Peptides (v.28, #3)
Editorial Board (CO2).
Novel peptides with tyrosinase inhibitory activity by Marloes Schurink; Willem J.H. van Berkel; Harry J. Wichers; Carmen G. Boeriu (485-495).
Tyrosinase inhibition by peptides may find its application in food, cosmetics or medicine. In order to identify novel tyrosinase inhibitory peptides, protein-based peptide libraries made by SPOT synthesis were used to screen for peptides that show direct interaction with tyrosinase. One of the peptide libraries studied consists of overlapping, octameric peptides derived from industrial proteins as β-casein, α-lactalbumin, β-lactoglobulin, ovalbumin, gliadin, glycinin, and β-conglycinin. On-membrane activity staining resulted in a set of peptides that are not only able to bind to tyrosinase, but are able to inhibit tyrosinase as well. Peptides containing aspartic or glutamic acid residues usually do not bind very well to tyrosinase. Strong tyrosinase-binding peptides always contain one or more arginine residues, often in combination with phenylalanine, while lysine residues can be found equally among nonbinding peptides as well as moderate tyrosinase-binding peptides. The presence of the hydrophobic, aliphatic residues valine, alanine or leucine appears to be important for tyrosinase inhibition. Therefore, good tyrosinase inhibitory peptides preferably contain arginine and/or phenylalanine in combination with valine, alanine and/or leucine.
Keywords: Tyrosinase; Inhibitors; Protein–protein interaction; Peptide SPOT synthesis; Fluorescent protein labeling;
Antibodies against a multiple-peptide conjugate comprising chemically modified human immunodeficiency virus type-1 functional Tat peptides inhibit infection by Krishnakumar Devadas; Robert A. Boykins; Indira K. Hewlett; Owen L. Wood; Kathleen A. Clouse; Kenneth M. Yamada; Subhash Dhawan (496-504).
We demonstrated recently that selective side-chain modification of functional cysteine-rich (Tat21–40) and arginine-rich (Tat53–68) domains of the HIV-1 Tat protein blocks pathogenic activities of these peptides while retaining their immunological characteristics. In the present study, we have synthesized a multiple-peptide conjugate system comprising modified Tat21–40 and Tat53–68 peptides (HIV-1-Tat-MPC). Immunization of mice with this highly homogeneous 10.7 kDa HIV-1-Tat-MPC synthetic construct induced an effective immune response in mice. The antibodies generated against HIV-1-Tat-MPC efficiently suppressed Tat-induced viral replication and significantly reduced HIV-associated cytopathic effects in human monocytes. These results indicate that epitope-specific antibodies directed against functional sites of Tat protein using non-pathogenic peptides inhibit HIV pathogenesis. The HIV-1-Tat-MPC, therefore, has potential for the development of a safe, effective, and economical therapeutic vaccine to reduce the progression of HIV infection.
Keywords: HIV-Tat; Peptides; AIDS; Vaccination;
Expression of genes encoding antimicrobial and bradykinin-related peptides in skin of the stream brown frog Rana sakuraii by Hiroe Suzuki; Shawichi Iwamuro; Aya Ohnuma; Laurent Coquet; Jérôme Leprince; Thierry Jouenne; Hubert Vaudry; Christopher K. Taylor; Peter W. Abel; J. Michael Conlon (505-514).
Peptidomic analysis of an extract of the skin of the stream brown frog Rana sakuraii Matsui and Matsui, 1990 led to the isolation of a C-terminally α-amidated peptide (VR-23; VIGSILGALASGLPTLISWIKNR·NH2) with broad-spectrum antimicrobial activity that shows structural similarity to the bee venom peptide, melittin together with two peptides belonging to the temporin family (temporin-1SKa; FLPVILPVIGKLLNGIL·NH2 and temporin-1SKb; FLPVILPVIGKLLSGIL·NH2), and peptides whose primary structures identified them as belonging to the brevinin-2 (2 peptides) and ranatuerin-2 (1 peptide) families. Using a forward primer that was designed from a conserved region of the 5′-untranslated regions of Rana temporaria preprotemporins in a 3′-RACE procedure, a cDNA clone encoding preprotemporin-1SKa was prepared from R. sakuraii skin total RNA. Further preprotemporin cDNAs encoding temporin-1SKc (AVDLAKIANIAN KVLSSL F·NH2) and temporin-1SKd (FLPMLAKLLSGFL·NH2) were obtained by RT-PCR. Unexpectedly, the 3′-RACE procedure using the same primer led to amplification of a cDNA encoding a preprobradykinin whose signal peptide region was identical to that of preprotemporin-1SKa except for the substitution Ser18 → Asn. R. sakuraii bradykinin ([Arg0,Leu1,Thr6,Trp8] BK) was 28-fold less potent than mammalian BK in effecting B2 receptor-mediated relaxation of mouse trachea and the des[Arg0] derivative was only a weak partial agonist. The evolutionary history of the Japanese brown frogs is incompletely understood but a comparison of the primary structures of the R. sakuraii dermal peptides with those of Tago's brown frog Rana tagoi provides evidence for a close phylogenetic relationship between these species.
Keywords: Frog skin; Melittin; Temporin; Brevinin-2; Ranatuerin-2; Bradykinin;
Isolation and characterization of a novel bradykinin potentiating peptide (BPP) from the skin secretion of Phyllomedusa hypochondrialis by Katia Conceição; Katsuhiro Konno; Robson Lopes de Melo; Marta M. Antoniazzi; Carlos Jared; Juliana M. Sciani; Isaltino M. Conceição; Benedito C. Prezoto; Antônio Carlos Martins de Camargo; Daniel C. Pimenta (515-523).
Bradykinin potentiating peptides (BPPs) from Bothrops jararaca venom were first described in the middle of 1960s and were the first natural inhibitors of the angiotensin-converting enzyme (ACE). BPPs present a classical motif and can be recognized by their typical pyroglutamyl (Pyr)/proline rich sequences presenting, invariably, a proline residue at the C-terminus. In the present study, we describe the isolation and biological characterization of a novel BPP isolated from the skin secretion of the Brazilian tree-frog Phyllomedusa hypochondrialis. This new BPP, named Phypo Xa presents the sequence Pyr-Phe-Arg-Pro-Ser-Tyr-Gln-Ile-Pro-Pro and is able to potentiate bradykinin activities in vivo and in vitro, as well as efficiently and competitively inhibit ACE. This is the first canonical BPP (i.e. Pyr-Aaa n -Gln-Ile-Pro-Pro) to be found not only in the frog skin but also in any other natural source other than the snake venoms.
Keywords: Phyllomedusa hypochondrialis; Bradykinin-potentiating peptide; Mass spectrometry; De novo sequencing; Natural peptides; Secretion; Bradykinin;
Antimicrobial peptides from the skin of the Japanese mountain brown frog Rana ornativentris: Evidence for polymorphism among preprotemporin mRNAs by Aya Ohnuma; J. Michael Conlon; Keiko Yamaguchi; Hiroaki Kawasaki; Laurent Coquet; Jérôme Leprince; Thierry Jouenne; Hubert Vaudry; Shawichi Iwamuro (524-532).
A previous study led to the isolation of antimicrobial peptides belonging to the temporin and brevinin-2 families from a pooled extract of the skin of adult specimens of the Japanese mountain brown frog Rana ornativentris Werner 1903. In order to ascertain whether individual frogs expressed the full complement of temporin genes, we individually cloned cDNAs encoding the temporin precursors from total RNA extracted from the skins of 12 frogs by RT-PCR using a set of preprotemporin-specific primers. All the specimens examined contained mRNAs directing the synthesis of the novel, but inactive, temporin-1Oe (ILPLLGNLLNGLL·NH2). Nucleotide sequence analysis revealed marked polymorphism among individual frogs. Twenty-seven distinct preprotemporin-1Oe mRNAs were identified that contained synonymous substitutions in the antimicrobial peptide region and both synonymous and non-synonymous substitutions in the signal peptide and intervening sequence regions. Up to eight preprotemporin-1Oe mRNA variants were found within a single frog. In addition, several cDNAs encoding preprotemporin-1Oa and -1Ob and a single cDNA encoding preprotemporin-1Oc were characterized. Peptidomic analysis of norepinephrine-stimulated skin secretions revealed the presence of temporin-1Oe, temporin-1Of (SLILKGLASIAKLF·NH2), temporin-1Og (FLSSLLSKVVSLFT·NH2), four members of the ranatuerin-2 family and one member of the palustrin-2 family in addition to previously characterized temporin and brevinin-2 peptides.
Keywords: Antimicrobial peptide; Frog skin; Preprotemporin; Gene polymorphism;
Purification and characterization of eight peptides from Galleria mellonella immune hemolymph by Małgorzata Cytryńska; Paweł Mak; Agnieszka Zdybicka-Barabas; Piotr Suder; Teresa Jakubowicz (533-546).
Defense peptides play a crucial role in insect innate immunity against invading pathogens. From the hemolymph of immune-challenged greater wax moth, Galleria mellonella (Gm) larvae, eight peptides were isolated and characterized. Purified Gm peptides differ considerably in amino acid sequences, isoelectric point values and antimicrobial activity spectrum. Five of them, Gm proline-rich peptide 2, Gm defensin-like peptide, Gm anionic peptides 1 and 2 and Gm apolipophoricin, were not described earlier in G. mellonella. Three others, Gm proline-rich peptide 1, Gm cecropin D-like peptide and Galleria defensin, were identical with known G. mellonella peptides. Gm proline-rich peptides 1 and 2 and Gm anionic peptide 2, had unique amino acid sequences and no homologs have been found for these peptides. Antimicrobial activity of purified peptides was tested against Gram-negative and Gram-positive bacteria, yeast and filamentous fungi. The most effective was Gm defensin-like peptide which inhibited fungal and sensitive bacteria growth in a concentration of 2.9 and 1.9 μM, respectively. This is the first report describing at least a part of defense peptide repertoire of G. mellonella immune hemolymph.
Keywords: Galleria mellonella; Insect immunity; Antibacterial/antimicrobial peptides; Hemolymph; Peptide purification;
An antifungal peptide from red lentil seeds by H.X. Wang; T.B. Ng (547-552).
An antifungal peptide, with a molecular mass of 11 kDa, was isolated from dry seeds of the red lentil (Lens culinaris) using a procedure that involved four chromatographic steps. The antifungal peptide was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and S-Sepharose. The final chromatographic step involved gel filtration by fast protein liquid chromatography on Superdex 75. The antifungal peptide inhibited mycelial growth in Mycosphaerella arachidicola with an IC50 of 36 μM. It also exhibited antifungal activity against Fusarium oxysporum, but there was no inhibitory activity toward tumor cell lines and human immunodeficiency virus type 1 reverse transcriptase (RT).
Keywords: Antifungal peptide; Lens culinaris; Isolation;
Bacisubin, an antifungal protein with ribonuclease and hemagglutinating activities from Bacillus subtilis strain B-916 by Yongfeng Liu; Zhiyi Chen; T.B. Ng; Jie Zhang; Mingguo Zhou; Fuping Song; Fan Lu; Youzhou Liu (553-559).
An antifungal protein, with a molecular mass of 41.9 kDa, and designated as bacisubin, was isolated from a culture of Bacillus subtilis strain B-916. The isolation procedure consisted of ion exchange chromatography on DEAE-Sepharose Fast Flow, and fast protein liquid chromatography on Phenyl Sepharose 6 Fast Flow and hydroxyapatite columns. The protein was adsorbed on all three chromatographic media. Bacisubin exhibited inhibitory activity on mycelial growth in Magnaporthe grisease, Sclerotinia sclerotiorum, Rhizoctonia solani, Alternaria oleracea, A. brassicae and Botrytis cinerea. The IC50 values of its antifungal activity toward the last four fungal species were 4.01 μM, 0.087 μM, 0.055 μM and 2.74 μM, respectively. Bacisubin demonstrated neither protease activity, nor protease inhibitory activity. However, it manifested ribonuclease and hemagglutinating activities.
Keywords: Bacillus subtilis; Antifungal protein; Purification;
A peptide with HIV-1 reverse transcriptase inhibitory activity from the medicinal mushroom Russula paludosa by Jianbin Wang; H.X. Wang; T.B. Ng (560-565).
Hot water extracts of 16 species of mushrooms, including both edible and medicinal mushrooms, were screened for human immunodeficiency virus (HIV)-1 reverse transcriptase (RT) inhibitory activity. Extracts of Lactarius camphoratus, Trametes suaveolens, Sparassis crispa, Pleurotus sajor-caju, Pleurotus pulmonarius, and Russula paludosa elicited over 50% inhibition when tested at the concentration of 1 mg/ml. The extract of R. paludosa demonstrated the highest inhibitory activity on HIV-1 RT (97.6%). Fraction SU2, purified from R. paludosa extract by anion exchange chromatography on DEAE-cellulose and gel filtration on Superdex 75, exhibited potent inhibitory activity on HIV-1 RT. At the concentrations of 1 mg/ml, 0.2 mg/ml, and 0.04 mg/ml, the inhibition ratios were 99.2%, 89.3%, and 41.8%, respectively, giving an IC50 of 11 μM. The molecular mass of SU2 was 4.5 kDa and its N-terminal amino acid sequence was determined to be KREHGQHCEF. The peptide was devoid of hemagglutinating, ribonuclease, antifungal, protease, protease inhibitory, and laccase activities.
Keywords: AIDS; HIV; Reverse transcriptase (RT); Purification; Edible and medicinal mushrooms;
Significance of the N- and C-terminal regions of CAP-1, a cuticle calcification-associated peptide from the exoskeleton of the crayfish, for calcification by Hirotaka Inoue; Tsuyoshi Ohira; Hiromichi Nagasawa (566-573).
Calcification-associated peptide (CAP)-1 is considered to play an important role in calcification of the exoskeleton of the crayfish, Procambarus clarkii. In this study, in order to clarify the molecular mechanism of calcification, we constructed expression systems of recombinant molecules of CAP-1 and its related peptides in Escherichia coli, and verified the structure–activity relationship of these peptides. The inhibitory assay on calcium carbonate precipitation and the calcium-binding assay showed that the C-terminal acidic region was most important for both activities. The CD spectra of these peptides varied depending on calcium concentration and showed that the N-terminal region is associated with the peptide conformation. These results indicate that the C-terminal part of CAP-1 may concentrate calcium ions for nucleation and/or interact with calcium carbonate precipitate to control the growth and that the N-terminal part contribute to maintaining the peptide conformation.
Keywords: Biomineralization; Calcification; Calcium binding; Cuticle peptide; Exoskeleton;
Inhibition of PK/PBAN-mediated functions in insects: Discovery of selective and non-selective inhibitors by Miriam Altstein; Orna Ben-Aziz; Irina Zeltser; Kalpana Bhargava; Michael Davidovitch; Allison Strey; Nan Pryor; Ronald J. Nachman (574-584).
The antagonistic properties of a few linear and backbone cyclic (BBC) conformationally constraint peptide libraries and their analogs, were tested for the ability to inhibit pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) mediated functions: sex pheromone biosynthesis in Heliothis peltigera female moths, cuticular melanization in Spodoptera littoralis larvae, pupariation in the fleshfly Neobellieria bullata and hindgut contraction in Leucophaea maderae, elicited by exogenously injected PBAN, pheromonotropin (PT), leucopyrokinin (LPK), myotropin (MT) or by the endogenous peptides. The data revealed differential inhibitory patterns within the same assay with different elicitors (in both the pheromonotropic and melanotropic assays) and among the different functions and disclosed selective antagonists, hinting at the possibility that the receptors that mediate those functions may differ from one another structurally.
Keywords: PBAN; Sex pheromone biosynthesis; Cuticular melanization; Pupariation; Hindgut contraction; Insect neuropeptide antagonists;
Evidence dromyosuppressin acts at posterior and anterior pacemakers to decrease the fast and the slow cardiac activity in the blowfly Protophormia terraenovae by Anna Maria Angioy; Patrizia Muroni; Iole Tomassini Barbarossa; Jennifer McCormick; Ruthann Nichols (585-593).
The molecular complexity of the simple blowfly heart makes it an attractive preparation to delineate cardiovascular mechanisms. Blowfly cardiac activity consists of a fast, high-frequency signal phase alternating with a slow, low-frequency signal phase triggered by pacemakers located in the posterior abdominal heart and anterior thoracocephalic aorta, respectively. Mechanisms underlying FMRFamide-related peptides (FaRPs) effects on heart contractions are not well understood. Here, we report antisera generated to a FaRP, dromyosuppressin (DMS, TDVDHVFLRFamide), recognized neuronal processes that innervated the blowfly Protophormia terraenovae heart and aorta. Dromyosuppressin caused a reversible cardiac arrest. High- and low-frequency signals were abolished after which they resumed; however, the concentration-dependent resumption of the fast phase differed from the slow phase. Dromyosuppressin decreased the frequency of cardiac activity in a dose-dependent manner with threshold values between 5 fM and 0.5 fM (fast phase), and 0.5 fM and 0.1 fM (slow phase). Dromyosuppressin structure–activity relationship (SAR) for the decrease of the fast-phase frequency was not the same as the SAR for the decrease of the slow-phase frequency. The alanyl-substituted analog TDVDHVFLAFamide ([Ala9] DMS) was inactive on the fast phase, but active on the slow phase, a novel finding. FaRPs including myosuppressins are reported to require the C-terminal RFamide for activity. Our data are consistent with the conclusions DMS acts on posterior and anterior cardiac tissue to play a role in regulating the fast and slow phases of cardiac activity, respectively, and ligand–receptor binding requirements of the abdominal and thoracocephalic pacemakers are different.
Keywords: Aorta; DMS; FMRFamide; FaRP; Heart; Neurosecretion;
A novel adipokinetic peptide in a water boatman (Heteroptera, Corixidae) and its bioanalogue in a saucer bug (Heteroptera, Naucoridae) by Gerd Gäde; Petr Šimek; Heather G. Marco (594-601).
The corpora cardiaca (CC) of two water bug species, the water boatman Corixa punctata and the saucer bug Ilyocoris cimicoides, contain a substance that cause hyperlipemia in the migratory locust. The primary sequence of one octapeptide belonging to the adipokinetic hormone (AKH)/red pigment-concentrating hormone (RPCH) family was deduced from the multiple MSN electrospray mass data of CC material from each species. Whereas the saucer bug contains the known octapeptide pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp amide, code-named Anaim-AKH, the water boatman has a novel peptide identified as pGlu-Leu/Ile-Asn-Phe-Ser-Pro-Ser-Trp amide, code-named Corpu-AKH. The ambiguity about the amino acid at position 2, i.e. Leu or Ile, in Corpu-AKH was solved by isolating the peptide in a single-step by reversed-phase HPLC and establishing co-elution with the synthetic peptide containing Leu at position 2. Functionally, the peptides regulate lipid mobilization, as evidenced by an adipokinetic effect after injecting synthetic Anaim-AKH and Corpu-AKH into the respective acceptor species. Swimming activity of I. cimicoides also causes hyperlipemia.
Keywords: Insects; Heteroptera; AKH/RPCH family; Mass spectrometry;
Suppression of food intake by a complement C3a agonist [Trp5]-oryzatensin(5–9) by Kousaku Ohinata; Kentaro Suetsugu; Yoko Fujiwara; Masaaki Yoshikawa (602-606).
[Trp5]-oryzatensin(5–9) (WPLPR), an agonist peptide for complement C3a receptor, has been designed based on the C-terminal region of ileum-contracting peptide oryzatensin derived from rice protein. We previously reported that WPLPR has anti-analgesic and anti-amnesic activities after central or oral administration. In this study, we found a novel function of WPLPR on food intake. WPLPR suppressed food intake after intracerebroventricular or intraperitoneal (i.p.) administration at a dose of 3–30 nmol/mouse or 30–300 mg/kg, respectively, in fasted mice. Orally administered WPLPR at a dose of 300 mg/kg also decreased food intake. WPLPR decreased gastric emptying after i.p. injection at a dose of 300 mg/kg. The anorexigenic activity of WPLPR was blocked by cyclooxygenase inhibitor or antagonist for prostaglandin (PG) E receptor EP4 subtype. These results suggest that WPLPR decreases food intake through PGE2 production followed by EP4 receptor activation.
Keywords: Food intake; WPLPR; Complement C3a; Prostaglandin E2; EP4 receptor;
Intravenous CCK-8, but not GLP-1, suppresses ghrelin and stimulates PYY release in healthy men by Ixchel M. Brennan; Bärbel Otto; Kate L. Feltrin; James H. Meyer; Michael Horowitz; Christine Feinle-Bisset (607-611).
We have investigated the effects of exogenous CCK-8 and GLP-1, alone and in combination, on ghrelin and PYY secretion. Nine healthy males were studied on four occasions. Plasma ghrelin and PYY concentrations were measured during 150 min intravenous infusions of: (i) isotonic saline, (ii) CCK-8 at 1.8 pmol/kg/min, (iii) GLP-1 at 0.9 pmol/kg/min or (iv) CCK-8 and GLP-1 combined. CCK-8 markedly suppressed ghrelin and stimulated PYY when compared with control between t = 0–120 min (P < 0.001 for both). GLP-1 had no effect on ghrelin, but decreased PYY slightly at 120 min (P < 0.05). During infusion of CCK-8 + GLP-1, there was comparable suppression of ghrelin (P < 0.001), but the stimulation of PYY was less (P < 0.001), than that induced by CCK-8, between t = 20–120 min. In conclusion, in healthy subjects, in the doses evaluated, exogenous CCK-8 suppresses ghrelin and stimulates PYY, and exogenous GLP-1 has no effect on ghrelin and attenuates the effect of CCK-8 on PYY.
Keywords: Cholecystokinin; Glucagon-like peptide-1; Peptide YY; Ghrelin;
Intraventricular (i3vt) ghrelin increases food intake in fatty Zucker rats by Lynda M. Brown; Stephen C. Benoit; Stephen C. Woods; Deborah J. Clegg (612-616).
Ghrelin is an orexigenic peptide secreted from the stomach and also made in the brain. Ghrelin receptors are expressed on hypothalamic cells important in appetite and energy balance. We determined that intra-3rd-ventricular (i3vt) ghrelin dose-dependently increases acute (1 and 2 h) food intake in lean and fatty Zucker rats (0, 0.01, 0.1 and 1.0 nmol ghrelin). The percentage increase of food intake in fatty Zucker rats was significantly greater than that in lean rats. Fatty Zucker rats had 4.5 times more ghrelin receptor mRNA in the hypothalamus than lean Zucker rats, suggesting a possible mechanism for the increased sensitivity.
Keywords: Zucker fatty rats; Energy balance; Ghrelin receptor; Hypothalamus; Appetite control;
Contractile effects of ghrelin-related peptides on the chicken gastrointestinal tract in vitro by Takio Kitazawa; Hiroyuki Kaiya; Tetsuro Taneike (617-624).
Ghrelin is an endogenous ligand for growth hormone secretagogue receptor (GHS-R), and it stimulates growth hormone (GH) release, food intake and gastrointestinal motility in mammals. Ghrelin has also been identified in the chicken, but this peptide inhibits food intake in the chicken. We examined the effects of ghrelin and related peptides on contractility of the isolated chicken gastrointestinal tract in vitro. Among ghrelin-related peptides examined (1 μM of rat ghrelin, human ghrelin, chicken ghrelin and growth hormone releasing peptide-6 (GHRP-6)), only chicken ghrelin was effective on contraction of the chicken gastrointestinal tract. Des-acyl chicken ghrelin was ineffective, suggesting that octanoylation at Ser3 residue of chicken ghrelin was essential for inducing the contraction. Amplitude of chicken ghrelin-induced contraction was region-specific: highest in the crop and colon, moderate in the esophagus and proventriculus, and weak in the small intestine. The contractile response to chicken ghrelin in the crop was not affected by tetrodotoxin (TTX), but that in the proventriculus was decreased by TTX and atropine to the same extents. d-Lys3-GHRP-6 (a GHS-R antagonist) caused a transient contraction and inhibited the effect of chicken ghrelin without affecting the high-K+-induced contraction. Chicken ghrelin potentiated electrical field stimulation-induced cholinergic contraction without affecting the responsiveness to bath-applied carbachol in the proventriculus. The location of GHS-R differs in the crop (smooth muscle) and proventriculus (smooth muscle and enteric neurons). These results indicate that ghrelin has contractile activity on gastrointestinal tract in the chicken in vitro, and the effect was region-specific. The action would be mediated through the GHS-R, which is highly sensitive to chicken ghrelin.
Keywords: Ghrelin; Motilin; Growth hormone secretagogue receptor; Chicken gastrointestinal tract; Contraction;
Motilin activates neurons in the rat amygdala and increases gastric motility by Xin Feng; Theo Louis Peeters; Ming Tang (625-631).
Motilin and motilin receptors have been found in most regions of the brain, including the amygdala, one of the most important parts of the limbic system. Our previous study found that administration of motilin in the hippocampus stimulates gastric motility. We now explore the effect of motilin in the amygdala on gastric motility. In conscious rats, gastric motility was recorded after microinjection of motilin, motilin receptor antagonist (GM-109) or a mixture of the two into the basomedial amygdala nucleus (BMA). In anesthetized rats the changes of spontaneous discharges of gastric distention sensitive neurons (GDSN) in the BMA were recorded after intracerebroventricular (i.c.v.) microinjection of motilin or GM-109. In conscious rats the amplitude of gastric contractions increased dose-dependently after microinjection of motilin in the BMA, and decreased after microinjection of GM-109. The excitatory or inhibitory effects induced by motilin or GM-109 alone, were weakened by microinjection of a mixture solution of both. The spontaneous discharge frequency of gastric distention excitatory neuron (GDEN) was mainly inhibited by i.c.v. microinjection of motilin but excited by GM-109. In contrast, the spontaneous discharge frequency of gastric distention inhibitory neuron (GDIN) was mainly excited by motilin, but inhibited by GM-109. Our findings suggest that motilin may regulate gastric motility by modulating neural pathways in the BMA.
Keywords: Motilin; Motilin receptor antagonist; Gastric motility; Basomedial amygdala nucleus; Electrophysiology;
Gastrin 1–6 promotes growth of colon cancer cells through non-CCK receptors by Jeffrey Copps; Shawn Ahmed; Richard F. Murphy; Sándor Lovas (632-635).
Our previous studies have shown that stimulation of proliferation of DLD-1 and HT29 human colonic cancer cells by nanomolar gastrin (G17) and carboxymethyl gastrin (G17Gly) and reversal of growth by micromolar G17 and G17Gly involves binding sites which can neither be CCK1 nor CCK2 receptors; the N terminal fragment, G17(1–12), is sufficient to increase the number of HT-29 cells by binding the higher affinity binding site but is without a suppressing effect through the lower affinity site. In this study with DLD-1 cells, competitive binding using 125I-G17(1–12) showed that G17(1–12) binds both high and low affinity sites, as do G17 and G17Gly. G17(1–6)-NH2, even without the central-to-C-terminal portion of G17, was still able to bind a single site and to promote a dose-dependent increase in cell number at nanomolar concentrations. The results indicate the presence of a non-CCK receptor on human colonic cancer cells which could mediate the tumor-promoting activity of the N-terminal-to-central portion of G17Gly which, unlike G17, is produced by such cells.
Keywords: Gastrin; Glycine-extended gastrin; N-terminal fragments; Colon cancer; Cell growth; Receptor binding;
Central infusion of the melanocortin receptor antagonist agouti-related peptide (AgRP(83-132)) prevents cachexia-related symptoms induced by radiation and colon-26 tumors in mice by M.A. Joppa; K.R. Gogas; A.C. Foster; S. Markison (636-642).
Cachexia is a clinical wasting syndrome that occurs in multiple disease states, and is associated with anorexia and a progressive loss of body fat and lean mass. The development of new therapeutics for this disorder is needed due to poor efficacy and multiple side effects of current therapies. The pivotal role played by the central melanocortin system in regulating body weight has made this an attractive target for novel cachexia therapies. The mixed melanocortin receptor antagonist AgRP is an endogenous peptide that induces hyperphagia. Here, we used AgRP(83-132) to investigate the ability of melanocortin antagonism to protect against clinical features of cachexia in two distinct animal models. In an acute model, food intake and body weight gain were reduced in mice exposed to radiation (300 RAD), and delivery of AgRP(83-132) into the lateral cerebral ventricle prevented these effects. In a chronic tumor cachexia model, adult mice were injected subcutaneously with a cell line derived from murine colon-26 adenocarcinoma. Typical of cachexia, tumor-bearing mice progressively reduced body weight and food intake, and gained significantly less muscle mass than controls. Administration of AgRP(83-132) into the lateral ventricles significantly increased body weight and food intake, and changes in muscle mass were similar to the tumor-free control mice. These findings support the idea that antagonism of the central melanocortin system can reduce the negative impact of cachexia and radiation therapy.
Keywords: Feeding; Anorexia; Cytokine; Tumor; Melanocortin; AgRP;
Enterostatin inhibition of dietary fat intake is modulated through the melanocortin system by Ling Lin; MieJung Park; David A. York (643-649).
Enterostatin injected into the amygdala selectively reduces dietary fat intake by an action that involves a serotonergic component in the paraventricular nucleus. We have investigated the role of melanocortin signaling in the response to enterostatin by studies in melanocortin 4 receptor (MC4R) knock out mice and by the use of the MC4R and MC3R antagonist SHU9119, and by neurochemical phenotyping of enterostatin activated cells. We also determined the effect of enterostatin in vivo on the expression of AgRP in the hypothalamus and amygdala of rats and in culture on a GT1-7 neuronal cell line. Enterostatin had no effect on food intake in MC4R knock out mice. SHU9119 icv blocked the feeding response to amygdala enterostatin in rats. Amygdala enterostatin induced fos activation in α-melanocyte stimulating hormone (α-MSH) neurons in the arcuate nucleus. Enterostatin also reduced the expression of AgRP in the hypothalamus and amygdala and in GT1-7 cells. These data suggest enterostatin inhibits dietary fat intake through a melanocortin signaling pathway.
Keywords: Melanocortin receptors; Melanocyte stimulating hormone; Dietary fat; Amygdala;
The selective orexin 1 receptor antagonist SB-334867-A impairs acquisition and consolidation but not retrieval of spatial memory in Morris water maze by Esmaeil Akbari; Nasser Naghdi; Fereshteh Motamedi (650-656).
The novel neuropeptides orexin-A and orexin-B derive from a common 130-amino acid precursor molecule (prepro-orexin), are mainly localized to neurons within and around the lateral hypothalamus, and exhibit high affinity to the closely related G-Protein-coupled receptors orexin 1 and 2 receptor (OX1R, OX2R). Orexinergic neurons send their axons to the hippocampal formation (CA1, CA2 and dentate gyrus), which expresses OX1Rs. Recent studies have shown that central administration of orexin-A and orexin-B have effects on learning and memory but literature concerning the role of orexinergic system in cognition remains controversial. More recently, antagonists have been described. The most potent and selective is SB-334867-A, which has an affinity of 40 nM at OX1R which is at least 50-fold selective over OX2R. It is likely that the intracerebroventricular (i.c.v.) administration may block OX1Rs in many brain regions. Previously we have shown that intra-CA1 injection of SB-334867-A impairs acquisition, consolidation and retrieval of spatial memory in MWM task. In the present study, the effect of pre-training, post-training and pre-probe of trial intra-DG (dentate gyrus) administration of SB-334867-A (1.5, 3, 6 μg/0.5 μl) on acquisition, consolidation and retrieval in a single-day testing version of MWM (Morris water maze) task was examined. Our results show impaired acquisition and consolidation of MWM task for SB-334867-A as compared with the control group. However, SB-334867-A had no effect on retrieval in spatial memory. Also, this antagonist had no effect on escape latency of a non-spatial visual discrimination task. Therefore, it seems that endogenous orexin-A and orexin-B, through DG OX1Rs, play an important role in spatial learning and memory in the rat.
Keywords: Spatial learning and memory; Orexin 1 receptor; SB-334867-A; Dentate gyrus; Morris water maze;
Periaqueductal gray cholecystokinin infusions block morphine-induced disruption of maternal behavior by Claudia M. Miranda-Paiva; Newton S. Canteras; Marcia H. Sukikara; Antonia G. Nasello; Ivone I. Mackowiak; Luciano F. Felicio (657-662).
Cholecystokinin (CCK) and opiates interaction is critical for maintaining maternal behavior during lactation. Morphine inhibits while CCK restores maternal behavior. Recently we have shown that periaqueductal gray (PAG) is a region critically involved in the opioidergic blockade of maternal behavior. A critical level of morphine-induced activation of the rostral lateral PAG is required to inhibit maternal behavior in lactating rats. Since central CCK injections reverted morphine-induced inhibition of maternal behavior, we tested whether this peptide would act similarly in the PAG. This hypothesis was confirmed in experiments showing that morphine's inhibitory effect on maternal responsiveness was blocked by 1.0 and 0.2 nmol CCK injections into the rostral PAG, but not in nearby regions of the mesencephalic reticular nucleus. To test for possible compensatory changes the CCK 2 receptor due to morphine treatments the expression of CCK 2 receptor mRNA was evaluated in the PAG. PAG CCK 2 receptor cDNA amplification revealed no difference in morphine treated animals. These results broaden understanding of the role played by CCK in the PAG. This CCK action might not depend on changes in its receptor.
Keywords: CCK 2 receptor mRNA; PAG; Morphine; Lactation; Sensitization;
UFP-101 antagonizes the spinal antinociceptive effects of nociceptin/orphanin FQ: Behavioral and electrophysiological studies in mice by Cristiano Nazzaro; Anna Rizzi; Severo Salvadori; Remo Guerrini; Domenico Regoli; Hanns Ulrich Zeilhofer; Girolamo Calo (663-669).
Nociceptin/orphanin FQ (N/OFQ) modulates various biological functions, including nociception, via selective stimulation of the N/OFQ peptide receptor (NOP). Here we used the NOP selective antagonist UFP-101 to characterize the receptor involved in the spinal antinociceptive effects of N/OFQ evaluated in the mouse tail withdrawal assay and to investigate the mechanism underlying this action by assessing excitatory postsynaptic currents (EPSC) in laminas I and II of the mouse spinal cord dorsal horn with patch-clamp techniques. Intrathecal (i.t.) injection of N/OFQ in the range of 0.1–10 nmol produced a dose dependent antinociceptive effect, which was prevented by UFP-101, but not by naloxone. In contrast the antinociceptive effect of the mu-opioid peptide receptor agonist endomorphin-1 was blocked by naloxone but not by UFP-101. Moreover, N/OFQ and endomorphin-1 induced a significant antinociceptive effect in wild type mice while in mice knockout for the NOP receptor gene only endomorphin-1 was found to be active. In mouse spinal cord slices 1 μM N/OFQ reduced EPSC to 60 ± 4% of control values. This inhibitory effect was reversed in a concentration dependent manner by UFP-101 (pA2 value 6.44). The present results demonstrate that N/OFQ-induced spinal antinociception in vivo and inhibition of spinal excitatory transmission in vitro are mediated by receptors of the NOP type.
Keywords: Nociceptin/orphanin FQ; NOP receptors; UFP-101; Tail withdrawal; Patch-clamp; Mouse;
Possible involvement of endogenous nociceptin/orphanin FQ in the pain-related behavioral responses induced by its own metabolite, nociceptin/orphanin FQ(14–17) by Hiroyuki Watanabe; Hirokazu Mizoguchi; Tohru Orito; Sou Katsuyama; Akihiko Yonezawa; Chizuko Watanabe; Tsukasa Sakurada; Shinobu Sakurada (670-677).
Nociceptin/orphanin FQ(14–17) (N/OFQ(14–17)) is one of the major fragments that are released from N/OFQ, an endogenous ligand for the opioid receptor like-1 (ORL-1) receptor by endopeptidase 24.11. In the present study, we determined the pharmacological profiles of N/OFQ(14–17) on pain-related behavioral responses in the mouse. Intrathecal (i.t.) administration of N/OFQ(14–17) (5–160 pmol) evoked pain-related behaviors, and these behavioral responses were reduced by i.t. co-administration of an ORL-1 receptor antagonist, [Nphe1]N/OFQ(1–13)NH2 (4 pmol). However, in the ligand-binding receptor assay, N/OFQ(14–17) had no affinity for the ORL-1 receptor. Furthermore, i.t. pretreatment with an antiserum against N/OFQ (1:50) diminished the N/OFQ(14–17)-induced pain-related behaviors, suggesting that endogenous N/OFQ is involved in their expression. Therefore, N/OFQ(14–17)-induced pain-related behaviors may be mediated through the release of endogenous N/OFQ in the mouse spinal cord.
Keywords: Nociceptin; Orphanin FQ; ORL-1 receptor; Pain; Endopeptidase; Spinal cord;
Serum activity of dipeptidyl peptidase IV (DPPIV; EC 220.127.116.11) in breast-fed infants with symptoms of allergy by Beata Jarmołowska; Krzysztof Bielikowicz; Małgorzata Iwan; Katarzyna Sidor; Elżbieta Kostyra; Maciej Kaczmarski (678-682).
β-Casomorphins, opioid peptides present in mother's milk, are a good substrate for DPPIV (EC 18.104.22.168) which is a major factor limiting the half-life of biologically active peptides. Serum DPPIV activity of two groups of infants (healthy and atopic dermatitis) and contents of β-casomorphin-5 and -7 in their mothers’ milk were determined in the study. We have found correlation between those two parameters in the group of children with atopic dermatitis syndromes, while no such a correlation was found in the control group.
Keywords: β-Casomorphin; Dipeptidyl peptidase IV (DPPIV); Atopic dermatitis;
High glucose promotes the release and expression of novel vasoactive peptide, coupling factor 6, in human umbilical vein endothelial cells by Xiaolu Li; Shanshan Xing; Like Zhang; Qichong Xing; Suhua Yan; Hongyan Dai; Shuling You; Yongzheng Pang; Chaoshu Tang (683-690).
As a novel vasoactive peptide, plasma coupling factor 6 (CF6) was shown to be elevated in patients with diabetes mellitus, yet the mechanism involved is unknown. We studied CF6 protein release and its potential mechanism in human umbilical vein endothelial cells (HUVECs) incubated with high glucose levels. High glucose level enhanced CF6 expression and peptide secretion in HUVECs in a time- and concentration-dependent manner, which was independent of increased osmolarity. PKC or p38 MAPK inhibition significantly suppressed high glucose-mediated CF6 release in HUVECs, and the inhibition rate was −45% and −30%, respectively. Also, high glucose-induced CF6 production was antagonized by insulin treatment. Hence, high glucose increases the expression and secretion of CF6 in endothelial cells and appears to be mediated by PKC and p38 MAPK activity.
Keywords: Coupling factor 6; High glucose; Endothelial cells; p38 Mitogen activated protein kinase; Protein kinase C;
Angiogenesis induced by novel peptides selected from a phage display library by screening human vascular endothelial cells under different physiological conditions by Britta Hardy; Annat Raiter; Chana Weiss; Boris Kaplan; Ariel Tenenbaum; Alexander Battler (691-701).
Angiogenesis is a process modulated by several endogenous vascular growth factors as well as by oxygen conditions. For example VEGF failed to induce useful therapeutic angiogenesis in clinical trials. We used a combinatory phage display peptide library screening on human umbilical endothelial cells under normoxia and hypoxia conditions in order to identify novel peptides that bind endothelial cells. The identified peptides induced angiogenesis as demonstrated by endothelial cell proliferation, migration and tube formation. Injection of peptides into the ears of mice resulted in increased numbers of blood vessels. Peptides did not induce VEGF receptor gene expression indicating a possible VEGF unrelated mechanism.
Keywords: Endothelial cell; Angiogenesis; Peptides; Phage-display library; VEGF receptors;
Evidence for a new angiotensin-(1–7) receptor subtype in the aorta of Sprague–Dawley rats by D.M.R. Silva; H.R. Vianna; S.F. Cortes; M.J. Campagnole-Santos; R.A.S. Santos; V.S. Lemos (702-707).
We have recently described, in the mouse aorta, the vasodilator effect of angiotensin-(1–7) (Ang-(1–7)) was mediated by activation of the Mas Ang-(1–7) receptor and that A-779 and D-Pro7-Ang-(1–7) act as Mas receptor antagonists. In this work we show pharmacological evidence for the existence of a different Ang-(1–7) receptor subtype mediating the vasodilator effect of Ang-(1–7) in the aorta from Sprague–Dawley (SD) rats. Ang-(1–7) induced an endothelium-dependent vasodilator effect in aortic rings from SD rats which was inhibited by removal of the endothelium and by l-NAME (100 μM) but not by indomethacin (10 μM). The Ang-(1–7) receptor antagonist D-Pro7-Ang-(1–7) (0.1 μM) abolished the vasodilator effect of the peptide. However, the other specific Ang-(1–7) receptor antagonist, A-779 in concentrations up to 10 μM, did not affect vasodilation induced by Ang-(1–7). The Ang II AT1 and AT2 receptors antagonists CV11974 (0.01 μM) and PD123319 (1 μM), respectively, the bradykinin B2 receptor antagonist HOE 140 (1 μM) and the inhibitor of ACE captopril (10 μM) did not change the effect of Ang-(1–7). Our results show that in the aorta of SD rats, the vasodilator effect of Ang-(1–7) is dependent on endothelium-derived nitric oxide. This effect is mediated by the activation of Ang-(1–7) receptors sensitive to D-Pro7-Ang-(1–7), but not to A-779, which suggests the existence of a different Ang-(1–7) receptor subtype.
Keywords: Ang-(1–7); Mas receptor; Rat aorta; Vasodilator effect;
Apelin stimulates proliferation and suppresses apoptosis of mouse osteoblastic cell line MC3T3-E1 via JNK and PI3-K/Akt signaling pathways by Si-Yuan Tang; Hui Xie; Ling-Qing Yuan; Xiang-Hang Luo; Jiao Huang; Rong-Rong Cui; Hou-De Zhou; Xian-Ping Wu; Er-Yuan Liao (708-718).
The aim of this study was to investigate the effects of apelin on proliferation and apoptosis of mouse osteoblastic MC3T3-E1 cells. APJ was expressed in MC3T3-E1 cells. Apelin did not affect Runx2 expression, alkaline phosphatase (ALP) activity, osteocalcin and type I collagen secretion, suggesting that it has no effect on osteoblastic differentiation of MC3T3-E1 cells. However, apelin stimulated MC3T3-E1 cell proliferation and inhibited cell apoptosis induced by serum deprivation. Our study also shows that apelin decreased cytochrome c release and caspase-3, capase-8 and caspase-9 activation in serum-deprived MC3T3-E1 cells. Apelin activated c-Jun N-terminal kinase (JNK) and Akt (phosphatidylinositol 3-kinase downstream effector), and the JNK inhibitor SP600125, the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 or the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO) inhibited its effects on proliferation and serum deprivation-induced apoptosis. Furthermore, apelin protected against apoptosis induced by the glucocorticoid dexamethasone or TNF-α. Apelin stimulates proliferation and suppresses serum deprivation-induced apoptosis of MC3T3-E1 cells and these actions are mediated via JNK and PI3-K/Akt signaling pathways.
Keywords: Apelin; Osteoblast; Proliferation; Apoptosis;
Enterostatin (APGPR) enhances memory consolidation in mice by Kousaku Ohinata; Soushi Sonoda; Tomoko Shimano; Masaaki Yoshikawa (719-721).
Enterostatin (APGPR) is a pentapeptide released from its precursor protein, procolipase. We found for the first time that enterostatin has memory-enhancing activity. Enterostatin enhanced memory consolidation after central or oral administration at a dose of 10 nmol/mouse or 300 mg/kg, respectively, in a step-through type passive avoidance test in mice. The memory-enhancing activity of enterostatin was inhibited by pretreatment with lorglumide, an antagonist for cholecystokinin 1 (CCK1) receptor. However, enterostatin had no affinity for CCK receptors. These results suggest that enterostatin improves memory retention through CCK release.
Erratum to “The possible involvement of endogenous ligands for mu-, delta- and kappa-opioid receptors in modulating morphine-induced CPP expression in rats” [Peptides 27 (12) (2006) 3307–3314] by Jing Liang; Yijing Li; Xingjie Ping; Peng Yu; Yanfang Zuo; Liuzhen Wu; Ji-Sheng Han; Cailian Cui (722-723).