglobin alpha-chain: KYR by Lucie Catiau; Johnatan Traisnel; Véronique Delval-Dubois; Nour-Eddine Chihib; Didier Guillochon; Naïma Nedjar-Arroume (633-638).
► Peptides derived from α 107–141 were antibacterial whatever the bacterial species. ► The most active peptide is the shortest one: α 137–141. ► Shortest the sequence of the peptide is, lowest the MIC is. ► KYR sequence is the minimal antimicrobial sequence from the hemoglobin α-chain. ► These active peptides were able to interact with cellular membrane.Hemoglobin is an animal protein described as a source of biologically active peptides. Peptic digestion of bovine hemoglobin alpha-chain allowed obtaining peptide fractions with antimicrobial activity. These peptides were purified by reverse-phase High-Performance Liquid Chromatography (HPLC) and characterized by mass spectrometry. The minimal inhibitory concentration and mode of action of these peptides were studied against five bacterial strains including Escherichia coli and Salmonella enteritidis as Gram-negative bacteria and Listeria innocua, Micrococcus luteus and Staphylococcus aureus as Gram-positive bacteria. The action aforementioned peptides were studied on artificial membranes as well. The most active peptides resulted to be the short ones. Consequently, the minimal peptidic sequence necessary for the antibacterial activity was clearly determined: KYR.
Keywords: Hemoglobin; Antimicrobial; MIC; Liposome; Minimal sequence;

► Suggesting that the administration of SPPs had a reducing effect on ROS generation. ► SPPs exerted protective effects on hydroxyl radical-induced DNA damage. ► SPPs inhibit the NF-κB pathway without altering MAP kinase activation. ► That H2O2 induced IκB phosphorylation was increased in cells treated with SPPs, where as phosphorylation of IKK is decreased. ► p38 kinase was suppressed by cotreatment with SPPs. ► SPP suppress activation of H2O2 induced NF-κB pathway.Two new peptides derived from seaweed pipefish Syngnathus schlegeli, SPP-1(QLGNLGV) and SPP-2 (SVMPVVA) were assessed for their ability to prevent hydrogen peroxide induced oxidative stress in human dermal fibroblasts (HDFs). Both peptides showed a significant hydroxyl radical scavenging activity when tested by ESR technique. And also the peptides effectively suppressed the hydrogen peroxide induced ROS production and DNA damage in HDF cells. Furthermore the two peptides increase the protein expression levels of intracellular antioxidant enzymes SOD1, GSH and catalase in hydrogen peroxide stressed HDF cells. At the cellular signaling level, SPPs block the NF-κB activation which may lead to the reduction of oxidative stress mediated damage of HDF cells. These finding indicate the potential antioxidant effects of SPPs as response to H2O2 stimulation.
Keywords: Seaweed pipefish; Oxidative stress; Antioxidant; Reactive oxygen species; NF-κB;

Purification and identification of a novel primitive secretory enzyme catalyzing the hydrolysis of imidazole-related dipeptides in the jawless vertebrate Lethenteron reissneri by Takahiro Oku; Seiichi Ando; Takehiko Hayakawa; Kyoko Baba; Ryuichiro Nishi; Kazuhiro Shiozaki; Shoji Yamada (648-655).
► An imidazole dipeptide-hydrolyzing enzyme is purified from brook lamprey muscle. ► The enzyme is a primitive M20A metallopeptidase distributed in jawless vertebrate. ► The lamprey secretory-type enzyme may be classified as a new peptidase of M20A.Imidazole-related dipeptides, such as carnosine and anserine, occur widely in skeletal muscles of jawed vertebrates. All of the known enzymes that catalyze the hydrolysis of these dipeptides belong to the M20A metallopeptidase subfamily; two secretory enzymes, serum carnosinase (EC and anserinase (EC, and one non-secretory enzyme, cytosolic nonspecific dipeptidase (EC Here we report the enzymatic characterization and molecular identification of an unidentified enzyme, which catalyzes the hydrolysis of these dipeptides, from the skeletal muscle of Far Eastern brook lamprey (Lethenteron reissneri). A 60-kDa subunit protein of the enzyme was purified to near homogeneity. We cloned two M20A genes from the skeletal muscle of Far Eastern brook lamprey; one was a secretory-type gene encoding for the 60-kD protein, and another was a non-secretory-type gene presumably encoding for cytosolic nonspecific dipeptidase. Our findings indicate that the purified enzyme is a N-glycosylated secretory M20A dipeptidase distributed specifically in the jawless vertebrate group, and may be derived from a common ancestor gene between serum carnosinase and anserinase. We propose that this dipeptidase is a novel secretory M20A enzyme and is classified as neither serum carnosinase nor anserinase.
Keywords: Anserinase; Anserine; Carnosine; Dipeptidase; Jawless vertebrate; Serum carnosinase; Lethenteron reissneri;

Purification, synthesis and characterization of AaCtx, the first chlorotoxin-like peptide from Androctonus australis scorpion venom by Ilhem Rjeibi; Kamel Mabrouk; Hend Mosrati; Caroline Berenguer; Hafedh Mejdoub; Claude Villard; Daniel Laffitte; Denis Bertin; L’Houcine Ouafik; José Luis; Mohamed ElAyeb; Najet Srairi-Abid (656-663).
► In this study we purified a new chlorotoxin-like peptide. ► We examined its effect on the U87 glioblastoma cell line. ► We find that this peptide is less active than chlorotoxin. ► The missing of acidic aminoacids is likely responsible for the lower activity. ► This will allow to design highly active anti-glioma peptides.AaCtx is the first chlorotoxin-like peptide isolated from Androctonus australis scorpion venom. Its amino acid sequence shares 70% similarity with chlorotoxin from Leiurus quinquestriatus scorpion venom, from which it differs by twelve amino acids. Due to its very low concentration in venom (0.05%), AaCtx was chemically synthesized. Both native and synthetic AaCtx were active on invasion and migration of human glioma cells. However, their activity was found to be lower than that of chlorotoxin. The molecular model of AaCtx shows that most of amino acids differing between AaCtx and chlorotoxin are localized on the N-terminal loop and the α-helix. Based on known compounds that block chloride channels, we suggest that the absence of negative charged amino acids on AaCtx structure may be responsible for its weak activity on glioma cells migration and invasion. This finding serves as a starting point for structure–function relationship studies leading to design high specific anti-glioma drugs.
Keywords: Chlorotoxin-like peptide; Human glioma; Scorpion venom; Androctonus australis; Structure–activity relationship;

Characterization of antimicrobial peptides in skin secretions from discrete populations of Lithobates chiricahuensis (Ranidae) from central and southern Arizona by J. Michael Conlon; Milena Mechkarska; Laurent Coquet; Thierry Jouenne; Jérôme Leprince; Hubert Vaudry; Jolanta Kolodziejek; Norbert Nowotny; Jay D. King (664-669).
► Characterization of six antimicrobial peptides present in skin secretions of populations of the leopard frog Lithobates chiricahuensis from distinct ranges in southern and central Arizona. ► Characterization of two peptides (brevinin-1CHc and palustrin-2CHa) present only in secretions of frogs from the southern range. ► Identification of esculentin-2CHa as a potent, broad spectrum antimicrobial peptide with therapeutic potential. ► Phylogenetic analysis of the North American leopard frogs based upon the primary structures of brevinin-1 peptides.Populations of the Chiricahua leopard frog Lithobates chiricahuensis (Ranidae) occupying regions in southern Arizona (southern range) are morphologically distinct from those from the Mogollon Rim of central Arizona (northern range) and a comparison of DNA sequences of mitochondrial genes has suggested that they may represent separate species. Peptidomic analysis of norepinephrine-stimulated skin secretions has led to the identification of six peptides with antimicrobial activity in samples from specimens from both groups. The primary structure of the peptides (esculentin-2CHa, ranatuerin-2CHa, -CHb, and -CHc, and brevinin-1CHa and -CHb) isolated from both southern and northern range frogs are identical consistent with the proposal that the two populations are conspecific. However, palustrin-2CHa and the atypical brevinin-1CHc (FFPTIAG*****LTKLFCA ITKKC), containing a five amino acid residue deletion, were identified only in secretions from southern range specimens. Consequently, there is some support for the proposal that the two populations are closely related but separate species but this support is relatively weak. Esculentin-2CHa (GFSSIFRGVAKFASKGLG KDLAKLGVDLVACKISKQC) displayed the highest antimicrobial potency (MIC ≤ 10 μM) against a variety of microorganisms and was only moderately hemolytic (LC50  = 150 μM). Cladistic analysis based upon the primary structures of brevinin-1 peptides indicates a close phylogenetic relationship between L. chiricahuensis, L. onca, and L. yavapaiensis.
Keywords: Frog skin; Antimicrobial peptide; Brevinin-1; Esculentin-2; Ranatuerin-2; Palustrin-2; Taxonomy;

Identification and characterization of antimicrobial peptides from the skin of the endangered frog Odorrana ishikawae by Eiko Iwakoshi-Ukena; Kazuyoshi Ukena; Aiko Okimoto; Miyuki Soga; Genya Okada; Naomi Sano; Tamotsu Fujii; Yoshiaki Sugawara; Masayuki Sumida (670-676).
▶ Nine antimicrobial peptides were isolated from the skin of the endangered frog Odorrana ishikawae. ▶These peptides belonging to five known families of antimicrobial peptides showed broad-spectrum of growth inhibitory activities against several microorganisms. ▶Each precursor protein has a common feature structure; a putative signal peptide, an N-terminal acidic spacer domain, a Lys-Arg-processing site, and an antimicrobial peptide at the C-terminus.The endangered anuran species, Odorrana ishikawae, is endemic to only two small Japanese Islands, Amami and Okinawa. To assess the innate immune system in this frog, we investigated antimicrobial peptides in the skin using artificially bred animals. Nine novel antimicrobial peptides containing the C-terminal cyclic heptapeptide domain were isolated on the basis of antimicrobial activity against Escherichia coli. The peptides were members of the esculentin-1 (two peptides), esculentin-2 (one peptide), palustrin-2 (one peptide), brevinin-2 (three peptides) and nigrocin-2 (two peptides) antimicrobial peptide families. They were named esculentin-1ISa, esculentin-1ISb, esculentin-2ISa, palustrin-2ISa, brevinin-2ISa, brevinin-2ISb, brevinin-2ISc, nigrocin-2ISa and nigrocin-2ISb. Peptide primary structures suggest a close relationship with the Asian odorous frogs, Odorrana grahami and Odorrana hosii. These antimicrobial peptides possessed a broad-spectrum of growth inhibition against five microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis and Candida albicans). Nine different cDNAs encoding the precursor proteins were also cloned and showed that the precursor proteins exhibited a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and an antimicrobial peptide at the C-terminus.
Keywords: Antimicrobial peptide; Innate immunity; Frog skin; Odorrana; Microorganism;

Effect of proline position on the antimicrobial mechanism of buforin II by Yang Xie; Eleanor Fleming; Jessica L. Chen; Donald E. Elmore (677-682).
► First study to consider importance of proline position in buforin II mechanism. ► Sole proline is in ideal position for buforin II membrane translocation. ► Sole proline is in ideal position for bufoirn II antimicrobial activity. ► Results emphasize interplay of translocation and DNA binding in buforin II mechanism. ► Results highlight importance of considering proline position in other peptides.Buforin II (BF2) is a histone-derived antimicrobial peptide that causes cell death by translocating across membranes and interacting with nucleic acids. It contains one proline residue critical for its function. Previous research found that mutations replacing proline lead to decreased membrane translocation and antimicrobial activity as well as increased membrane permeabilization. This study further investigates the role of proline in BF2's antimicrobial mechanism by considering the effect of changing proline position on membrane translocation, membrane permeabilization, and antimicrobial activity. For this purpose, four mutants were made with proline substitution (P11A) or relocation (P11A/G7P, P11A/V12P, P11A/V15P). These mutations altered the amount of helical content. Although antimicrobial activity correlated with the α-helical content for the peptides containing proline, membrane translocation did not. This observation suggests that factors in BF2's bactericidal mechanism other than translocation must be altered by these mutations. To better explain these trends we also measured the nucleic acid binding and membrane permeabilization of the mutant peptides. A comparison of mutant and wild type BF2 activity revealed that BF2 relies principally on membrane translocation and nucleic acid binding for antimicrobial activity, although membrane permeabilization may play a secondary role for some BF2 variants. A better understanding of the role of proline in the BF2 antimicrobial mechanism will contribute to the further design and development of BF2 analogs. Moreover, since proline residues are prevalent among other antimicrobial peptides, this systematic characterization of BF2 provides general insights that can promote our understanding of other systems.
Keywords: Buforin II; Histone-derived antimicrobial peptide; Proline; Membrane translocation; Nucleic acid binding;

Leishmanicidal activity of synthetic antimicrobial peptides in an infection model with human dendritic cells by José Julián Pérez-Cordero; José Manuel Lozano; Jimena Cortés; Gabriela Delgado (683-690).
► In this study we search of new therapeutic alternatives for leishmaniasis. ► We examine antimicrobial synthetic peptides as a molecules with pharmacological potential. ► We use dendritic cells in an in vitro model to study antileishmanial peptides. ► Dermaseptin shows a relevant antileishmanial activity related with nitric oxide.Different species of Leishmania are responsible for cutaneous, mucocutaneous or visceral leishmaniasis infections in millions of people around the world . The adverse reactions caused by antileishmanial drugs, emergence of resistance and lack of a vaccine have motivated the search for new therapeutic options to control this disease. Different sources of antimicrobial molecules are under study as antileishmanial agents, including peptides with antimicrobial and/or immunomodulatory activity, which have been considered to be potentially active against diverse species of Leishmania. This study evaluated the cytotoxicity on dendritic cells, hemolytic activity, leishmanicidal properties on Leishmania panamensis and Leishmania major promastigotes and effectiveness on parasite intracellular forms (dendritic cells infected with L. panamensis and L. major promastigotes), when each parasite in culture was exposed to different concentrations of a group of synthetic peptides with previously reported antimicrobial properties, which were synthesized based on their naturally occurring reported sequences. Dermaseptin, Pr-2 and Pr-3 showed inhibitory activity on the growth of L. panamensis promastigotes, while Andropin and Cecropin A (with a selectivity index of 4 and 40, respectively) showed specific activity against intracellular forms of this species. The activities of Andropin and Cecropin A were exclusively against the intracellular forms of the parasite, therefore indicating the relevance of these two peptides as potential antileishmanial agents. In the case of L. major promastigotes, Melittin and Dermaseptin showed inhibitory activity, the latter also showed a selectivity index of 8 against intracellular forms. These findings suggest Andropin, Cecropin A and Dermaseptin as potential therapeutic tools to treat New and Old World cutaneous leishmaniasis.
Keywords: Leishmania; Antimicrobial peptides; Dendritic cells;

► A novel antimicrobial peptide of 1676 Da was purified from venom of Bellamya bengalensis. Sequence of the peptide was determined by tandem mass spectrometry (MS/MS). ► The MIC and MBC values were 8 μg/ml and 16 μg/ml against Staphylococcus epidermidis resistant to ampicillin and chloramphenicol. ► Peptide increased the staphylococcal membrane permeability.Increasing tendency of clinical bacterial strains resistant to conventional antibiotics has being a great challenge to the public's health. Antimicrobial peptides, a new class of antibiotics is known to have the activity against a wide range of bacteria resistant to conventional antibiotics. An antimicrobial peptide of 1676 Da was purified from Bellamya bengalensis, a fresh water snail, using ultrafiltration and reversed phase liquid chromatography. The effect of this peptide on Staphylococcus epidermidis resistant to ampicillin and chloramphenicol was investigated; the MIC and MBC values were 8 μg/ml and 16 μg/ml, respectively. Complete sequence of the peptide was determined by tandem mass spectrometry (MS/MS). Further, peptide net charge, hydrophobicity and molecular modeling were evaluated in silico for better understanding the probable mechanisms of action. The peptide showed the specificity to bacterial membranes. Hence, this reported peptide revealed a promising candidate to contribute in the development of therapeutic agent for Staphylococcal infections.
Keywords: Antimicrobial peptides; Antibiotic resistant; Mass spectrometry; Staphylococcus epidermidis; Bellamya bengalensis;

S-thanatin in vitro prevents colistin resistance and improves its efficacy in an animal model of Pseudomonas aeruginosa sepsis by Oscar Cirioni; Guoqiu Wu; Linxian Li; Fiorenza Orlando; Carmela Silvestri; Roberto Ghiselli; Zilong Shen; Eleonora Gabrielli; Lucia Brescini; Giovanni Lezoche; Mauro Provinciali; Mario Guerrieri; Andrea Giacometti (697-701).
► Colistin and s-thanatin showed synergy. ► Serial exposure of bacterial strains to colistin alone resulted in enrichment of resistant derivatives. ► Addition of s-thanatin in the cultures reduced the increase in colistin MIC. ► Combination between colistin and s-thanatin showed significantly lowest lethality rates in vivo.An experimental study was performed to evaluate the interaction between s-thanatin and colistin both in vitro and in vivo, using two Pseudomonas aeruginosa strains with different patterns of susceptibilities. We evaluated whether selecting for colistin-resistant P. aeruginosa could be prevented in vitro by combining colistin with s-thanatin. The strains were serially exposed in broth to twofold stepwise increasing concentrations of colistin alone or in combination with a fixed concentration [0.25× minimum inhibitory concentration (MIC)] of s-thanatin. We also performed an in vitro synergy study. For in vivo studies, a mouse model of Pseudomonas sepsis has been used. Main outcome measures were lethality and quantitative blood cultures. Exposure to colistin alone gradually selected for Pseudomonas strains with an increased MIC. In vitro studies, s-thanatin showed a positive interaction with colistin, and was able to prevent its resistance. In vivo studies, s-thanatin combined with colistin exhibited the highest efficacy on all main outcome measurements. These results highlight the potential usefulness of this combination and provide a future therapeutic alternative in severe Pseudomonas infections.
Keywords: Pseudomonas; Colistin; Antimicrobial peptides; Sepsis;

Neuropeptide FF receptor antagonist, RF9, attenuates the fever induced by central injection of LPS in mice by Yi-qing Wang; Sheng-bin Wang; Jing-lin Ma; Jia Guo; Quan Fang; Tao Sun; Yan Zhuang; Rui Wang (702-706).
▶ NPFF does not modify the fever induced by central injection of LPS in mice. ▶ Neuropeptide FF receptors antagonist, RF9, attenuates the LPS-induced fever in mice. ▶ NPFF receptors activation is required for LPS to produce fever. ▶ NPFF systems participate in the control of acute neuroinflammation in conscious animals.The endogenous opioid system has been found to be involved in the fever caused by lipopolysaccharide (LPS). Neuropeptide FF (NPFF, FLFQPQRF-NH2) is an endogenous peptide known to modulate opioid activity, mainly in the central nervous system. Therefore, those data suggested a link between LPS-induced fever and NPFF systems. Using a model of acute neuroinflammation, we sought to determine the effects of NPFF systems on the fever induced by i.c.v. injection of LPS. Coinjected with different doses of NPFF (10 and 30 nmol), the fever of LPS (125 ng) was not modified. Interestingly, the selective NPFF receptors antagonist RF9 (30 nmol) injected into the third ventricle failed to induce significant effect, but it decreased the fever of LPS (125 ng) after cerebral administration in mice. These results suggest that NPFF receptors activation is required for LPS to produce fever. This interaction is the first evidence that NPFF systems participate in the control of acute neuroinflammation in conscious animals.
Keywords: Neuropeptide FF (NPFF); RF9; Lipopolysaccharide (LPS); Neuroinflammation; Fever; Mice;

The influence of μ-opioid receptor agonist and antagonist peptides on peripheral blood mononuclear cells (PBMCs) by E. Fiedorowicz; B. Jarmołowska; M. Iwan; E. Kostyra; R. Obuchowicz; M. Obuchowicz (707-712).
▶ All examined peptides caused no significant changes to PBMCs proliferation measured by the enzymatic activity of the cells in the long-term incubation. ▶ Amounts of incorporated BrdU reveal a stimulation of PBMCs proliferation by bovine and human β-casomorphin-7, which indicates a short-term influence of opioid peptides on PBMCs. ▶ The μ-opioid receptor agonist peptides were found to cause a significant increase in IL-4 secretion, an insignificant increase in IL-13, and a decrease of IFN-γ secretion, which suggest an activation of the Th2 pathway.Milk is one of the main source of biologically-active peptides that may function as regulatory substances called food hormones. After passing the gut–blood barrier, the μ-opioid receptor agonist and antagonist peptides may become the new factors influencing various functions of the human organism. The aim of the conducted research was to determine the influence of μ-opioid receptor agonist peptides: human and bovine β-casomorphin-7 (h/bBCM-7) and antagonistic peptides: casoxin-6 and- D (CXN-6/D) on proliferation and cytokine secretion of human peripheral blood mononuclear cells (PBMCs). The PBMCs proliferation was measured by the use of the BrdU test, which assesses the DNA synthesis activity and the WST-1 test which assesses the activity of mitochondrial dehydrogenase enzymes. The influence of all the investigated peptides on secretion of IL-4, IL-8, IL-13 and IFN-γ was determined by the use of the ELISA tests. Incubating the cells with the peptides has not caused any changes to their enzymatic activity, which has been proved by a WST-1 test. When using a BrdU test, however, it has been observed that there appear changes to proliferation of PBMCs correlated to amounts of bromodeoxyuridine incorporated into the cellular DNA. Moreover, changes to secretion of IL-4 and IL-13 by the cells under the influence of agonists were detected, as well as changes to secretion of IFN-gamma under the influence of all the examined substances. The obtained results provide information on immunomodulatory effects of food-derived opioid peptides, which may be of clinical significance especially in the case of allergic diseases in newborns.
Keywords: Opioid peptides; β-Casomorphin-7; μ-Opioid receptor; Peripheral blood mononuclear cells;

▶ Morphine withdrawal induces down-regulation of PPD mRNA and up-regulation of p-CREB. ▶ Multiple 100 Hz EA shows cumulative effect for suppressing opioid withdrawal syndrome. ▶ 100 Hz EA normalizes the PPD mRNA level by inhibiting the phosphorylation of CREB.Alleviating opiate withdrawal syndrome in addicts is a critical precondition to break away from drug and further to prevent reuse. Electroacupuncture (EA) was claimed to be effective for alleviating withdrawal syndrome, but the optimal protocol remained unclear. In the present study we found that (1) 100 Hz EA administered 12–24 h after the last morphine injection suppressed the withdrawal syndrome in rats, multiple sessions of EA were more effective than single session, with the after-effect lasting for at least 7 days. (2) A down-regulation of preprodynorphin (PPD) mRNA level was observed in spinal cord, PAG and hypothalamus 60 h after the last morphine injection, which could be reversed by multiple sessions, but not a single session of EA. (3) Accompanied with the decrease of PPD mRNA level, there was an up-regulation of p-CREB in the three CNS regions, which was abolished by 100 Hz EA treatment. The findings suggest that down-regulation of p-CREB and acceleration of dynorphin synthesis in spinal cord, PAG and hypothalamus may be implicated in the cumulative effect of multiple 100 Hz EA treatment for opioid detoxification.
Keywords: Morphine dependence; Withdrawal; Electroacupuncture; Preprodynorphin; Preproenkephalin; p-CREB;

Pharmacology of a new tritiated endomorphin-2 analog containing the proline mimetic cis-2-aminocyclohexanecarboxylic acid by Attila Keresztes; Erika Birkás; Annamária Páhi; Géza Tóth; Lidia Bakota; Károly Gulya; Mária Szücs (722-728).
► A new tritiated endomorphin-2 analog was synthesized in which Pro2 is replaced by 2-aminocyclohexanecarboxylic acid. ► It shows high specific radioactivity, μ-opioid receptor affinity, selectivity and enzymatic resistance. ► It is a good tool to envisage the distribution of μ-opioid receptors by using receptor autoradiography. ► It may be more suitable than the parent ligand for receptor–ligand interaction and topographic studies.As part of ongoing work aimed at generating proteolytically stable, readily applicable, radiolabeled endomorphin-2 (EM-2) analogs for elucidation of the topological requirements of peptide binding to μ-opioid receptors, we report here on the synthesis, radiolabeling, binding kinetics and binding site distribution of an EM-2 analog in which Pro2 is replaced by 2-aminocyclohexanecarboxylic acid, ACHC. [3H][(1S,2R)ACHC]2EM-2 (specific activity 63.49 Ci × mmol−1) bound specifically to its binding sites with high affinity (K D  = 0.55 ± 0.06 nM) and saturably, yielding a receptor density, B max of 151 ± 4 fmol × mg protein−1 in rat brain membranes. A similar affinity value was obtained in kinetic assays. Both Na+ and Gpp(NH)p decreased the affinity, proving the agonist character of the radioligand. Specific μ-opioid ligands displaced the radioligand with much higher affinities than did δ- and κ-ligands. The autoradiographic distribution of the binding sites of [3H][(1S,2R)ACHC]2EM-2 agreed well with the known locations of the μ-opioid receptors in the rat brain. In consequence of its high affinity, selectivity and enzymatic resistance , the new radioligand will be a good tool in studies of the topographical requirements of μ-opioid-specific peptide binding.
Keywords: Endomorphin; Peptide synthesis; 2-Aminocylcohexanecarboxylic acid (ACHC); Radiolabeling; Receptor binding; Autoradiography; Proteolytic stability;

► Peripheral nociceptin/orphanin FQ (N/OFQ) increases the density of mucosal mast cells in the gastric fundus ► N/OFQ promoted the activation of mucosal mast cells. ► N/OFQ decreased the contacts between mucosal mast cells and nerve fibers. ► The antagonist of N/OFQ receptor abolishes the stress-induced hyperplasia of mucosal mast cells.The endogenous neuropeptide nociceptin/orphanin FQ (N/OFQ) modulates behavioral and gastrointestinal responses to stress. Mucosal mast cells (MMCs) are primary mediators of stress-related responses in the gastrointestinal tract. We investigated the influence of N/OFQ and of the N/OFQ peptide (NOP) receptor antagonist, UFP-101, on MMCs in the rat gastric fundus. N/OFQ was infused subcutaneously for 52 h at 0.1, 1 and 10 μg/kg/h and at 1 μg/kg/h for 4 h, 52 h, 7 days and 14 days via Alzet osmotic minipumps. Density of MMCs and connective tissue mast cells (CTMCs) was assessed histochemically and immunohistochemically. Activation and location of MMCs were assessed by transmission electron microscopy. Contacts between MMCs and nerve elements were assessed by double immunofluorescence. N/OFQ (1 μg/kg/h) and UFP-101 (10 and 30 μg/kg/h) were infused subcutaneously in the absence and presence of acute cold-restraint stress and density of MMCs was assessed. Peripheral N/OFQ dose-dependently increased the density of MMCs, while not influencing CTMCs. The increasing effect was maintained up to 14 days following continuous infusion, while after termination of the 4-h infusion, the effect declined rapidly. The peptide promoted the activation of MMCs and their migration from the lamina propria toward the epithelial layer. The association between MMCs and nerve fibers was time-dependently down-regulated following N/OFQ infusion. The stress-induced hyperplasia of MMCs was not influenced by N/OFQ and abolished by UFP-101. UFP-101 alone was ineffective. The present results suggest that endogenous N/OFQ could be considered a potential component of the circuit neuropeptides-mast cells-stress.
Keywords: Nociceptin/orphanin FQ; UFP-101; Rat; Gastric fundus; Mucosal mast cells; Nerve fibers; Cold-restraint stress;

Modulation of gastric motility by brain-gut peptides using a novel non-invasive miniaturized pressure transducer method in anesthetized rodents by Guillaume Gourcerol; David W. Adelson; Mulugeta Million; Lixin Wang; Yvette Taché (737-746).
► Validation of a novel non-invasive method to monitor gastric motility in rodents. ► Chronic overexpression of CRF prevents central vagal activation of gastric contractions in mice. ► Laparotomy and cecal manipulation decrease phasic gastric contractions in both rats and mice.Acute in vivo measurements are often the initial, most practicable approach used to investigate the effects of novel compounds or genetic manipulations on the regulation of gastric motility. Such acute methods typically involve either surgical implantation of devices or require intragastric perfusion of solutions, which can substantially alter gastric activity and may require extended periods of time to allow stabilization or recovery of the preparation. We validated a simple, non-invasive novel method to measure acutely gastric contractility, using a solid-state catheter pressure transducer inserted orally into the gastric corpus, in fasted, anesthetized rats or mice. The area under the curve of the phasic component (pAUC) of intragastric pressure (IGP) was obtained from continuous manometric recordings of basal activity and in responses to central or peripheral activation of cholinergic pathways, or to abdominal surgery. In rats, intravenous ghrelin or intracisternal injection of the thyrotropin-releasing hormone agonist, RX-77368, significantly increased pAUC while coeliotomy and cacal palpation induced a rapid onset inhibition of phasic activity lasting for the 1-h recording period. In mice, RX-77368 injected into the lateral brain ventricle induced high-amplitude contractions, and carbachol injected intraperitoneally increased pAUC significantly, while coeliotomy and cecal palpation inhibited baseline contractile activity. In wild-type mice, cold exposure (15 min) increased gastric phasic activity and tone, while there was no gastric response in corticotropin releasing factor (CRF)-overexpressing mice, a model of chronic stress. Thus, the novel solid-state manometric approach provides a simple, reliable means for acute pharmacological studies of gastric motility effects in rodents. Using this method we established in mice that the gastric motility response to central vagal activation is impaired under chronic expression of CRF.
Keywords: Cold; CRF; Ghrelin; Intragastric pressure; Surgery; TRH agonist;

Uptake, Transport and Regulation of JBP485 by PEPT1 in vitro and in vivo by Zhihao Liu; Changyuan Wang; Qi Liu; Qiang Meng; Jian Cang; Lin Mei; Taiichi Kaku; Kexin Liu (747-754).
Display Omitted► JBP485 is a substrate of PEPT1 and its absorption is actively mediated by PEPT1. ► The mRNA expression of PEPT1 is increased when treated with JBP485. ► The uptake of JBP485 could be changed when coadministered with other drugs.Cyclo-trans-4-l-hydroxyprolyl-l-serine (JBP485) is a dipeptide with anti-hepatitis activity that has been chemically synthesized. Previous experiments in rats showed that JBP485 was well absorbed by the intestine after oral administration. The human peptide transporter (PEPT1) is expressed in the intestine and recognizes compounds such as dipeptides and tripeptides. The purposes of this study were to determine if JBP485 acted as a substrate for intestinal PEPT1, and to investigate the characteristics of JBP485 uptake and transepithelial transport by PEPT1. The uptake of JBP485 was pH dependent in human intestinal epithelial cells Caco-2. And JBP485 uptake was also significantly inhibited by glycylsarcosine (Gly-Sar, a typical substrate for PEPT1 transporters), JBP923 (a derivative of JBP485), and cephalexin (CEX, a β-lactam antibiotic and a known substrate of PEPT1) in Caco-2 cells. The rate of apical-to-basolateral transepithelial transport of JBP485 was 1.84 times higher than that for basolateral-to-apical transport. JBP485 transport was obviously inhibited by Gly-Sar, JBP923 and CEX in Caco-2 cells. The uptake of JBP485 was increased by verapamil but not by cyclosporin A (CsA) and inhibited by the presence of Zn2+ or the toxic metabolite of ethanol, acetaldehyde (AcH) in Caco-2 cells. The in vivo uptake of JBP485 was increased by verapamil and decreased by ethanol in vivo, which was consisted with the in vitro study. PEPT1 mRNA levels were enhanced after exposure of the cells to JBP485 for 24 h, compared to control. In conclusion, JBP485 was actively transported by the intestinal oligopeptide transporter PEPT1. This mechanism is likely to contribute to the rapid absorption of JBP485 by the gastrointestinal tract after oral administration.
Keywords: JBP485; Uptake; Transport; Regulation; PEPT1;

▶ N-terminally PEGylated obestatin is approximately 3 times more stable than native obestatin. ▶ Chronic treatment with N-terminally PEGylated obestatin reduced plasma triglycerides by 40%. ▶ Obestatin treatment did not alter food/fluid intake, body weight, or tissue morphometrics. ▶ Obestatin may play a role in modulating physiological lipid metabolism, but does not affect fat deposition.Obestatin (OB(1-23) is a 23 amino acid peptide encoded on the preproghrelin gene, originally reported to have metabolic actions related to food intake, gastric emptying and body weight. The biological instability of OB(1-23) has recently been highlighted by studies demonstrating its rapid enzymatic cleavage in a number of biological matrices. We assessed the stability of both OB(1-23) and an N-terminally PEGylated analog (PEG-OB(1-23)) before conducting chronic in vivo studies. Peptides were incubated in rat liver homogenate and degradation monitored by LC-MS. PEG-OB(1-23) was approximately 3-times more stable than OB(1-23). Following a 14 day infusion of Sprague–Dawley rats with 50 nmol/kg/day of OB(1-23) or a N-terminally PEGylated analog (PEG-OB(1-23)), we found no changes in food/fluid intake, body weight and plasma glucose or cholesterol between groups. Furthermore, morphometric liver, muscle and white adipose tissue (WAT) weights and tissue triglyceride concentrations remained unaltered between groups. However, with stabilized PEG-OB(1-23) we observed a 40% reduction in plasma triglycerides. These findings indicate that PEG-OB(1-23) is an OB(1-23) analog with significantly enhanced stability and suggest that obestatin could play a role in modulating physiological lipid metabolism, although it does not appear to be involved in regulation of food/fluid intake, body weight or fat deposition.
Keywords: Obestatin; Lipids; Peptide; Peptide analog; Triglycerides;

Repeated glucoprivation delayed hyperphagic responses while activating neuropeptide Y neurons in rats by Yoshiharu Ozawa; Hiroshi Arima; Minemori Watanabe; Hiroshi Shimizu; Yoshihiro Ito; Ryoichi Banno; Yoshihisa Sugimura; Nobuaki Ozaki; Hiroshi Nagasaki; Yutaka Oiso (763-769).
▶ Glucoprivation induced robust feeding in rats. ▶ Glucoprivation increased NPY gene expression. ▶ Repeated glucoprivation delayed feeding responses while enhancing NPY expression.It is well known that glucoprivation induces the release of counterregulatory hormones such as glucagon, and that the response is attenuated when the stimuli are repeated. Glucoprivation also activates orexigenic neurons and induces hyperphagic responses, although it remains unclear whether these responses are attenuated in repeated glucoprivation. In this study, we examined time course changes in feeding as well as activities of orexigenic neuropeptide Y (NPY) neurons in repeated glucoprivation in rats. Either 2-deoxy-d-glucose (2DG), which blocks glucose utilization, or isotonic saline (control) was injected subcutaneously to rats for 14 days, and food consumption for 1 and 2 h after injection was monitored throughout the experiment. While 2DG injection induced robust feeding responses during the first 1 h after injection, the response was gradually attenuated and the food consumption was significantly less on days 12–14 compared to that on day 1. On the other hand, food consumption during 2 h after 2DG injection was not changed significantly for 14 days. The transcriptional activities of NPY neurons in the arcuate nucleus and C1/A1 region of the hindbrain, measured by intronic in situ hybridization, were significantly enhanced after repeated 2DG injection for 14 days, while the feeding responses to intracerebroventricular injection of NPY were significantly less in the 2DG-repeated group compared to the saline-repeated group. It is thus demonstrated that repeated glucoprivation delayed hyperphagic responses while activating NPY neurons in rats. Our data also suggest that decreased feeding responses to NPY might be at least partially responsible for the delayed response.
Keywords: Repeated glucoprivation; Neuropeptide Y; Arcuate nucleus; Hindbrain; Food intake; c-fos;

Effects of glycine-extended and serine13-phosphorylated forms of peptide YY on food intake in rats by Roger Reidelberger; Alvin Haver; Prasanth Chelikani; David A. Keire; Joseph R. Reeve (770-775).
► PYY(3-36)-NH2 is significantly more potent than PYY(1-36)-NH2 in reducing food intake. ► Gly-extended forms of PYY are significantly less potent than non-extended forms. ► Ser13-phosphorylation decreases the anorexigenic potency of PYY(3-36)-NH2. ► Ser13-phosphorylation does not affect the anorexigenic potency of PYY(1-36)-NH2.The gut hormone peptide YY(3-36)-amide [PYY(3-36)-NH2] is significantly more potent than PYY(1-36)-NH2 in reducing food intake in rats and humans. Other Gly-extended and Ser13-phosphorylated PYY forms have been detected or predicted based upon known cellular processes of PYY synthesis and modification. Here we compared the effects of 3-h IV infusion of PYY(1-36)-NH2, PYY(3-36)-NH2, PYY(1-36)-Gly-OH, PYY(3-36)-Gly-OH, Ser13(PO3)-PYY(1-36)-NH2, Ser13(PO3)-PYY(3-36)-NH2, Ser13(PO3)-PYY(1-36)-Gly-OH, and Ser13(PO3)-PYY(3-36)-Gly-OH during the early dark period on food intake in freely feeding rats. PYY(3-36)-NH2 and Ser13(PO3)-PYY(3-36)-NH2 reduced food intake similarly at 50 pmol/kg/min, while only PYY(3-36)-NH2 reduced food intake at 15 pmol/kg/min. PYY(1-36)-NH2 and Ser13(PO3)-PYY(1-36)-NH2 reduced food intake similarly at 50 and 150 pmol/kg/min. In contrast, PYY(1-36)-Gly-OH, PYY(3-36)-Gly-OH, Ser13(PO3)-PYY(3-36)-Gly-OH, and Ser13(PO3)-PYY(1-36)-Gly-OH had no effect on food intake at doses of 50 or 150 pmol/kg/min. Taken together, these results indicate that (i) PYY(3-36)-NH2 is significantly more potent than PYY(1-36)-NH2 in reducing food intake, (ii) Gly-extended forms of PYY are significantly less potent than non-extended forms, and (iii) Ser13-phosphorylation of PYY(3-36)-NH2 decreases the anorexigenic potency PYY(3-36)-NH2, but not PYY(1-36)-NH2. Thus, PYY(3-36)-NH2 appears to be the most potent PYY form for reducing food intake in rats.
Keywords: Peptide YY(3-36); Molecular forms; Satiety;

Nitric oxide is a central component in neuropeptide regulation of appetite by John E. Morley; Susan A. Farr; Rebecca L. Sell; Stanley M. Hileman; William A. Banks (776-780).
► This study compares the effect of the food modulators ghrelin, NPY and CCK in NOS KO mice. ► Ghrelin did not increase food intake in the homozygous NOS KO mice while wild type controls did experience increased food intake. ► Nitric oxide did show to be a central mediator in food intake in this study.In recent years, there have been a large number of neuropeptides discovered that regulate food intake. Many of these peptides regulate food intake by increasing or decreasing nitric oxide (NO). In the current study, we compared the effect of the food modulators ghrelin, NPY and CCK in NOS KO mice. Satiated homozygous and heterozygous NOS KO mice and their wild type controls were administered ghrelin ICV. Food intake was measured for 2 h post injection. Ghrelin did not increase food intake in the homozygous NOS KO mice compared to vehicle treated NOS KO mice, whereas food intake was increased in the wild type controls compared to vehicle treated wild type controls. NPY was administered ICV and food intake measured for 2 h. Homozygous NOS KO mice showed no increase in food intake after NPY administration, whereas the wild type controls did. In our final study, we administered CCK intraperitoneally to homozygous and heterozygous NOS KO mice and their wild type controls after overnight food deprivation. Food intake was measured for 1 h after injection. CCK inhibited food intake in wild type mice after overnight food deprivation, however, CCK failed to inhibit food intake in the NOS KO mice. The heterozygous mice showed partial food inhibition after the CCK. The current results add further support to the theory that NO is a central mediator in food intake.
Keywords: Nitric oxide; NPY; CCK; Ghrelin; Appetite; Food intake; NOS KO mice;

Glucose-dependent insulinotropic peptide receptor expression in the hippocampus and neocortex of mesial temporal lobe epilepsy patients and rats undergoing pilocarpine induced status epilepticus by Cláudia P. Figueiredo; Victor L.S. Antunes; Eduardo L.G. Moreira; Nelson de Mello; Rodrigo Medeiros; Gabriella Di Giunta; Bruno Lobão-Soares; Marcelo Linhares; Katia Lin; Tânia L. Mazzuco; Rui D.S. Prediger; Roger Walz (781-789).
▶ GIPR present a ubiquitous distribution in the mammalian brain. ▶ The GIPR expression was inversely related to neuronal degeneration. ▶ Increase in GIPR expression was observed in rat brain after pilocarpine treatment. ▶ The increase in GIPR expression may be associated with epileptogenesis processes. ▶ The GIPR expression was found in human hippocampus and neocortex.The glucose-dependent insulinotropic peptide receptor (GIPR) has been implicated with neuroplasticity and may be related to epilepsy. GIPR expression was analyzed by immunohistochemistry in the hippocampus (HIP) and neocortex (Cx) of rats undergoing pilocarpine induced status epilepticus (Pilo-SE), and in three young male patients with left mesial temporal lobe epilepsy related to hippocampal sclerosis (MTLE-HS) treated surgically. A combined GIPR immunohistochemistry and Fluoro-Jade staining was carried out to investigate the association between the GIPR expression and neuronal degeneration induced by Pilo-SE. GIPR was expressed in the cytoplasm of neurons from the HIP CA subfields, dentate gyrus (DG) and Cx of animals and human samples. The GIPR expression after the Pilo-SE induction increases significantly in the HIP after 1 h and 5 days, but not after 12 h or 50 days. In the Cx, the GIPR expression increases after 1 h, 12 h and 5 days, but not 50 days after the Pilo-SE. The expression of GIPR 12 h after Pilo-SE was inversely proportional to the Fluoro-Jade staining intensity. In the human tissue, GIPR expression patterns were similar to those observed in chronic Pilo-SE animals. No Fluoro-Jade stained cells were observed in the human sample. GIPR is expressed in human HIP and Cx. There was a time and region dependent increase of GIPR expression in the HIP and Cx after Pilo-SE that was inversely associated to neuronal degeneration.
Keywords: Mesial temporal lobe epilepsy; Hippocampal sclerosis; Glucose-dependent insulinotropic peptide receptor;

▶ The chimeric ITF containing CETP B cell epitope can form the correct trefoil structure. ▶ The chimeric ITF can resist the proteases digestion in vitro assay. ▶ The chimeric ITF can induce both of mucosal and systemic immune response in rabbits after oral administration. ▶ The anti-CETP antibodies induced by oral vaccination with cITF can inhibit CETP activity, increase HDL-C, decrease LDL-C and inhibit atherosclerosis in rabbits.Vaccination against cholesteryl ester transfer protein (CETP) is proven to be effective for inhibiting atherosclerosis in animal models. In this study, the proteases-resistant intestinal trefoil factor (TFF3) was used as a molecular vehicle to construct chimeric TFF3 (cTFF3) containing CETP B cell epitope and tetanus toxin helper T cell epitope. It was found that cTFF3 still preserved a trefoil structure, and can resist proteases digestion in vitro. After oral immunization with cTFF3, the CETP-specific IgA and IgG could be found in intestine lavage fluid and serum, and the anti-CETP antibodies could inhibit partial CETP activity to increase high-density lipoprotein cholesterol, decrease low-density lipoprotein cholesterol, and inhibit atherosclerosis in animals. Therefore, TFF3 is a potential molecular vehicle for developing oral peptide vaccines. Our research highlights a novel strategy for developing oral peptide vaccines in the future.
Keywords: Intestinal trefoil factor; Oral peptide vaccine; CETP; Atherosclerosis;

Central and peripheral forms of C-type natriuretic peptide (CNP): Evidence for differential regulation in plasma and cerebrospinal fluid by Belinda J. Schouten; Timothy C.R. Prickett; Ashley A. Hooper; Gary J. Hooper; Timothy G. Yandle; A. Mark Richards; Eric A. Espiner (797-804).
► CSF concentrations of NTproCNP are greatly elevated (>140-fold over those) those of CNP in CSF. ► NTproCNP and CNP are more closely correlated in CSF than in plasma. ► Systemic and CSF CNP are inversely correlated suggesting differential regulation. ► CSF concentrations of CNP are unrelated to neprilysin activity or cGMP in CSF.Aminoterminal proCNP (NTproCNP), a stable product of CNP gene expression and readily measured in human plasma, provides a new approach to studies of CNP which is rapidly degraded at source. CNP is detectable in human CSF but the presence and proportions of NTproCNP in CSF are unknown. Since CNP is widely expressed throughout the CNS, we hypothesized that the ratio of NTproCNP to CNP in CSF is greatly increased when compared to plasma and that CSF CNP peptides may contribute to their concentrations in the systemic circulation. Concurrent plasma and CSF concentrations of CNP forms were measured in 51 subjects undergoing spinal anesthesia for arranged orthopedic procedures. Elevated concentrations of NTproCNP (1045 ± 359 pmol/L), characterized by HPLC-RIA, were found in CSF and greatly exceeded those of CNP (7.9 ± 3.2 pmol/L). The ratio of NTproCNP to CNP in CSF (145 ± 55) was much higher than in plasma (31 ± 27). A significant inverse relation was found between plasma and CSF CNP concentrations (r  = −0.29, p  < 0.05). cGMP and neprilysin were unrelated to CNP levels in CSF. We conclude that CNP is differentially regulated across the brain in normal health. Despite markedly elevated levels of NTproCNP in CSF, it is unlikely that these contribute to systemic levels in healthy adults. Identifying NTproCNP as the dominant CNP form in CSF opens up the possibility of its use in future studies exploring CNP regulation within the CNS and possible applications in the diagnosis and monitoring of subjects with central neural disorders.
Keywords: CSF; NTproCNP; cGMP; HPLC;

Distinct systemic distribution of salusin-α and salusin-β in the rat by Noriko Suzuki; Masayoshi Shichiri; Tae Tateno; Kengo Sato; Yukio Hirata (805-810).
▶ Immunoreactive salusin-α showed distinct distribution from salusin-β in the rat. ▶ Immunoreactive salusin-β was detected in rat urine and tissues. ▶ Salusin-α and salusin-β are conserved in the rat.Salusin-α and salusin-β are multifunctional bioactive peptides that were initially predicted using in silico analyses. These peptides should be concomitantly biosynthesized from prosalusin in humans. However, little information is available yet on the biosynthesis and mode of presence of salusin-α and salusin-β in non-human species. In the present study, we examined whether salusin-α and salusin-β are conserved in the rat and whether salusin-α and salusin-β show distinct systemic distributions. Immunohistochemical analysis of rat tissues using a specific anti-rat salusin-α antibody detected immunoreactivity extensively in neuronal cells and fibers, and abundantly in the epithelial tissues throughout the organs. This distribution contrasts sharply with that of salusin-β, which is mainly localized to the neuroendocrine and hematopoietic systems. Western blot analysis of rat spleen extracts showed the presence of cleaved fragments corresponding to putative rat salusin-α. Reverse-phase and gel filtration high performance liquid chromatography analyses coupled with radioimmunoassay detection of rat urine extracts revealed a major immunoreactive component that co-eluted with synthetic putative rat salusin-β. These data support the processing of rat prosalusin into salusin-α and salusin-β despite absent dibasic amino acids between the two.
Keywords: Salusin; Immunohistochemistry; Western blotting; Processing; High performance liquid chromatography; Rat;

Characterization of prolactin-releasing peptide: Binding, signaling and hormone secretion in rodent pituitary cell lines endogenously expressing its receptor by Jana Maixnerová; Andrea Špolcová; Miroslava Pýchová; Miroslava Blechová; Tomáš Elbert; Martina Řezáčová; Blanka Železná; Lenka Maletínská (811-817).
▶ Three rodent pituitary cell lines possessed high levels of PrRP receptor. ▶ PrRP31 and PrRP20 were equipotent in binding, while PrRP13 showed lower potency. ▶ All PrRP analogs stimulated prolactin release and MAPK/ERK1/2 and CREB signaling. ▶ Only PrRP31 and PrRP20 had a significant central anorexigenic effect in mice.The recently discovered prolactin-releasing peptide (PrRP) binds to the PrRP receptor and is involved in endocrine regulation and energy metabolism. However, its main physiological role is currently unknown. Two biologically active isoforms of PrRP exist: the 31 (PrRP31) and the 20 (PrRP20) amino acid forms, which both contain a C-terminal Phe amide sequence. In the present study, the PrRP receptor was immunodetected in three rodent tumor pituitary cell lines: GH3, AtT20 and RC-4B/C cells. The saturation binding of radioiodinated PrRP31 to intact cells demonstrated a K d in the 10−9  M range and a B max in the range of tens of thousands binding sites per cell. For binding to RC-4B/C cells, both PrRP31 and PrRP20 competed with 125I-PrRP31 with a similar K i. The C-terminal analog PrRP13 showed lower binding potency compared to PrRP31 and PrRP20. All PrRP analogs increased the phosphorylation of MAPK/ERK1/2 (mitogen-activated phosphorylase/extracellular-regulated kinase) and CREB (cAMP response element-binding protein) in RC-4B/C cells. Additionally, prolactin release was induced by the PrRP analogs in a dose-dependent manner in RC-4B/C cells. Finally, food intake after intracerebroventricular administration of PrRP analogs in fasted mice was followed. Both PrRP31 and PrRP20 decreased food intake, but PrRP13 did not show significant effect. Studies on pituitary cell lines expressing the PrRP receptor are more physiologically relevant than those on cells transfected with the receptor. This cell type can be used as a model system for pharmacological studies searching for PrRP antagonists and stable effective PrRP agonists, as these drugs may have potential as anti-obesity agents.
Keywords: PrRP; Pituitary cell lines; Binding; MAPK/ERK1/2; Prolactin; Food intake;

The role of neuropeptide S and neuropeptide S receptor 1 in regulation of respiratory function in mice by Hongyan Zhu; Charles Perkins; Melissa K. Mingler; Fred D. Finkelman; Marc E. Rothenberg (818-825).
Npsr1 does not have a dominant direct role in the development of experimental asthma. ▶ Intracerebroventricular administration of NPS increases respiratory frequency in an NPSR1-dependent manner at baseline or in response to methacholine challenge. ▶ Intracerebroventricular administration of NPS does not change airway mechanics in response to methacholine. ▶ Respiratory changes induced by intracerebroventricular administration of NPS are part of fight-or-flight response.Genome-wide screening and positional cloning have linked neuropeptide S receptor 1 (NPSR1) with asthma and airway hyperresponsiveness. However, the mechanism by which NPSR1 regulates pulmonary responses remains elusive. Because neuropeptide S and its receptor NPSR1 are expressed in brain regions that regulate respiratory rhythm, and Npsr1-deficient mice have impaired stress and anxiety responses, we aimed to investigate whether neuropeptide S and NPSR1 regulate respiratory function through a central-mediated pathway. After neuropeptide S intracerebroventricular administration, respiratory responses of wildtype and Npsr1-deficient mice were monitored by whole-body or invasive plethysmography with or without serial methacholine inhalation. Airway inflammatory and hyperresponsiveness were assessed in allergen-challenged (ovalbumin or Aspergillus fumigatus) Npsr1-deficient mice. Analysis of breathing patterns by whole-body plethysmography revealed that intracerebroventricular neuropeptide S, as compared with the artificial cerebral spinal fluid control, increased respiratory frequency and decreased tidal volume in an NPSR1-dependent manner but did not affect enhanced pause. Following serial methacholine inhalation, intracerebroventricular neuropeptide S increased respiratory frequency in wildtype mice, but not in Npsr1-deficient mice, and had no effect on tidal volume. Intracerebroventricular neuropeptide S significantly reduced airway responsiveness to methacholine as measured by whole-body plethysmography. Npsr1 deletion had no impact on airway inflammation or hyperresponsiveness in ovalbumin- or A. fumigatus-induced experimental asthma. Our results demonstrate that neuropeptide S and NPSR1 regulate respiratory function through a central nervous system-mediated pathway.
Keywords: Respiration; Brain; Neuropeptide S; Neuropeptide S receptor 1; Panting; Stress;

JmjC-domain-containing histone demethylases of the JMJD1B type as putative precursors of endogenous DSIP by Inessa I. Mikhaleva; Igor A. Prudchenko; Vadim T. Ivanov; Vladislav B. Voitenkov (826-831).
► The biological origin of delta-sleep inducing peptide (DSIP) remains enigmatic. ► DSIP-like sequences were searched in nucleotide and protein databases for mammals. ► The WKGGNASGE sequence was found at 324–332 sites of Lys-specific demethylase 3B. ► This human JmjC-domain-containing histone demethylase is encoded by JMJD1B gene. WKGGNASGE and DSIP exhibited similar potency in a preliminary biological study.Delta sleep inducing peptide (WAGGDASGE, DSIP) is a well known multifunctional regulatory peptide. Numerous studies have confirmed its stress-protective and adaptive activity which is independent of the origin or nature of the stress or other harmful factors. However, the biosynthetic origin of DSIP remains obscure, since nothing is known of its protein precursor(s) and their encoding gene(s). We have performed a comprehensive analysis of available gene and protein databases for homologous peptide sites within mammalian resources including man. A family of Jumonji C (JmjC)-domain-containing histone demethylases was shown to contain a sequence fragment closely homologous to DSIP. One type of these ubiquitous and phylogenetically ancient proteins encoded by JMJD1B gene includes the WKGGNASGE sequence that differs from DSIP by only 2 amino acid residues in positions 2 and 5. The respective peptide was synthesized and its biological effects were evaluated in a preliminary way in the forced swimming and antitoxic tests. We suggest that the histone demethylases of the JmjC-group containing DSIP-related region can be considered as possible protein precursors of endogenous peptides with DSIP-like activity.
Keywords: Delta sleep inducing peptide (DSIP); Origin; K,N-DSIP; Protein precursors; Encoding gene; Activity; Synthesis; JmjC-domain-containing histone demethylases;

Microcin J25-Ga induces apoptosis in mammalian cells by inhibiting mitochondrial RNA-polymerase by María V. Niklison-Chirou; Fernando Dupuy; Lucila Saavedra; Elvira Hebert; Claudia Banchio; Carlos Minahk; Roberto D. Morero (832-834).
► COS-7 cells undergo apoptosis upon incubation with MccJ25-Ga but not with native microcin J25. ► MccJ25-Ga displayed a strong inhibitory activity on mitochondrial RNA synthesis.MccJ25, an antimicrobial peptide, was unable to cause apoptosis of COS-7 cells in spite of inducing reactive-oxygen species overproduction as well as cytochrome c release from isolated mitochondria. Surprisingly, MccJ25-Ga, an amidated variant of MccJ25 that displays similar anti-mitochondrial effects, did induce apoptosis in COS-7. The only difference found between the activities of these peptides was the unpredicted inhibition of mitochondrial RNA synthesis by MccJ25-Ga. These results led us to hypothesize that both mitochondrial RNA polymerase and mitochondrial membrane might be the molecular targets of MccJ25-Ga in mitochondria and this combined effect may lead to apoptosis.
Keywords: Microcin; Mitochondria; RNA-polymerase; Apoptosis;

Whey proteins as source of dipeptidyl dipeptidase IV (dipeptidyl peptidase-4) inhibitors by Giovanni Tulipano; Valeria Sibilia; Anna Maria Caroli; Daniela Cocchi (835-838).
► Whey proteins can reduce postprandial glucose levels and stimulate insulin release. ► Administration of whey protein was associated with higher levels of intact incretins. ► Dipeptidyl peptidase-4 (DPP-4) is the main enzyme responsible for incretin degradation. ► β-Lactoglobulin is the major whey protein found in the milk of cows and other ruminants. ► Ile-Pro-Ala (IPA) is a bioactive peptide generated by proteolysis of bovine β-lactoglobulin. ► Our results showed that IPA can be regarded as a moderate DPP-4 inhibitor.Preclinical and clinical studies suggest that whey proteins can reduce postprandial glucose levels and stimulate insulin release in healthy subjects and in subjects with type 2 diabetes by reducing dipeptidyl peptidase-4 (DPP-4) activity in the proximal bowel and hence increasing intact incretin levels. Our aim was to identify DPP-4 inhibitors among short peptides occurring in hydrolysates of β-lactoglobulin, the major whey protein found in the milk of ruminants. We proved that the bioactive peptide Ile-Pro-Ala can be regarded as a moderate DPP-4 inhibitor.

Orexins/hypocretins increase the promoter activity of selective steroidogenic enzymes by Sonja M. Kagerer; Christine Eichholz; Olaf Jöhren (839-843).
► The increased promoter activities of CYP21, HSD3B2, CYP11B1, and CYP11B2 genes in human NCI-H295R-cells after orexin A and orexin B treatment demonstrate the regulation of these genes at the transcriptional level by orexins. ► In contrast, orexin has no effect on CYP21, HSD3B2, CYP11B1, and CYP11B2 mRNA stability. ► These results substantiate a role of orexins in adrenal gland regulation of steroid synthesis.Orexins (hypocretins) regulate multiple physiological functions, including central regulation of energy homeostasis and sleep–wake behavior but also peripheral hormonal actions. Recent data suggest specific effects of orexins at adrenal glands. To further assess the mechanism by which orexins regulate steroidogenesis we analyzed the effect of orexin A and B on the transcriptional activity of the luciferase reporter gene driven by the human steroid 21-hydroxylase (CYP21), 3β-hydroxysteroid dehydrogenase (HSD3B2), 11β-hydroxylase (CYP11B1), and aldosterone synthase (CYP11B2) gene promoter regions. After transient transfection of the reporter gene constructs into human NCI H295R cells, treatment with orexin A and B for 6 and 12 h increased the promoter activity of the CYP11B2, HSD3B2 and, to a lesser extend, CYP21 genes. The activity of the CYP11B1 was increased by both orexins after 3 h of treatment. Compared to the effects of forskolin or angiotensin II, however, the effect of orexins on the transcriptional activity of the steroidogenic enzyme genes was moderate. Our results suggest that orexins increase the expression of steroidogenic enzymes at the transcriptional level and that orexins play a role in the long term regulation of adrenal steroid production.