Peptides (v.27, #12)
Contents Continued (VI).
IFC Editorial Advisory Board (CO2).
CRF receptors in the rodent and human cardiovascular systems: Species differences by Beatrice Waser; Ruth Rehmann; Jean Rivier; Wylie Vale; Jean Claude Reubi (3029-3038).
CRF has powerful receptor-mediated cardiovascular actions. To evaluate the precise distribution of CRF receptors, in vitro CRF receptor autoradiography with 125I-[Tyr0, Glu1, Nle17]-sauvagine or [125I]-antisauvagine-30 was performed in the rodent and human cardiovascular system. An extremely high density of CRF2 receptors was detected with both tracers in vessels of rodent lung, intestine, pancreas, mesenterium, kidney, urinary bladder, testis, heart, brain, and in heart muscle. In humans, CRF2 receptors were detected with 125I- antisauvagine-30 at low levels in vessels of kidneys, intestine, urinary bladder, testis, heart and in heart muscle, while only heart vessels were detected with 125I-[Tyr0, Glu1, Nle17]-sauvagine. This is the first extensive morphological study reporting the extremely wide distribution of CRF2 receptors in the rodent cardiovascular system and a more limited expression in man, suggesting a species-selective CRF receptor expression.
Keywords: Cardiovascular system; CRF receptors; Species differences;
Orpotrin: A novel vasoconstrictor peptide from the venom of the Brazilian Stingray Potamotrygon gr. orbignyi by Katia Conceição; Katsuhiro Konno; Robson L. Melo; Elineide E. Marques; Clélia A. Hiruma-Lima; Carla Lima; Michael Richardson; Daniel C. Pimenta; Mônica Lopes-Ferreira (3039-3046).
Characterization of the peptide content of venoms has a number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. In order to analyze in detail the peptides and small proteins of crude samples, techniques such as chromatography and mass spectrometry have been employed. The present study describes the isolation, biochemical characterization, and sequence determination of a novel peptide, named Orpotrin from the venom of Potamotrygon gr. orbignyi. The natural peptide was shown to be effective in microcirculatory environment causing a strong vasoconstriction. The peptide was fully sequenced by de novo amino acid sequencing with mass spectrometry and identified as the novel peptide. Its amino acid sequence, HGGYKPTDK, aligns only with creatine kinase residues 97–105, but has no similarity to any bioactive peptide. Therefore, possible production of this peptide from creatine kinase by limited proteolysis is discussed. Taken together, the results indicate the usefulness of this single-step approach for low molecular mass compounds in complex samples such as venoms.
Keywords: Orpotrin; Potamotrygon; Venom; Stingrays; Vasoconstriction; De novo sequencing; Natural peptides; Creatine kinase;
Pathological effects of the mushroom toxin α-amanitin on BALB/c mice by Jian Zhao; Mei Cao; Jie Zhang; Qun Sun; Qian Chen; Zhi-rong Yang (3047-3052).
The pathological effects of α-amanitin on BALB/c mice after receiving intravenous injection were evaluated by RP-HPLC and mouse genome oligonucleotide microarray. The content of α-amanitin in Amanita virosa was about 2833.8 μg/g dry fruiting body. The liver and kidneys showed critical pathological changes after α-amanitin poisoning, and sera BUN, Crea, ALT, AST, TBIL and DBIL were the sensitive markers. The compound α-amanitin was detected in liver and kidney tissue homogenates by RP-HPLC after 48 h. The results of mouse genome oligonucleotide microarray showed 146 genes’ expression changed, which formed the alternant network. The expression of 66 genes decreased, while 80 ones increased with more than two-fold differential expression after 48 h. The compound α-amanitin influenced not only RNA polymerase II, but also the expression of its associated genes. The application of mouse oligo chip provided valuable data for further understanding the biological properties and molecular pathogenesis of α-amanitin, also might be helpful for screening the curative drug for α-amanitin intoxication.
Keywords: α-Amanitin; Reversed-phase high-performance liquid chromatography; Mouse genome oligonucleotide microarray; Differentially expressed genes;
The mastoparanogen from wasp by Xueqing Xu; Hailong Yang; Haining Yu; Jianxu Li; Ren Lai (3053-3057).
Mastoparans are a family of small peptides identified from the venom of hymenopteroid insects. Although they have been characterized as early as 1979, and so far are recognized as a leading biomolecule in potential drug therapy, their precursors, mastoparanogen, have still not been determined. In this paper, several mastoparans from the venom of the wasp Vespa magnifica (Smith) are reported. The cDNA of mastoparanogen is 236 base pairs in length, and encodes 40 amino acid residues, including a N-terminal acidic fragment and a C-terminal mature basic mastoparan, which contain multiple acidic amino acid residues and a tetradecapeptide with three lysines, INLKAIAALAKKLLG, respectively. The glycine at the tetradecapeptide end is the donator of –NH4 for the amidation of the leucine at the C-terminal. As far as we know, this is the first report of the precursor of animal mastoparan.
Keywords: Wasp venom; Vespa magnifica; Mastoparan; Mastoparanogen; Precursor; G-protein;
Sequence diversity of O-superfamily conopetides from Conus marmoreus native to Hainan by Sulan Luo; Dongting Zhangsun; Qiujin Lin; Lei Xie; Yong Wu; Xiaopeng Zhu (3058-3068).
The full-length cDNAs of six new O-superfamily conotoxins (CTX) were cloned and sequenced from Conus marmoreus native to Hainan in China South Sea using RT-PCR and 3′-RACE. Six novel conotoxin precursors encoded by these cDNAs consist of three typical regions of signal, pro-peptide and mature peptide. All the six toxin regions share a common O-superfamily cysteine pattern (C-C-CC-C-C, with three disulfide bridges). The predicted precursors are composed of 73–88 amino acids, and the predicted mature peptides consist of 26–34 amino acids. Phylogenetic analysis of new conotoxins from C. marmoreus from the present study and published homologue T-superfamily sequences from other Conus species was performed systematically. Patterns of sequence divergence for three regions of signal, pro-region and mature peptides, as well as Cys codon usage define the major O-superfamily branches and suggest how these separate branches arose. Percent identities of the amino acid sequences of the signal region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between inter- and intra-species. Notably, the diversity of the pro-region was also high with intermediate divergence between that observed in signal and toxin regions. Amino acid sequences and their mode of action (target) of previously identified conotoxins from molluscivorous C. marmoreus for the known conotoxins classes are discussed in detail. The data presented are new and should pave the way for chemical synthesis of these unique conotoxins for to allow determination of the molecular targets of these peptides, and also to provide clues for a better understanding of the phylogeny of these peptides.
Keywords: Sequence diversity; O-superfamily conotoxin; cDNA cloning; Conus marmoreus native to Hainan;
Molecular cloning of grammistins, peptide toxins from the soapfish Pogonoperca punctata, by hemolytic screening of a cDNA library by Tatsuyuki Kaji; Nami Sugiyama; Shoichiro Ishizaki; Yuji Nagashima; Kazuo Shiomi (3069-3076).
A novel method, based on the hemolytic screening of a cDNA phage library, was developed to isolate cDNAs encoding grammistins (antibacterial peptide toxins) of the soapfish Pogonoperca punctata. As a result, cDNAs encoding six grammistins were isolated and elucidated for their nucleotide sequences. In common with the grammistins, the precursor protein is composed of a highly conserved signal peptide, a considerably conserved propeptide that is characterized to contain a pair of basic residues (Lys-Arg) at plural positions including the C-terminus and one copy of a mature peptide. This precursor organization is similar to those of dermaseptins, antibacterial peptides from the frog skin.
Keywords: cDNA cloning; Grammistin; Hemolytic screening; Pogonoperca punctata; Soapfish;
Purification and characterization of novel antimicrobial peptides from the skin secretion of Hylarana guentheri by Jianwu Zhou; Stephen McClean; Alan Thompson; Yang Zhang; Chris Shaw; Pingfan Rao; Anthony J. Bjourson (3077-3084).
Linear, amphipathic and cationic antimicrobial peptides have been previously reported from a wide range of amphibian species especially frogs of the genus Rana. Such antimicrobial peptides are attracting increasing attention in pharmacological applications because they mainly act by permeabilizing and disrupting the target cell or virion membranes with a low degree of resistance. The Guenther's frog, Hylarana guentheri, is a Chinese frog of the genus Rana that is widely distributed in Southern China. It is commonly the dominant amphibian species even where the amphibian population is declining. In this study, we describe the isolation, purification, structural and biological characterization of five novel peptides from H. guentheri frog skin secretions that possess antimicrobial activity, including brevinin-2GHa, brevinin-2GHb, brevinin-2GHc, temporin-GH and a novel antimicrobial peptide named guentherin. The cDNAs encoding two novel members of the brevinin-2 family, brevinin-2GHb and brevinin-2GHc were also subsequently cloned and sequenced.
Keywords: Antimicrobial; Frog; Venom; Brevinin; Temporin; Guentherin;
Two families of antimicrobial peptides with multiple functions from skin of rufous-spotted torrent frog, Amolops loloensis by Yi Lu; Jianxu Li; Haining Yu; Xueqing Xu; Jianguo Liang; Yongqiang Tian; Dongying Ma; Guoqing Lin; Guoqiang Huang; Ren Lai (3085-3091).
There are around 27 species of Amolops amphibian distributed in South-east of Asia. Seven antimicrobial peptides (AMPs) belonging to two different families were purified from skin of rufous-spotted torrent frog, Amolops loloensis, and designated brevinins-ALa, b, c, and d, and temporins-ALa, b, and c. The brevinins-AL family which is structurally related to brevinins-1 from skin secretions of the European frog, Rana brevipoda, is composed of 24 amino acids and has an intra-disulfide bridge at the C-terminus. The temporins-AL family, composed of 13 or 16 amino acid residues, is related with temporins from the skin secretions of R. temporaria. The findings of this study will facilitate the solutions to the taxonomic questions of the ranid genus Amolops and Staurois. In the work of this paper, both brevinins-ALb and temporin-Ma induced mast cell degranulation and histamine release, and had cytotoxic activity toward solid tumor cell line HepG2. Brevinins-ALb also exerted strong hemolytic activity while temporin-Ma had no such activity.
Keywords: Amphibian; Antimicrobial peptide; Diversity; Brevinins-AL; Temporins-AL; Amolops loloensis;
Isolation and biochemical characterization of peptides presenting antimicrobial activity from the skin of Phyllomedusa hypochondrialis by Katia Conceição; Katsuhiro Konno; Michael Richardson; Marta M. Antoniazzi; Carlos Jared; Sirlei Daffre; Antonio Carlos M. Camargo; Daniel C. Pimenta (3092-3099).
Amphibian antimicrobial peptides have been known for many decades and several of them have been isolated from anuran species. Dermaseptins are among the most studied antimicrobial peptides and are found in the skin secretion of tree frogs from the Phyllomedusinae subfamily. These peptides exert a lytic action on bacteria, protozoa, yeast, and filamentous fungi at micromolar concentrations, but unlike polylysines, present little hemolytic activity. In this work, two antimicrobial peptides were isolated from the crude skin secretion of Phyllomedusa hypochondrialis and tested against Gram-positive and Gram-negative bacteria, presenting no hemolytic activity at the tested concentrations. One of them was identified with the recently reported peptide PS-7 belonging to the phylloseptin family, and another was a novel peptide, named DPh-1, which was fully purified, sequenced by ‘de novo’ mass spectrometry and grouped into Dermaseptins (DPh-1).
Keywords: Phyllomedusa hypochondrialis; De novo sequencing; Natural peptides; Antimicrobial peptides; Dermaseptins; Phylloseptins;
The antimicrobial peptide cathelicidin interacts with airway mucus by Kerstin Felgentreff; Christoph Beisswenger; Matthias Griese; Tanja Gulder; Gerhard Bringmann; Robert Bals (3100-3106).
Antimicrobial peptides (AMPs) and mucins are components of airway secretions and both contribute to the innate host defense system. At neutral pH, AMPs are positively charged, mucins negatively. It was the aim of the study to test whether these opposite charges result in interactions between AMPs and mucins. We measured binding of mucins isolated from porcine gastric mucosa to the cathelicidin LL-37 coated to multiwell plates and found that LL-37 electrostatically interacts with mucins. Circular dichroism spectra of the peptide revealed the induction of α-helical conformation by mucins. Addition of mucins to solutions of LL-37 significantly decreased the antimicrobial activity of the peptide against Pseudomonas aeruginosa and Streptococcus pneumoniae. We then tested whether LL-37 is bound to mucins in airway secretions from human subjects and found that a significant proportion of the peptide and its propeptide are bound to high molecular weight components. Together these data show that cationic AMPs interact with anionic mucins in airway secretions. Functions of AMPs are modulated by this interaction.
Keywords: Mucin; Innate immunity; Antimicrobial peptide; Cathelicidin; Host defense;
Vasoactive intestinal peptide enhances wound healing and proliferation of human bronchial epithelial cells by Cha-Xiang Guan; Min Zhang; Xiao-Qun Qin; Yan-Ru Cui; Zi-Qiang Luo; Hong-Bo Bai; Xiang Fang (3107-3114).
In the present study, we investigated the effects of vasoactive intestinal peptide (VIP) on wound healing of bronchial epithelium. Wound healing of the mechanical damaged human bronchial epithelial cells (HBEC) was observed in the absence or presence of VIP. Effects of VIP on chemotactic migration, cell proliferation of HBEC were also tested. HBEC chemotaxis was assessed by the blind well chamber technique, the cell cycle was determined by flow cytometry, and cell proliferation was determined by measuring the expression of proliferating cell nuclear antigen Ki67. Effects of VIP on epithelial E-cadherins protein and mRNA were also measured by immunohistochemistry and RT-PCR. The results showed that VIP accelerated the recovery of wound area of HBEC. VIP increased the migration and proliferation of HBEC, and these effects were blocked by a VPAC1 receptor antagonist. VIP also increased the expression of E-cadherin mRNA and protein in HBEC, suggesting that protective effects of VIP on wound healing may be related to its ability to increase the expression of E-cadherin. In conclusion, VIP has protective effects against human bronchial epithelial cell damage, and the beneficial effects of VIP might be mediated, at least in part, by VPAC1, and associated with increased expression of E-cadherin.
Keywords: Vasoactive intestinal peptide; Human bronchial epithelial cells; Wound healing; Proliferation;
A peptide inhibitor of MurA UDP-N-acetylglucosamine enolpyruvyl transferase: The first committed step in peptidoglycan biosynthesis by José Molina-López; François Sanschagrin; Roger C. Levesque (3115-3121).
The MurA enzyme from Pseudomonas aeruginosa was purified to homogeneity and found to be biologically active as a UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase in a coupled enzyme assay where ATPase activity was measured by the release of inorganic phosphate. A microtiter plate assay coupled to competitive biopanning using the UDP-N-acetylglucosamine was used to screen 109 C-7-C and 12-mers peptides from phage display libraries. From 60 phage-encoded peptides identified after the fourth round of biopanning, deduced amino acid sequences were aligned and two peptides were synthesized and tested for inhibition of the MurA-catalyzed reaction. The PEP 1354 peptide inhibited the ATPase activity of MurA with an IC50 value of 200 μM and was found to be a competitive inhibitor of UNAG. The pre-incubation of MurA with inhibitor indicated a time-independent inhibition. This time-dependent inhibition is the first report of peptide inhibitors of MurA, which represent the scaffold for the synthesis of inhibitory peptidomimetic molecules.
Keywords: MurA; Phage display; UDP-N-acetylglucosamine; Peptide inhibitors;
Isolation and characterization of a novel thermostable non-specific lipid transfer protein-like antimicrobial protein from motherwort (Leonurus japonicus Houtt) seeds by Xingyong Yang; Jun Li; Xianbi Li; Rong She; Yan Pei (3122-3128).
In screening for potent antimicrobial proteins from plant seeds, a novel heat-stable antimicrobial protein, designated LJAMP2, was purified from seeds of the motherwort (Leonurus japonicus Houtt), a medicine herb, with a procedure involving cation exchange chromatography on a CM FF column, and reverse phase HPLCs on C8 column and C18 column. LJAMP2 exhibited a molecular mass of 6.2 kDa determined. Automated Edman degradation determined the partial N-terminal sequence of LJAMP2 to be NH2-AIGCNTVASKMAPCLPYVTGKGPLGGCCGGVKGLIDAARTTPDRQAVCNCLKTLAKSYSG, which displays homology with plant non-specific lipid transfer proteins (nsLTPs). In vitro bioassays showed that LJAMP2 inhibits the growth of a variety of microbes, including filamentous fungi, bacteria and yeast. The growth of three phytopathogenic fungi, Alternaria brassicae, Botrytis maydis, and Rhizoctonia cerealis, are inhibited at 7.5 μM of LJAMP2, whereas Bacillus subtilis is about 15 μM. The IC50 of LJAMP2 for Aspergillus niger, B. maydis, Fusarium oxysporum, Penicillium digitatum and Saccharomyces cerevisiae are 5.5, 6.1, 9.3, 40.0, and 76.0 μM, respectively.
Keywords: Motherwort; Antifungal; Antibacterial; nsLTPs; Seeds; IC50;
Isolation and characterization of a novel mung bean protease inhibitor with antipathogenic and anti-proliferative activities by Shaoyun Wang; Juan Lin; Mingyu Ye; Tzi Bun Ng; Pingfan Rao; Xiuyun Ye (3129-3136).
A novel protease inhibitor, designated mungoin, with both antifungal and antibacterial activities, and exhibiting a molecular mass of 10 kDa in SDS-polyacrylamide gel electrophoresis, was isolated from mung bean (Phaseolus mungo) seeds. The isolation procedure involved a combination of extraction, ammonium sulfate precipitation, ion exchange chromatography on CM-Sephadex, and high-performance liquid chromatography (HPLC) on SP-Toyopearl. Its isoelectric point was estimated to be 9.8 by isoelectric focusing. Its N-terminal amino acid sequence was determined to be EMPGKPACLDTDDFCYKP, demonstrating some resemblance to the C-terminal sequences of other protease inhibitors and inhibitor precursors from leguminous plants. It exerted a potent inhibitory action toward a variety of fungal species including Physalospora piricola, Mycosphaerella arachidicola, Botrytis cinerea, Pythium aphanidermatum, Sclerotium rolfsii and Fusarium oxysporum, as well as an antibacterial action against Staphylococcus aureus. In addition, this novel plant protease inhibitor displayed anti-proliferative activity toward tumor cells.
Keywords: Mung bean; Protease inhibitor; Antifungal protein;
Uncovering conserved patterns in bioactive peptides in Metazoa by Feng Liu; Geert Baggerman; Liliane Schoofs; Geert Wets (3137-3153).
Bioactive (neuro)peptides play critical roles in regulating most biological processes in animals. Peptides belonging to the same family are characterized by a typical sequence pattern that is conserved among the family's peptide members. Such a conserved pattern or motif usually corresponds to the functionally important part of the biologically active peptide. In this paper, all known bioactive (neuro)peptides annotated in Swiss-Prot and TrEMBL protein databases are collected, and the pattern searching program Pratt is used to search these unaligned peptide sequences for conserved patterns. The obtained patterns are then refined by combining the information on amino acids at important functional sites collected from the literature. All the identified patterns are further tested by scanning them against Swiss-Prot and TrEMBL protein databases. The diagnostic power of each pattern is validated by the fact that any annotated protein from Swiss-Prot and TrEMBL that contains one of the established patterns, is indeed a known (neuro)peptide precursor. We discovered 155 novel peptide patterns in addition to the 56 established ones in the PROSITE database. All the patterns cover 110 peptide families. Fifty-five of these families are not characterized by the PROSITE signatures, and 12 are also not identified by other existing motif databases, such as Pfam and SMART. Using the newly identified peptide signatures as a search tool, we predicted 95 hypothetical proteins as putative peptide precursors.
Keywords: Neuropeptide; Toxin; Growth factor; Peptide precursor; Conserved pattern; PROSITE database; Pratt; Motif database; Peptide signature;
Evolutionary history of the calcitonin gene-related peptide family in vertebrates revealed by comparative genomic analyses by Maho Ogoshi; Koji Inoue; Kiyoshi Naruse; Yoshio Takei (3154-3164).
The calcitonin gene-related peptide (CGRP) family is composed of CGRP, amylin and adrenomedullin (AM) in mammals. In teleost fish, AM forms an independent subfamily of five members (AM1–5), which inspired us to trace the evolutionary history of the CGRP family throughout vertebrates by comparative genomic approach. Linkage mapping and synteny analyses of the CGRP family genes in medaka, Oryzias latipes, revealed that AM1/CGRP, AM2/amylin, and AM5 genes were located on respective proto-chromosomes before the divergence of teleost lineage. In teleost fish, additional whole genome duplication generated AM1/4, CGRP1/2, AM2/3, but one of the duplicated amylin and AM5 genes was silenced. In mammals, the amylin or AM2 gene was translocated to different chromosomes, while the CGRP gene was multiplied in tandem to generate CGRP-α,β, and recently identified calcitonin receptor-stimulating peptide genes. Based on these data, we identified a novel AM5 gene in several mammalian species as we previously did for AM2.
Keywords: CGRP; Amylin; Adrenomedullin; Linkage analyses; Molecular evolution;
Characterization and tissue distribution of multiple agouti-family genes in pufferfish, Takifugu rubripes by Tadahide Kurokawa; Koji Murashita; Susumu Uji (3165-3175).
Four types of agouti-family genes (AGRP1, AGRP2, ASIP1 and ASIP2) were obtained from torafugu, Takifugu rubripes. Their characterization and structure were analyzed to elucidate the relationship among the torafugu agouti-family genes. Both AGRP1 and AGRP2 showed genomic synteny with the human AGRP gene. Phylogenetic tree analysis showed that AGRP1 formed a cluster with human AGRP. We inferred that torafugu AGRP1 and AGRP2 are orthologs of human AGRP and that they are paralogous genes derived from genome duplication occurred in the teleost phylogeny. Torafugu ASIP1 showed genomic synteny with the human ASIP, but ASIP2 did not. The ASIP1 expression level was about five times higher in the white ventral skin than in the black dorsal skin. Therefore, we concluded that torafugu ASIP1 is an ortholog of human ASIP, nevertheless, we are unable to determine if torafugu ASIP2 is a paralog of ASIP1 or not.
Keywords: Takifugu; Multiple gene; Agouti; AGRP; mRNA; Tissue distribution;
Effect of β-lactotensin on acute stress and fear memory by Rena Yamauchi; Etsuko Wada; Daisuke Yamada; Masaaki Yoshikawa; Keiji Wada (3176-3182).
β-Lactotensin (β-LT) is a bioactive peptide derived from bovine milk β-lactoglobulin and is a natural ligand for neurotensin receptors. We examined the effect of β-LT on restraint stress and fear memory in mice. Mice subjected to acute restraint stress exhibited a decreased number of head-dips and increased head-dip latency compared to non-stressed controls in the hole-board test, reflecting increased stress-induced behaviors. However, prior administration of β-LT improved the behaviors caused by stress. The anti-stress effect of β-LT was blocked by levocabastine, a neurotensin receptor subtype 2 (NTR2) antagonist. In the fear-conditioning test, the duration of freezing responses by cued fear conditioning was significantly reduced in mice administered β-LT compared with control mice. These results suggest that β-LT has an anti-stress effect and promotes the extinction of fear memory, which may be mediated by NTR2.
Keywords: Neurotensin; β-Lactotensin; Anti-stress; Hole-board test; Fear memory;
CART (85–102)—Inhibition of psychostimulant-induced hyperlocomotion: Importance of cyclization by Tomasz Dylag; Piotr Rafalski; Jolanta Kotlinska; Jerzy Silberring (3183-3192).
Synthetic derivative of C-terminal fragment of CART (55–102) with reduced thiol groups, [Abu86,94]CART (85–102)red, given together with amphetamine (5 mg/kg, s.c.) or cocaine (15 mg/kg, s.c.), reversed hyperlocomotion induced by these drugs at a dose of 0.1 μg but not at a higher dose. In the cerebral cortex homogenate, [Abu86,94]CART (85–102)red was nonspecifically cleaved from N- and C-termini. This peptide contains two chemically blocked Cys residues, and two others in reduced form. Concomitant with cleavage, rapid cyclization occurred. The newly formed cyclic peptides were stable. The cyclic peptide [Abu86,94]CART (85–102)ox failed to inhibit amphetamine- and cocaine-induced locomotor activity. The ability to inhibit the locomotor-stimulant activity of amphetamine was retained in [Abu86,88,94,101]CART (85–102), in which all Cys were replaced with 2-aminobutyric acid to prevent their pairing. Disulfide bridge formation may be an interesting mechanism that prevents proteolysis of [Abu86,94]CART (85–102)red and terminates its ability to reverse amphetamine-induced hyperlocomotion.
Keywords: CART; Peptide; Amphetamine; Cocaine; Locomotor activity; Mouse; Metabolism; Mass spectrometry;
Dose-dependent effects of peptide YY(3-36) on conditioned taste aversion in rats by Prasanth K. Chelikani; Alvin C. Haver; Roger D. Reidelberger (3193-3201).
We used a conditioned taste aversion test to assess whether PYY(3-36) reduces food intake by producing malaise. Two-hour IV infusion of PYY(3-36) (8, 15, and 30 pmol/kg/min) at dark onset in non-food-deprived rats produced a dose-dependent inhibition of feeding and a conditioned aversion to the flavored chow paired with PYY(3-36) infusion. In food-deprived rats, PYY(3-36) at 2 and 4 pmol/kg/min inhibited intake of a flavored saccharin solution without producing conditioned taste aversion, whereas higher doses (8 and 15 pmol/kg/min) inhibited saccharin intake and produced taste aversion. These results suggest that anorexic doses of PYY(3-36) may produce a dose-dependent malaise in rats, which is similar to that reported for PYY(3-36) infusion in humans. Previous studies have shown that PYY(3-36) potently inhibits gastric emptying, and that gut distention can produce a conditioned taste aversion. Thus, PYY(3-36) may produce conditioned taste aversion in part by slowing gastric emptying.
Keywords: Satiety; Peptide; Intravenous infusion; Conditioned aversion;
Anxiolytic-like effect of the selective Neuropeptide Y Y2 receptor antagonist BIIE0246 in the elevated plus-maze by Fabrizio Bacchi; Aleksander A. Mathé; Patricia Jiménez; Luigi Stasi; Roberto Arban; Philip Gerrard; Laura Caberlotto (3202-3207).
The involvement of Neuropeptide Y (NPY) in the pathophysiology of mood disorders has been suggested by clinical and preclinical evidence. NPY Y1 and Y2 receptors have been proposed to mediate the NPY modulation of stress responses and anxiety related behaviors. To further investigate the role of Y2 receptors in anxiety we studied the effect of BIIE0246, a selective Y2 receptor antagonist, in the elevated plus-maze test. Rats treated with 1.0 nmol BIIE0246 showed an increase in the time spent on the open arm of the maze. In addition, to study the effects of the Y2 antagonism on NPY protein level, NPY-like immunoreactivity was measured in different brain regions following treatment with BIIE0246, but no statistically significant effects were observed. These results suggest that BIIE0246 has an anxiolytic-like profile in the elevated plus-maze.
Keywords: NPY receptors; Behavior; Radioimmunoassay; Anxiety; Elevated plus-maze; Rats;
Neuropeptide Y (NPY) modulates oxidative burst and nitric oxide production in carrageenan-elicited granulocytes from rat air pouch by Mirjana Dimitrijević; Stanislava Stanojević; Stana Mićić; Vesna Vujić; Vesna Kovačević-Jovanović; Katarina Mitić; Stephan von Hörsten; Duško Kosec (3208-3215).
We studied the effects of neuropeptide Y (NPY) and NPY-related receptor specific peptides on functions of carrageenan-elicited granulocytes in vitro and ability of NPY to modulate carrageenan-induced air pouch inflammation in rats in vivo. Anti-inflammatory effect of NPY comprises reduced granulocyte accumulation into the air pouch, to some extent attenuation of phagocytosis, attained via Y1 receptor, and considerable decrease in peroxide production, albeit mediated via Y2 and Y5 receptors activation. Conversely, NPY increases nitric oxide production and this potentiation is mediated via Y1 receptor. It is concluded that NPY Y1 and Y2/Y5 receptors’ interaction participates in NPY-induced modulation of granulocyte functions related to inflammation.
Keywords: Neuropeptide Y (NPY); NPY receptors; Inflammation; Granulocytes; Rat;
Down-regulation of the beacon gene expression in the regenerating rat adrenal cortex by Agnieszka Ziolkowska; Marcin Rucinski; Marianna Tyczewska; Anna Sandra Belloni; Magdalena Nowak; Gastone G. Nussdorfer; Ludwik K. Malendowicz (3216-3219).
Beacon, a hypothalamic peptide involved in the regulation of food intake, has been recently shown to be expressed in the adrenal cortex, and to inhibit its secretion and growth. To further characterize the role of beacon in the control of adrenal growth, we investigated the level of beacon gene expression in the regenerating rat adrenal cortex. Conventional reverse transcription-polymerase chain reaction (PCR) and immunocytochemistry demonstrated the expression of beacon mRNA and protein in the adrenals at both days 5 and 8 of regeneration after enucleation and contralateral adrenalectomy. Semiquantitative real time-PCR revealed a net down-regulation of beacon mRNA in the regenerating glands, as compared to the intact adrenal cortex of sham-operated animals. Beacon gene expression was higher at day 8 than at day 5 of regeneration. Mitotic index, as assayed by the stachmokinetic method with vincristin, was negligible in the intact adrenal, but greatly elevated in regenerating gland, with a higher index found at day 5 than at day 8 after surgery. Taken together our findings indicate that the level of beacon gene expression is inversely correlated with the proliferative activity of adrenocortical cells, and suggest that beacon might act as an endogenous inhibitor of adrenocortical growth in the rat.
Keywords: Beacon; Ubiquitin-like proteins; Regenerating adrenal cortex; Rat;
Interleukin-1β-induced anorexia is reversed by ghrelin by Patricia Verónica Gonzalez; Andrea Beatriz Cragnolini; Helgi Birgir Schiöth; Teresa Nieves Scimonelli (3220-3225).
Interleukins, in particular interleukin-1β (IL-1β), reduce food intake after peripheral and central administration, which suggests that they contribute to anorexia during various infectious, neoplastic, and autoimmune diseases. On the other hand, ghrelin stimulates food intake by acting on the central nervous system (CNS) and is considered an important regulator of food intake in both rodents and humans. In the present study, we investigated if ghrelin could reverse IL-1β-induced anorexia. Intracerebroventricular (i.c.v.) injection of 15, 30 or 45 ng/μl of IL-1β caused significant suppression of food intake in 20 h fasting animals. This effect lasted for a 24 h period. Ghrelin (0.15 nmol or 1.5 nmol/μl) produced a significant increase in cumulative food intake in normally fed animals. However, it did not alter food intake in 20 h fasting animals. Central administration of ghrelin reduced the anorexic effect of IL-1β (15 ng/μl). The effect was observed 30 min after injection and lasted for the next 24 h. This study provides evidence that ghrelin is an orexigenic peptide capable of antagonizing IL-1β-induced anorexia.
Keywords: Ghrelin; IL-1β; Food intake; Central nervous system;
Effects of oral preload, CCK or bombesin administration on short term food intake of melanocortin 4-receptor knockout (MC4RKO) mice by C.H. Vaughan; C. Haskell-Luevano; A. Andreasen; N.E. Rowland (3226-3233).
We investigated whether either heterozygous (HET) or homozygous (knockout, KO) disruption of the melanocortin type 4 receptor (MC4R) gene alters post ingestive responsiveness of mice. Specifically, we tested the hypothesis that hyperphagia in MC4RKO mice might be due to a deficit in processes that sustain intermeal intervals (satiety) and/or processes that terminate ongoing episodes of eating (satiation). To test satiety, mice drank an oral preload and then we monitored intake of a subsequent liquid diet test meal. To test satiation, we examined the effect of exogenous administration of cholecystokinin (CCK) and bombesin (BN) on the size of a liquid diet meal. Experiment 1 was comprised of two studies. In the first, we determined that the intake of all three genotypes following fasts of either 6, 12, or 24 h were comparable, and so chose 12 h deprivation for the subsequent studies. In the second, 12 h fasted mice were allowed to consume a fixed preload, approximately 50% of their expected mean intake and, following delays of either 30 or 60 min, were allowed to consume to satiation. Compared with no preload, the preload significantly reduced meal size comparably in all three genotypes. The reduction in intake was greater when the test meal was presented 30 compared with 60 min after the preload, again with no genotype differences in this decay of satiety. In experiment 2, we administered either CCK or BN and examined suppression of meal size after a 12 h fast. Mice were tested repeatedly with CCK-8 (2, 6, or 18 μg/kg ip) or BN (2, 4 or 8 μg/kg ip) with vehicle injection days intervening. The 30 min intakes of HET and KO mice were suppressed more than those of WT following either CCK or BN. These experiments suggest that diminished responsiveness to nutrients or gut satiety hormones is not responsible for hyperphagia in MC4RKO mice.
Keywords: Cholecystokinin; Bombesin; MC4RKO mice; Preload; Gut; Satiety;
H2O2 and Src-dependent transactivation of the EGF receptor mediates the stimulatory effect of leptin on renal ERK and Na+, K+-ATPase by Jerzy Bełtowski; Grażyna Wójcicka; Jadwiga Trzeciak; Andrzej Marciniak (3234-3244).
We examined the mechanism through which leptin increases Na+, K+-ATPase activity in the rat kidney. Leptin was infused under anaesthesia into the abdominal aorta proximally to the renal arteries and then Na+, K+-ATPase activity was measured in the renal cortex and medulla. Leptin (1 μg/kg min) increased Na+, K+-ATPase activity after 3 h of infusion, which was accompanied by the increase in urinary H2O2 excretion and phosphorylation level of extracellular signal regulated kinase (ERK). The effect of leptin on ERK and Na+, K+-ATPase was abolished by catalase, specific inhibitors of epidermal growth factor (EGF) receptor, AG1478 and PD158780, as well as by ERK inhibitor, PD98059, and was mimicked by both exogenous H2O2 and EGF. The effect of leptin was also prevented by the inhibitor of Src tyrosine kinase, PP2. Leptin and H2O2 increased Src phosphorylation at Tyr418. We conclude that leptin-induced stimulation of renal Na+, K+-ATPase involves H2O2 generation, Src kinase, transactivation of the EGF receptor, and stimulation of ERK.
Keywords: Leptin; Obesity; Arterial hypertension; Na+, K+-ATPase; Epidermal growth factor;
Hypothalamic clamp on insulin release by leptin-transgene expression by Stéphane Boghossian; Michael G. Dube; Rita Torto; Pushpa S. Kalra; Satya P. Kalra (3245-3254).
The effects of sustained leptin action locally in the hypothalamus on the functional link between fat accrual and insulin secretion after chronic high fat diet (HFD) consumption in leptin-deficient ob/ob mice, and on the post-prandial insulin response in rats consuming regular chow diet (RCD), was examined in this study. A single intracerebroventricular (icv) injection of recombinant adeno-associated virus vector encoding leptin gene (rAAV-lep) enhanced hypothalamic leptin-transgene expression in ob/ob mice consuming RCD and suppressed the time-related weight gain and fat accumulation concomitant with abrogation of hyperinsulinemia and enhanced glucose tolerance. This increased hypothalamic leptin-transgene expression continued to impose insulinopenia and increased glucose tolerance but was ineffective in suppressing weight gain and fat accumulation after these mice were switched to chronic HFD consumption. A similar icv rAAV-lep pretreatment in rats consuming RCD markedly attenuated the post-prandial rise in insulin release concomitant with suppressed weight and fat depots. These results show for the first time that a sustained hypothalamic leptin action can stably clamp pancreatic insulin secretion independent of the status of fat accrual engendered by diets of varying caloric enrichment. Thus, the efficacy of increased leptin afferent signaling in the hypothalamus to persistently restrain pancreatic insulin release and insulin resistance can be explored as an adjunct therapeutic modality to alleviate pathophysiological derrangements that confer type 2 diabetes.
Keywords: Leptin gene therapy; Hypothalamus; Insulin release; Insulin resistance;
Stimulated release of calcitonin gene-related peptide from the human right atrium in patients with and without diabetes mellitus by Thomas Strecker; Anne Dieterle; Peter W. Reeh; Michael Weyand; Karl Messlinger (3255-3260).
Calcitonin gene-related peptide (CGRP), a potent vasodilator released during activation from a subset of sensory Aδ- and C-fiber afferents, has been suggested to play a beneficial role in myocardial ischemia. Variations in CGRP release can possibly be correlated with diseases that involve changes in activity or degeneration of cardiac afferent fibers. The aim of the present study was to examine basal and stimulated CGRP release from cardiac tissue in patients who underwent cardiopulmonary bypass surgery and to compare patients with and without known history of diabetes mellitus. Small pieces of the right atrium routinely excised during the bypass operations were passed through series of oxygenated solutions. The TRPV1 receptor agonist capsaicin and the nitric oxide donor NONOate were added for stimulation of cardiac afferent fibers. The eluates were processed using an enzyme immuno-assay (EIA) for measurement of CGRP concentrations. Both capsaicin and NONOate caused significant increases in CGRP release. No significant differences in CGRP release between patients with and without diabetes mellitus were examined. The present study evaluates a simple and reproducible model for measuring stimulated CGRP release from the human right atrium.
Keywords: Calcitonin gene-related peptide (CGRP); Heart; Diabetes mellitus; Capsaicin; Nitric oxide; Nociceptors;
Cardiovascular effects of native and non-native urotensin II and urotensin II-related peptide on rat and salmon hearts by H.C.G. Prosser; J. Leprince; H. Vaudry; A.M. Richards; M.E. Forster; C.J. Pemberton (3261-3268).
Urotensin II (UII) was first discovered in the urophyses of goby fish and later identified in mammals, while urotensin II-related peptide (URP) was recently isolated from rat brain. We studied the effects of UII on isolated heart preparations of Chinook salmon and Sprague–Dawley rats. Native rat UII caused potent and sustained, dose-dependent dilation of the coronary arteries in the rat, whereas non-native UII (human and trout UII) showed attenuated vasodilation. Rat URP dilated rat coronary arteries, with 10-fold less potency compared with rUII. In salmon, native trout UII caused sustained dilation of the coronary arteries, while rat UII and URP caused significant constriction. Nω-nitro- l -arginine methyl (l-NAME) and indomethacin significantly attenuated the URP and rat UII-induced vasodilation in the rat heart. We conclude that UII is a coronary vasodilator, an action that is species form specific. We also provide the first evidence for cardiac actions of URP, possibly via mechanisms common with UII.
Keywords: Urotensin II; URP; Isolated rat heart; Salmon coronary arteries; Vasodilation;
Angiotensin II induces NF-κB activation in HUVEC via the p38MAPK pathway by Rui-Wei Guo; Li-Xia Yang; Mao-Quan Li; Bei Liu; Xian-Mei Wang (3269-3275).
Angiotensin II (Ang II) is the main active peptide of the renin–angiotensin system (RAS), producing a number of inflammatory mediators that lead to endothelial dysfunction and the progression of atherosclerosis. Ang II-induced NF-κB nuclear translocation plays a pivotal role in this response. This study examines the NF-κB activation mechanism elicited by Ang II in human umbilical vein endothelial cells (HUVEC). Electrophoretic mobility shift assays and Western blotting revealed that Ang II, signaling via AT1, produces a time-dependent increase in NF-κB DNA binding and IκBα degradation. These results also demonstrate that Ang II leads to MAPK phosphorylation and p38MAPK pathway-induced NF-κB activation. Furthermore, AT1 is required for p38MAPK phosphorylation induced by Ang II. This study provides evidence that Ang II elicits NF-κB activation via the p38MAPK pathway in HUVEC.
Keywords: Angiotensin II; NF-κB; MAPK; Human umbilical vein endothelial cell;
CRH inhibits NF-κB signaling in human melanocytes by Blazej Zbytek; Lawrence M. Pfeffer; Andrzej T. Slominski (3276-3283).
Corticotropin releasing hormone (CRH), a messenger of stress at the central level, is expressed in the epidermis where it operates within local equivalent of hypothalamo-pituitary axis. CRH inhibits NF-κB activity in human immortalized epidermal (PIG1) melanocytes. In melanocytes CRH stimulates pro-opiomelanocortin (POMC) mRNA and adrenocorticotropin (ACTH) peptide production. Knockdown of POMC levels by transfecting cells with antisense oligonucleotides blocks the effect of CRH on NF-κB signaling indicating that the above inhibition is indirect, e.g. through activation of POMC. We suggest that induction of POMC by CRH serves as a feedback mechanism to self-restrict inflammatory response in the skin.
Keywords: CRH; Melanocyte; NF-κB; POMC;
Activation of the Nociceptin/Orphanin FQ system is unable to reverse CRF2 receptor mediated anorexia in the rat by Amalia Fedeli; Federica Policani; Massimo Ubaldi; Andrea Cippitelli; Maurizio Massi; Roberto Ciccocioppo (3284-3291).
Central injection of Nociceptin/Orphanin FQ (N/OFQ), inhibits the anorectic effect of corticotropin-relasing factor (CRF) and stress in rats. Recently, Urocortin II (Ucn II) and Urocortin III (Ucn III), two selective CRF2 receptor agonists, have been identified. Here, we investigated the effect of intracerebroventricular (ICV) injection of 0.25, 0.75, 1.50 or 3 nmol/rat of Ucn II or Ucn III on food and water intake in food deprived rats. The effect of N/OFQ on Ucn II and UCNIII-induced anorexia was also studied. Results showed a greater inhibition of food consumption by Ucn II than Ucn III. Pretreatment with N/OFQ (0.25–2.0 nmol/rat) did not block the effects of Ucn II and UCNIII. Conversely, injection of N/OFQ (0.25–2.0 nmol/rat) blocked the anorectic effect of CRF (0.1 nmol/rat). These findings suggest that N/OFQ selectively prevent the anorectic effect mediated by activation of the CRF1 receptor system.
Keywords: Urocortin II; Urocortin III; Nociceptin/Orphanin FQ; NOP receptors; CRF receptors; Food intake;
Increased expression of mu opioid receptors in animals susceptible to diet-induced obesity by Maria J. Barnes; Gregory Holmes; Stefany D. Primeaux; David A. York; George A. Bray (3292-3298).
Stimulation of mu opioid receptors preferentially increases the intake of a high fat diet. In this paper we investigated whether there was a difference in the expression of mu opioid receptors between animals susceptible (Osborne–Mendel) or resistant (S5B/Pl) to obesity induced by eating a high fat diet. Immunohistochemical studies demonstrated that Osborne–Mendel rats eating a chow diet had an increased number of mu opioid receptors in the arcuate nucleus when compared to S5B/Pl rats. These immunohistochemical findings were supported by Real Time-PCR which demonstrated that the mRNA level of mu opioid receptors was also increased in the hypothalamus of Osborne–Mendel rats compared to S5B/Pl rats. Low doses of the mu opioid receptor agonist DAMGO [d-Ala2-N-Me-Phe4-Glycol5]-enkephalin administered to Osborne–Mendel rats caused a significant increase in the preference for a diet high in fat. The same doses of DAMGO switched the diet preference of S5B/Pl rats to high fat but did not significantly increase food intake. The combination of these findings suggests that the increased levels of hypothalamic mu opioid receptors in Osborne–Mendel rats may contribute to their preference for a diet high in fat and increase their susceptibility to becoming obese.
Keywords: Mu opioid receptors; Diet-induced obesity; Arcuate nucleus;
Effect of novel nociceptin/orphanin FQ–NOP receptor ligands on ethanol drinking in alcohol-preferring msP rats by D. Economidou; A. Fedeli; R. Martin Fardon; F. Weiss; M. Massi; R. Ciccocioppo (3299-3306).
Activation of the NOP receptor by the endogenous ligand nociceptin/orphanin FQ (N/OFQ) reduces alcohol consumption in genetically selected alcohol-preferring Marchigian Sardinian (msP) rats. The present study evaluated the effect of three newly synthesized peptidergic and one brain-penetrating heterocyclic NOP receptor agonists on alcohol drinking in the two bottle choice paradigm. MsP rats were intracerebroventricularly (ICV) injected with the NOP receptor agonists OS-462 (0.5 and 1.0 μg), UFP-102 (0.25 and 1.0 μg) or UFP-112 (0.01 and 0.05 μg), or with Ro 64-6198 (0.3 and 1.0 mg/kg) given intraperitoneally (i.p.) and tested for 10% alcohol consumption. Results showed decreased alcohol consumption after treatment with all three peptidergic NOP receptor agonists (OS-462, UFP-102 and UFP-112). OS-462 (at the 1.0 μg dose) and UFP-102 (at the 0.25 μg dose) induced a significant increase in food intake as well. Surprisingly, Ro 64-6198 was ineffective at the 0.3 mg/kg dose, whereas it increased ethanol and food consumption at the 1.0 mg/kg dose. Pre-treatment with the selective μ-receptor antagonist naloxone (0.5 mg/kg, i.p.) reduced these effects of 1.0 mg/kg of Ro 64-6198. These findings confirm that activation of brain NOP receptors reduces alcohol drinking in msP rats and demonstrate that OS-462, UFP-102 and UFP-112 act as potent NOP receptor agonists. On the other hand, Ro 64-6198 increased alcohol drinking, an effect probably induced by a residual agonist activity of this compound at μ-opioid receptors. Overall, the results indicate that OS-462, UFP-102 and UFP-112 may represent valuable pharmacological tools to investigate the functional role of the brain N/OFQ system.
Keywords: Nociceptin/orphanin FQ; NOP receptor; Alcohol intake;
The possible involvement of endogenous ligands for mu-, delta- and kappa-opioid receptors in modulating morphine-induced CPP expression in rats by Jing Liang; Yijing Li; Xingjie Ping; Peng Yu; Yanfang Zuo; Liuzhen Wu; Ji-Sheng Han; Cailian Cui (3307-3314).
Previous studies suggested that electroacupuncture (EA) can suppress opioid dependence by the release of endogenous opioid peptides. To explore the site of action and the receptors involved, we tried to inject highly specific agonists for μ-, δ- and κ-opioid receptors into the CNS to test whether it can suppress morphine-induced conditioned place preference (CPP) in the rat. Male Sprague–Dawley rats were trained with 4 mg/kg morphine, i.p. for 4 days to establish the CPP model. This CPP can be prevented by (a) i.p. injection of 3 mg/kg dose of morphine, (b) intracerebroventricular (i.c.v.) injection of micrograms doses of the selective μ-opioid receptor agonist DAMGO, δ-agonist DPDPE or κ-agonist U-50,488H or (c) microinjection of DAMGO, DPDPE or U50488H into the shell of the nucleus accumbens (NAc). The results suggest that the release of endogenous μ-, δ- and κ-opioid agonists in the NAc shell may play a role for EA suppression of opiate addiction.
Keywords: Morphine; Conditioned place preference (CPP); Opioid receptors; Nucleus accumbens (NAc);
Synthesis, radiolabeling and receptor binding of [3H][(1S,2R)ACPC2]endomorphin-2 by Attila Keresztes; Géza Tóth; Ferenc Fülöp; Mária Szűcs (3315-3321).
Previously, we have shown that substitution of Pro2 for cis-2-aminocyclopentanecarboxylic acid, ACPC in endomorphin-2 results in an analogue with greatly augmented proteolytic stability, high μ-opioid receptor affinity and selectivity. We now report the synthesis and biochemical characterization of [3H][(1S,2R)ACPC2]endomorphin-2 with a specific activity of 1.41 TBq/mmol (38.17 Ci/mmol). Specific binding of [3H][(1S,2R)ACPC2]endomorphin-2 was saturable and of high affinity with an equilibrium dissociation constant, K d = 1.80 ± 0.21 nM and receptor density, B max = 345 ± 27 fmol × mg protein−1 at 25 °C in rat brain membranes. Similar affinity values were obtained in kinetic and displacement assays. Both Na+ and Gpp(NH)p decreased the affinity proving the agonist character of the radioligand. [3H][(1S,2R)ACPC2]endomorphin-2 retained the μ-specificity of the parent peptide. The new radioligand will be a useful tool to map the topographical requirements of μ-opioid peptide binding due to its high affinity, selectivity and enzymatic stability.
Keywords: Endomorphin; 2-Aminocylcopentanecarboxylic acid; Radiolabeling; Receptor binding; Conformational constrain;
Dmt-Tic-NH-CH2-Bid (UFP-502), a potent DOP receptor agonist: In vitro and in vivo studies by Raffaella Vergura; Elena Valenti; Christopher P. Hebbes; Elaine C. Gavioli; Barbara Spagnolo; John McDonald; David G. Lambert; Gianfranco Balboni; Severo Salvadori; Domenico Regoli; Girolamo Calo’ (3322-3330).
Knockout and pharmacological studies demonstrated that the activation of delta opioid peptide (DOP) receptors produces antidepressant-like effects in rodents. Here we report the results obtained with the novel DOP ligand H-Dmt-Tic-NH-CH2-Bid (UFP-502). UFP-502 bound with high affinity (pK i 9.43) to recombinant DOP receptors displaying moderate selectivity over MOP and KOP. In CHOhDOP [35S]GTPγS binding and mouse vas deferens experiments, UFP-502 behaved as a potent (pEC50 10.09 and 10.70, respectively) full agonist. In these preparations, naloxone, naltrindole and N,N(CH3)2Dmt-Tic-OH showed similar pA 2 values against UFP-502 and DPDPE and the same rank order of potency. In vivo in mice, UFP-502 mimicked DPDPE actions, producing a significant reduction of immobility time after intracerebroventricular administration in the forced swimming test and a clear antinociceptive effect after intrathecal injection in the tail withdrawal assay. However, while the effects of DPDPE were fully prevented by naltrindole those evoked by UFP-502 were unaffected (tail withdrawal assay) or only partially reversed (forced swimming test). In conclusion, UFP-502 represents a novel and useful chemical template for the design of selective agonists for the DOP receptor.
Keywords: Opioids; Delta opioid peptide receptor; UFP-502; Receptor binding; Bioassay; Forced swimming and tail withdrawal assays; Mice;
Brain processing of hemorphin-7 peptides in various subcellular fractions from rats by Laurence Murillo; Jean-Marie Piot; Corinne Coitoux; Ingrid Fruitier-Arnaudin (3331-3340).
Hemorphins are multifunctional peptides derived from hemoglobin or blood processing. They have been found at high levels within the central nervous system where they have a direct effect on neuronal cells via peptidergic receptors. As relatively few studies have examined their metabolic stability in the brain, such investigation was performed to locate the cellular distribution of enzymatic activity against these peptides. High-performance liquid chromatography (HPLC) combined with electrospray ionisation mass spectrometry (ESI-MS) allows identification of degradation products resulting from incubation of hemorphin-7 peptides (LVV-hemorphin-7, VV-hemorphin-7 and hemorphin-7) with subcellular fractions isolated from rat brain tissue. Metabolic activities were found against the three peptides in brain homogenate and subcellular fractions with the highest metabolic activity (<3% peptide remaining after 10 min) observed in the microsomal fraction which processed hemorphin-7 peptides mainly into N-terminal fragments (giving LVVH5) suggesting action of brain-membrane enzymes with C-terminal specificity. Incubation of the ACE inhibitor captopril (0.2 μM) with microsomal fraction, together with LVVH7, decreased the processing of LVVH7 to form LVVH5 by 85%.
Keywords: Hemorphins; Mass spectrometry; Peptide catabolism; Brain subcellular fractions; Brain aminopeptidase; ACE;
Only through the brain nuclei, arginine vasopressin regulates antinociception in the rat by Jun Yang; Cao-you Song; Wen-yan Liu; Bao-cheng Lin (3341-3346).
The effect of arginine vasopressin (AVP) on rat antinociception was investigated. Intraventricular injection of 50 or 100 ng AVP dose-dependently increased the pain threshold; in contrast, intraventricular injection of 10 μl anti-AVP serum decreased the pain threshold; both intrathecal injection of 200 ng AVP or 10 μl anti-AVP serum and intravenous injection of 5 μg AVP or 200 μl anti-AVP serum did not influence the pain threshold. Pain stimulation reduced AVP concentration in hypothalamic paraventricular nucleus (PVN), and elevated AVP concentration in hypothalamic supraoptical nucleus (SON) and periaqueductal gray (PAG), but no change in AVP concentration was detected in pituitary, spinal cord and serum. The results indicated that AVP regulation of antinociception was limited to the brain nuclei.
Keywords: Arginine vasopressin; Anti-arginine vasopressin serum; Antinociception; Hypothalamic paraventricular nucleus; Periaqueductal gray; Rat;
Molecular cloning and functional characterization of a prolactin-releasing peptide homolog from Xenopus laevis by Tatsuya Sakamoto; Aiko Oda; Kazutoshi Yamamoto; Miyoko Kaneko; Sakae Kikuyama; Akio Nishikawa; Akiyoshi Takahashi; Hiroshi Kawauchi; Kazuyoshi Tsutsui; Masaaki Fujimoto (3347-3351).
Amino acid sequences for identified prolactin (PRL)-releasing peptides (PrRPs) were conserved in mammals (>90%) or teleost fishes (100%), but there were considerable differences between these classes in the sequence (<65%) as well as in the role of PrRP. In species other than fishes and mammals, we have identified frog PrRP. The cDNA encoding Xenopus laevis prepro-PrRP, which can generate putative PrRPs, was cloned and sequenced. Sequences for the coding region showed higher identity with teleost PrRPs than mammalian homologues, but suggested the occurrence of putative PrRPs of 20 and 31 residues as in mammals. The amino acid sequence of PrRP20 was only one residue different from teleost PrRP20, but shared 70% identity with mammalian PrRP20s. In primary cultures of bullfrog (Rana catesbeiana) pituitary cells, Xenopus PrRPs increased prolactin concentrations in culture medium to 130–160% of the control, but PrRPs was much less potent than thyrotropin-releasing hormone (TRH) causing a three- to four-fold increase in prolactin concentrations. PrRP mRNA levels in the developing Xenopus brain peak in early prometamorphosis, different from prolactin levels. PrRP may not be a major prolactin-releasing factor (PRF), at least in adult frogs, as in mammals.
Keywords: Prolactin-releasing peptide; Prolactin; Brain; Amphibian; Evolution; Gene expression;
Synthesis and characterization of novel biotinylated carboxyl-terminal parathyroid hormone peptides that specifically crosslink to the CPTH-receptor by Santanu Banerjee; Hafez Selim; Gihan Suliman; Andrew I. Geller; Harald Jüppner; F. Richard Bringhurst; Paola Divieti (3352-3362).
Parathyroid hormone (PTH) regulates calcium, phosphorous and skeletal homeostasis via interaction with the G protein-coupled PTH/PTHrP receptor, which is fully activated by the amino-terminal 34 amino-acid portion of the hormone. Recent evidence points to the existence of another class of receptors for PTH that recognize the carboxyl (C)-terminal region of intact PTH (1–84) (CPTHRs) and are highly expressed by osteocytes. Here we report the synthesis and characterization of two novel bifunctional CPTH ligands that include benzoylphenylalanine (Bpa) substitutions near their amino-termini and carboxyl-terminal biotin moieties, as well as a tyrosine34 substitution to enable radioiodination. These peptides are shown to bind to CPTHRs with affinity similar to that of PTH (1–84) and to be specifically and covalently crosslinked to CPTHRs upon exposure to ultraviolet light. Crosslinking to osteocytes or osteoblastic cells generates complexes of 80 and 220 kDa, of which the larger form represents an aggregate that can be resolved into the 80 kDa. The crosslinked products can be further purified using immunoaffinity and avidin-based affinity procedures. While the molecular structure of the CPTHR(s) remains undefined, these bifunctional ligands represent powerful new tools for use in isolating and characterizing CPTHR protein(s).
Keywords: Parathyroid hormone; Carboxy-terminal parathyroid hormone; Parathyroid hormone receptor type-1; Benzoylphenylalanine;
Bradykinin release and inactivation in brain of rats submitted to an experimental model of Alzheimer's disease by Lígia M. Iores-Marçal; Tânia A. Viel; Hudson Sousa Buck; Viviane A. Nunes; Andrezza J. Gozzo; Ilana Cruz-Silva; Antonio Miranda; Kazuaki Shimamoto; Nobuyuki Ura; Mariana S. Araujo (3363-3369).
The kallikrein-kinin system is involved in a variety of physiological and pathological processes. Components of this system, identified in rat and human brains, can be altered in neurodegenerative processes such as Alzheimer's disease. Here, we studied kinin release and its inactivation in rats submitted to chronic cerebroventricular infusion of β-amyloid (Aβ) peptide. Neurodegeneration was confirmed by histological analysis of brain samples. In cerebrospinal fluid of animals infused with Aβ, bradykinin concentration was increased, as determined by radioimmunoassay. However, in the brain of Aβ group, we only detected the tripeptide Arg-Pro-Pro, purified by reversed-phase chromatography and characterized by liquid chromatography-electrospray ionization mass spectrometry. This fragment of bradykinin indicated the possible participation of kinin-processing enzymes in the brain such as a prolyl oligopeptidase.
Keywords: β-Amyloid; Bradykinin; Kallikrein-kinin system; Kininase;
Neurokinin A and neurokinin B in the human retina by Eduard Schmid; Johannes Leierer; Gerhard Kieselbach; Barbara Teuchner; Martina Kralinger; Reiner Fischer-Colbrie; James E. Krause; Quynh Anh Nguyen; Gertrud Haas; Katrin Stemberger; Josef Troger (3370-3376).
Very recently, the authors found levels of neurokinin (NK) A-like immunoreactivities in the human retina which were more than five times higher than those of substance P (SP). The present study aimed to find out how many of these immunoreactivities can be attributed to NKA and NKB and then the exact distribution pattern of both NKA and NKB was evaluated in the human retina and compared with that of SP. For this purpose, NKA-like immunoreactivities were characterized in the human retina by reversed phase HPLC followed by radioimmunoassay using the K12 antibody which recognizes both NKA and NKB. Furthermore, the retinae from both a 22- and 70-year-old donor were processed for double-immunofluorescence NKA/SP and NKB/SP. The results showed that NKA contributes to approximately two thirds and NKB to approximately one third of the immunoreactivities measured with the K12 antibody. NKA was found to be localized in sparse amacrine cells in the proximal inner nuclear layer, in displaced amacrine cells in the ganglion cell layer with processes ramifying in stratum 3 of the inner plexiform layer and also in sparse ganglion cells. By contrast, staining for NKB was only observed in ganglion cells and in the nerve fiber layer. Double-immunofluorescence revealed cellular colocalization of NKA with SP and also of NKB with SP. Thus, the levels of NKA and NKB are more than three and two times higher than those of SP, respectively. Whereas the distribution pattern of NKA is typical for neuropeptides, the localization of NKB exclusively in ganglion cells is atypical and unique.
Keywords: Neurokinin A; Neurokinin B; Retina; Immunofluorescence;
Disruption of the kinin B1 receptor gene affects potentiating effect of captopril on BK-induced contraction in mice stomach fundus by Ana M.R.B. Barbosa; Sandra A. Felipe; João B. Pesquero; Antonio C.M. Paiva; Suma I. Shimuta (3377-3382).
A transgenic mouse model, deficient in kinin B1 receptor (B1 −/−) was used to evaluate the role of B2 receptor in the smooth muscle stomach fundus. The results showed that the potency of bradykinin (BK) to induce contraction in the gastric tissue was maintained whereas the efficacy was markedly reduced. The angiotensin converting enzyme (ACE) inhibitor captopril potentiated BK-induced effect in wild type (WT) but not in B1 −/− fundus. However, ACE activity detected by the convertion of Ang I to Ang II was inhibited by captopril in both types of gastric tissues. Taking into account the hypothesis that captopril and ACE bind to the B2 receptor, we suggest that this complex was not formed in the stomach deficient in B1 receptor. Therefore, our finding strongly support the hypothesis that in smooth muscles that constitutively express the kinin B1 and B2 receptors, an interaction between captopril and ACE, B1 and B2 receptors should occur forming a complex protein interaction for the potentiating effect of ACE on kinin receptors.
Keywords: Kinin B1 receptor knockout; Bradykinin; Angiotensin converting enzyme inhibitor;
TFF3-peptide increases transepithelial resistance in epithelial cells by modulating claudin-1 and -2 expression by Dirk Meyer zum Büschenfelde; Rudolf Tauber; Otmar Huber (3383-3390).
TFF3 plays an important role in the protection and repair of the gastrointestinal mucosa. The molecular mechanisms of TFF function, however, are still largely unknown. Increasing evidence indicates that apart from stabilizing mucosal mucins TFF3 induces cellular signals that modulate cell–cell junctions of epithelia. In transfected HT29/B6 and MDCK cells stably expressing FLAG-tagged human TFF3 we have recently shown that TFF3 down-regulates E-cadherin, impairs the function of adherens junctions and thus facilitates cell migration in wounded epithelial cell layers. Here we investigate TFF3-induced effects on the composition and function of tight junctions in these cells. TFF3 increased the cellular level of tightening claudin-1 and decreased the amount of claudin-2 known to form cation-selective channels. Expression of ZO-1, ZO-2 and occludin was not altered. The change in claudin-1 and -2 expression in TFF3-expressing HT29/B6 cells was accompanied by an increase in the transepithelial resistance in confluent monolayers of these cells. These data suggest that TFF3 plays a role in the regulation of intestinal barrier function by altering the claudin composition within tight junctions thus decreasing paracellular permeability of the intestinal mucosa.
Keywords: Claudin; Occludin; TFF3; Tight junction; Epithelial barrier;
Endogenous opiates and behavior: 2005 by Richard J. Bodnar; Gad E. Klein (3391-3478).
This paper is the 28th consecutive installment of the annual review of research concerning the endogenous opioid system, now spanning over a quarter-century of research. It summarizes papers published during 2005 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity, neurophysiology and transmitter release (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); immunological responses (Section 17).
Keywords: Opioids; mu Receptor; kappa Receptor; delta Receptor; Enkephalins; Endomorphins; Dynorphin; beta-Endorphin;
Biophysical delivery of peptides: Applicability for cancer therapy by Crispin R. Dass; Peter F.M. Choong (3479-3488).
There is a current trend towards evaluation of molecular agents for treatment of a variety of ailments, including cancer. One class of such biomolecules is proteins, and their shortened versions, peptides. Use of peptidic entities has been hindered by poor bioavailability in vivo and the high cost involved in mass-producing these macromolecular drugs. The need for localized delivery is being met with the development of various biophysical means, which include devices and aids, mainly transdermal and invasive implants. In addition, various cell-based delivery modalities, which include the use of spore-forming bacteria and stem cells, are being explored. This review discusses these methods in turn, and examines ways by which these can be enhanced for peptide delivery to tumors.
Keywords: Peptide; Protein; Drug delivery; Cancer; Device; Cell therapy;