Peptides (v.28, #8)
Editorial Board (CO2).
Hemodynamic effects of chronic urotensin II administration in animals with and without aorto-caval fistula by Gregory S. Harris; Robert M. Lust; Laxmansa C. Katwa (1483-1489).
Urotensin II (UTII) is a potent vasoactive peptide. Recent studies have demonstrated increased expression of both UTII and its receptor (UTR) expression in end-stage congestive heart failure (CHF), but it is unclear whether UTII and UTR are late stage markers of decompensation, or earlier adaptive responses. The purpose of this study was to measure the effects of chronic UTII administration in normal and volume overloaded animals. Chronic 4 weeks administration of UTII produced decreases in hemodynamic function in animals not subjected to volume overload while returning function to control levels in animals with overload. Expression levels of calcium regulatory proteins phospholamban (PLN), sarcoplasmic reticulum Ca2+ ATPase (SERCA2), and Na+/Ca2+ exchanger (NCX) were measured to determine if administration of UTII resulted in aberrant Ca2+ handling. Changes in protein expression revealed that UTII influenced Ca2+ handling proteins in normal animals although these changes are not seen in the volume overload.
Keywords: Urotensin II; Volume overload; Congestive heart failure; Hemodynamic function;
Isolation and sequencing of two FMRFamide-related peptides from the gut of Locusta migratoria L. by Sharon R. Hill; Ian Orchard (1490-1497).
Two FMRFamide-related peptides (FaRPs) have been isolated and sequenced from the whole gut of Locusta migratoria L. Peptides were extracted from 500 locust whole guts and separated using reversed-phase high performance liquid chromatography (RP-HPLC). Fractions containing FMRFamide-like immunoreactive (FLI) material were identified using radioimmunoassay (RIA). Sequencing of fractions, using tandem mass spectrometry (MALDI-TOF MS/MS), revealed the myosuppressin previously isolated from the locust CNS, SchistoFLRFamide (PDVDHVFLRFamide), and a novel extended RFamide (LWENLRFamide). The isolation of SchistoFLRFamide from midgut tissue supports the hypothesis that this myosuppressin is released locally from FLI processes over the gut and/or from endocrine-like midgut cells to play a role in the regulation of digestion.
Keywords: FaRP; Myosuppressin; SchistoFLRFamide; LWENLRFamide; Midgut; Circular muscle assay; RP-HPLC; Tandem MALDI-TOF MS;
Structure–function studies of peptides for cell adhesion inhibition: Identification of key residues by alanine mutation and peptide-truncation approach by Cheng Li; Seetharama D. Satyanarayanajois (1498-1508).
Blockage of the interaction of CD2 with its ligand CD58 is expected to bring out potential therapeutic value for autoimmune diseases and organ transplantation. Three series of peptides (cVL, cIL and cAQ series) were designed from ratCD2 and humanCD2 to modulate CD2–CD58 interaction. To determine the specific segments in parent peptides responsible for inhibitory activity as lead sequence, we generated shorter fragments of the parent peptides and evaluated their biological activity with cell adhesion assay. The structure–activity relationship studies indicated that small cyclic peptides derived from CD2 ligand binding epitopes could mimic native β-turn structure, and thus modulate CD2–CD58 interaction.
Keywords: CD2; CD2–CD58 interaction; Minimum inhibitory sequence; Alanine mutation; β-Turn;
In vitro activity of the synthetic lipopeptide PAL-Lys-Lys-NH2 alone and in combination with antifungal agents against clinical isolates of Cryptococcus neoformans by Francesco Barchiesi; Andrea Giacometti; Oscar Cirioni; Daniela Arzeni; Carmela Silvestri; Wojciech Kamysz; Alessandra Abbruzzetti; Alessandra Riva; Elzbieta Kamysz; Giorgio Scalise (1509-1513).
The in vitro activity of the lipopeptide PAL-Lys-Lys-NH2 (PAL), alone or combined with either fluconazole (FLU) or amphotericin B (AMB), was investigated against 14 Cryptococcus neoformans isolates. PAL MICs ranged from 1.0 to 4.0 μg/ml. Fungicidal activity was observed. Synergy, defined as a fractional inhibitory concentration (FIC) index of ≤0.5, was observed in 21.4% of PAL/AMB interactions. Antagonism (FIC index > 4) was never observed. The broad antifungal activity and the positive interactions with AMB suggest that PAL can represent a promising candidate in infections due to C. neoformans.
Keywords: Lipopeptides; Cryptococcus neoformans; Synergy; PAL-Lys-Lys-NH2;
First isolation of an antifungal lipid transfer peptide from seeds of a Brassica species by Peng Lin; Lixin Xia; T.B. Ng (1514-1519).
An antifungal peptide with a molecular mass of 9412 and an N-terminal sequence exhibiting notable homology to those of lipid transfer proteins was isolated from seeds of the vegetable Brassica campestris. The purification protocol entailed ion exchange chromatography on Q-Sepharose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and gel filtration by FPLC on a Superdex peptide column. The antifungal peptide was adsorbed on Affi-gel blue gel and Mono S. It inhibited mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola with an IC50 value of 8.3 μM and 4.5 μM, respectively. It exhibited dose-dependent binding to lyso-α-lauroyl phosphatidylcholine. The present findings constitute the first report on a non-specific lipid transfer protein from the seeds of a Brassica species.
Keywords: Isolation; Antifungal; Non-specific lipid transfer protein; Brassica campestris; Seeds;
Immunological and structural characterization of an epitope from the Trypanosoma cruzi KMP-11 protein by H. Díez; F. Guzmán; M.P. Alba; A. Cuéllar; M.C. Thomas; M.C. López; F. Rosas; V. Velasco; J.M. González; M.E. Patarroyo; C.J. Puerta (1520-1526).
The K1 peptide is an HLA-A*0201-restricted cytotoxic epitope derived from the Trypanosoma cruzi KMP-11 protein, this being the etiological agent of Chagas’ disease. This work describes the K1 peptide's secondary structure and its recognition by sera from chagasic patients. Circular dichroism and NMR spectroscopy analysis revealed that the K1 peptide adopts an α-helical conformation. Fifty-six percent of individuals had anti-K1 and 86% anti-KMP-11 antibodies by ELISA in the chronic Chagas’ group and 28 and 68% in the indeterminate Chagas’ group, respectively. By contrast, no reactivity was observed in sera from healthy individuals and tuberculosis patients. Antibody response subclass specificity to the K1 peptide was IgG1 and IgG3. Taken together these results support the idea that the K1 peptide acts as a B-cell-inducer epitope during Chagas’ disease.
Keywords: KMP-11 protein; K1 peptide; α-Helix; Humoral immune response; Chagas’ disease; Trypanosoma cruzi;
An antimicrobial peptide with antimicrobial activity against Helicobacter pylori by Lihua Chen; Yuhong Li; Jianxu Li; Xueqing Xu; Ren Lai; Quanming Zou (1527-1531).
An antimicrobial peptide named odorranain-HP was identified from skin secretions of the diskless odorous frog, Odorrana grahami. It is composed of 23 amino acids with an amino acid sequence of GLLRASSVWGRKYYVDLAGCAKA. By BLAST search, odorranain-HP had similarity to antimicrobial peptide odorranain-W1 but it has a different GLLR N-terminus. The cDNA encoding odorranain-HP was cloned from the cDNA library of the skin of O. grahami. This peptide showed antimicrobial activities against tested microorganisms. Interestingly, odorranain-HP could exert antimicrobial capability against Helicobacter pylori, along with its antimicrobial activities similar to odorranain-W1. This is the first report of naturally occurring peptide with anti-H. pylori activity from amphibian skins.
Keywords: Amphibian; Antimicrobial peptide; Odorrana grahami; Helicobacter pylori;
Purification and characterization of antimicrobial peptides from the skin secretion of Rana dybowskii by Sukwon S. Kim; Myoung Sup Shim; Jiyeol Chung; Doo-Yeon Lim; Byeong Jae Lee (1532-1539).
Six antimicrobial peptides designated dybowskins were isolated from the skin secretion of Rana dybowskii, an edible frog in Korea. Dybowskin-1 (FLIGMTHGLICLISRKC) and dybowskin-2 (FLIGMTQGLICLITRKC) were isoforms differing in only two amino acid residues at the 7th and 14th positions from the N-terminus, and they showed amino acid sequence similarities with ranalexin peptides. Dybowskin-3 (GLFDVVKGVLKGVGKNVAGSLLEQLKCKLSGGC), dybowskin-4 (VWPLGLVICKALKIC), dybowskin-5 (GLFSVVTGVLKAVGKNVAKNVGGSLLEQLKCKISGGC), and dybowskin-6 (FLPLLLAGLPLKLCFLFKKC) differed in both size and sequence, and they were, in terms of amino acid sequence similarities, related to brevinin-2, japonicin-2, esculentin-2, and brevinin-1 peptides, respectively. All the peptides presented in this paper contained Rana-box, the cyclic heptapeptide domain, which is conserved in other antimicrobial peptides derived from the genus Rana. All the dybowskin peptides showed a broad spectrum of antimicrobial activity against the Gram-positive and Gram-negative bacteria (minimum inhibition concentrations (MIC), 12.5 to >100 μg/ml) and against Candida albicans (MIC, 25 to >100 μg/ml). Especially, dybowskin-4 with valine at its N-terminus was the most abundant and showed the strongest antimicrobial activity among all the dybowskin peptides. This result indicates that the dybowskin peptides from R. dybowskii, whose main habitats are mountains or forests, have evolved differently from antimicrobial peptides isolated from other Korean frogs, whose habitats are plain fields.
Keywords: Peptide; Rana dybowskii; Dybowskin; Amphibian skin; Rana-box;
Isolation and cDNA cloning of cholecystokinin from the skin of Rana nigrovittata by Xiuhong Liu; Yipeng Wang; Lihua Cheng; Yuzhu Song; Ren Lai (1540-1544).
Keywords: Amphibian; Rana nigrovittata; Cholecystokinin; Skin–intestine–brain triangle peptide;
Presence of and stress-related changes in urocortin-like molecules in neurons and immune cells from the mussel Mytilus galloprovincialis by Davide Malagoli; Ivano Di Paolo; Enzo Ottaviani (1545-1552).
The distribution of urocortin (UCN)-like material is investigated in the bivalve mollusc Mytilus galloprovincialis. Immunocytochemical data demonstrate that UCN-like molecules are present in ganglionic neurons, microglial cells and immunocytes. Moreover, a co-localization of UCN- and corticotrophin-releasing hormone (CRH)-like molecules is found in microglial cells and in immunocytes, but not in neurons. Following high salinity-stress experiments, immunoreactivity for UCN and CRH increased in ganglionic neurons and immunocytes. Our findings extend the number of molecules potentially used by molluscan immunocytes to confront stress situations and strengthen the idea of functional conservation of stress-related molecules during evolution.
Keywords: Mytilus galloprovincialis; Urocortin; Corticotrophin; Stress response; Immunocytochemistry;
Leptin protects vital organ functions after sepsis through recovering tissue myeloperoxidase activity: An anti-inflammatory role resonating with indomethacin by Ji Lin; Guang-Tao Yan; Hui Xue; Xiu-Hua Hao; Kai Zhang; Lu-Huan Wang (1553-1560).
In this research, the role of leptin on sepsis-induced organ dysfunction was evaluated. Making use of a mice sepsis model, changes of alanine transaminase and uric acid in serum, myeloperoxidase activity, leptin levels and histological alterations in heart, lung, liver and kidney were determined. Results showed that sepsis induced significantly higher levels of serum alanine transaminase and uric acid, decreased tissue myeloperoxidase activity and leptin levels, and triggered distinct histological alterations. However, leptin and indomethacin injections reversed those impairments at 6 h and/or 12 h after injury. These data reveal a protective role of both leptin and indomethacin on vital organ functions after sepsis by recovering tissue myeloperoxidase activity.
Keywords: Leptin; Sepsis; Myeloperoxidase; Indomethacin; Recovery of function;
Evidence for a C-terminal structural motif in gastrin and its bioactive fragments in membrane mimetic media by Shane R. Stone; Dale F. Mierke; Graham E. Jackson (1561-1571).
The conformational preferences of human little gastrin, [Nle15] gastrin-17, and its short analogues, gastrin-4 and [β-Ala1] gastrin-5, which include the C-terminal tetrapeptide sequence Trp–Met–Asp–Phe–NH2 crucial for gastrin bioactivity, were determined by NMR spectroscopy in aqueous solutions of zwitterionic dodecylphosphocholine micelles.Backbone HN chemical shift temperature variance, Hα chemical shift deviations and complex non-sequential NOE patterns pointed to the C-terminal of [Nle15] gastrin-17 adopting an ordered conformation. Distance geometry calculations and NOE-restrained molecular dynamics simulations in membrane mimetic solvent boxes of decane and water indicated the C-terminal tetrapeptide sequence of all three peptides adopted a similar, well defined structure, with a general type IV β-turn observed for all three peptides. The conformation of [Nle15] gastrin-17 consisted of two short helices between Leu5–Glu9 and Ala11–Trp14, with the one helix terminating in a type I β-turn spanning Gly13–Asp16. The experimental evidence and conformational characteristics of the three peptides in micellar media support a membrane-associated mechanism of receptor recognition and activation for the gastrin hormone family and furthermore point to a possible biologically relevant structural motif for gastrin activity.
Keywords: Gastrin; Cholecystokinin; NMR; Membrane-mimetic media; Micelles; Dodecylphosphocholine; Conformation;
Nociceptin/orphanin FQ prevents gastric damage induced by cold-restraint stress in the rat by acting in the periphery by Daniela Grandi; Elvira Solenghi; Remo Guerrini; Carlo Polidori; Maurizio Massi; Giuseppina Morini (1572-1579).
The influence of peripheral nociceptin/orphanin FQ (N/OFQ) on cold restraint-induced gastric mucosal damage in the rat was investigated. Exposure to cold-restraint for 3 and 4 h caused the formation of hemorrhagic lesions in the glandular portion of the stomach. N/OFQ dose-dependently decreased lesion formation, in the range 0.03–1 μg/kg/h i.p. Its effect was reversed by the selective NOP receptor antagonist [Nphe1Arg14Lys15]N/OFQ–NH2 (UFP-101), 30 μg/kg/h ip. The selective NOP receptor agonist [(pF)Phe4Aib7Arg14Lys15]N/OFQ–NH2 (UFP-112), 0.01–0.3 μg/kg/h i.p., similarly reduced lesion formation. Light and scanning electron microscopy confirmed the protective activity of N/OFQ. Cold-restraint stress causes a reduction in mucus content and in adhering mucus layer, partly counteracted by N/OFQ. These results suggest that N/OFQ counteracts acute stress-induced gastric mucosal damage by interacting with NOP receptor and by influencing mucous cell activity.
Keywords: Nociceptin/orphanin FQ (N/OFQ); Cold-restraint stress; Gastric mucosal damage; Rat;
Increased hypothalamic angiotensin-(1–7) levels in rats with aortic coarctation-induced hypertension by Mariela M. Gironacci; K. Bridget Brosnihan; Carlos M. Ferrario; Susana Gorzalczany; María A. Lopez Verrilli; Mariano Pascual; Carlos Taira; Clara Peña (1580-1585).
Since angiotensin (Ang) (1–7) injected into the brain blocked Ang II pressor actions in rats made hypertensive by aortic coarctation (CH), we examined systemic and tissue angiotensin peptide levels, specifically concentrating on the hypothalamic Ang-(1–7) levels. Plasma, heart and kidney isolated from CH rats showed increased levels of Ang I, Ang II and Ang-(1–7) compared with the normotensive group, with Ang II being the predominant peptide in heart and kidney. In the hypothalamus, equimolar amounts of Ang II and Ang-(1–7) were found in the sham group, whereas only Ang-(1–7) levels increased in CH rats. We conclude that aortic coarctation activates systemic and tissue renin–angiotensin system. The increased central levels of Ang-(1–7) in the CH rats suggest a potential mitigating role of this peptide in central control of the hypertensive process.
Keywords: Angiotensin II; Angiotensin-(1–7); Aortic coarctation; Hypertensive rats; Tissue levels; Plasma levels; Hypothalamus;
Prolyl, cystyl and pyroglutamyl peptidase activities in the hippocampus and hypothalamus of streptozotocin-induced diabetic rats by Leonardo Zambotti-Villela; Simone C. Yamasaki; Joyce S. Villarroel; Cristiane Murena-Nunes; Paulo F. Silveira (1586-1595).
Prolyl, cystyl and pyroglutamyl peptidases are emerging targets for diabetes and cognitive deficit therapies. The present study is focused on the influence of diabetes mellitus induced by streptozotocin on levels of representative hydrolytic activities of these enzymes in the rat hypothalamus and hippocampus. Streptozotocin-diabetic rats presented about 348 mg glucose/dL blood, and a slightly increased hematocrit and plasma osmolality. The activities of soluble and membrane-bound dipeptidyl-peptidase IV, and soluble cystyl aminopeptidase did not differ between diabetic and control rats in both brain areas. Hippocampal soluble prolyl oligopeptidase presented similar activities between diabetic and controls. Increased activities in diabetics were observed for soluble prolyl oligopeptidase (1.78-fold) and membrane-bound cystyl aminopeptidase (2.55-fold) in the hypothalamus, and for membrane-bound cystyl aminopeptidase (5.14-fold) in the hippocampus. In both brain areas, the activities of membrane-bound and soluble pyroglutamyl aminopeptidase were slightly lower (<0.7-fold) in diabetics. All modifications (except hematocrit) observed in streptozotocin-treated rats were mitigated by the administration of insulin. Glucose and/or insulin were shown to alter in vitro the hypothalamic activities of soluble pyroglutamyl aminopeptidase and prolyl oligopeptidase, as well as membrane-bound cystyl aminopeptidase. These data provide the first evidence that diabetes mellitus generates direct and indirect effects on the activity levels of brain peptidases. The implied regional control of regulatory peptide activity by these peptidases suggests novel potential approaches to understand certain disruptions on mediator and modulatory functions in diabetes mellitus.
Keywords: STZ-diabetes; Hypothalamus; Hippocampus; Peptidases; Proteases;
Downregulation of prolactin-releasing peptide gene expression in the hypothalamus and brainstem of diabetic rats by Takashi Mera; Hiroaki Fujihara; Jun Saito; Makoto Kawasaki; Hirofumi Hashimoto; Takeshi Saito; Minori Shibata; Tatsushi Onaka; Yoshiya Tanaka; Takakazu Oka; Sadatoshi Tsuji; Yoichi Ueta (1596-1604).
We investigated the prolactin-releasing peptide (PrRP) mRNA levels in the hypothalamus and brainstem of streptozotocin (STZ)-induced diabetic rats and fa/fa Zucker diabetic rats, using in situ hybridization histochemistry. PrRP mRNA levels in the hypothalamus and brainstem of STZ-induced diabetic rats were significantly reduced in comparison with those of control rats. PrRP mRNA levels in the diabetic rats were reversed by both insulin and leptin. PrRP mRNA levels in the fa/fa diabetic rats were significantly reduced in comparison with those of Fa/? rats. PrRP mRNA levels in the fa/fa diabetic rats were significantly increased by insulin-treatment, but did not reach control levels in the Fa/? rats. We also investigated the effect of restraint stress on PrRP mRNA levels in STZ-induced diabetic rats. The PrRP mRNA levels in the control and the STZ-induced diabetic rats increased significantly after restraint stress. The diabetic condition and insulin-treatment may affect the regulation of PrRP gene expression via leptin and other factors, such as plasma glucose level. The diabetic condition may not impair the role of PrRP as a stress mediator.
Keywords: Prolactin-releasing peptide; Diabetes mellitus; Hyperglycemia; Insulin; Leptin; Restraint stress;
Rapid identification of precursor cDNAs encoding five structural classes of antimicrobial peptides from pickerel frog (Rana palustris) skin secretion by single step “shotgun” cloning by Mei Zhou; Lei Wang; Damian E. Owens; Tianbao Chen; Brian Walker; Chris Shaw (1605-1610).
The skin secretion of the North American pickerel frog (Rana palustris) has long been known to have pronounced noxious/toxic properties and to be highly effective in defence against predators and against other sympatric amphibians. As it consists largely of a complex mixture of peptides, it has been subjected to systematic peptidomic study but there has been little focus on molecular cloning of peptide-encoding cDNAs and by deduction, the biosynthetic precursors that they encode. Here, we demonstrate that the cDNAs encoding the five major structural families of antimicrobial peptides can be elucidated by a single step “shotgun” cloning approach using a cDNA library constructed from the source material of the peptidomic studies—the defensive skin secretion itself. Using a degenerate primer pool designed to a highly conserved nucleic acid sequence 5′ to the initiation codon of known antimicrobial peptide precursor transcripts, we amplified cDNA sequences representing five major classes of antimicrobial peptides, such as esculentins, brevinins, ranatuerins, palustrins and temporins. Bioinformatic comparisons of precursor open-reading frames and nucleic acid sequences revealed high degrees of structural similarities between analogous peptides of R. palustris and the Chinese bamboo odorous frog, Rana versabilis. This approach thus constitutes a robust technique that can be used either alone or ideally, in parallel with peptidomic analysis of skin secretion, to rapidly extract primary structural information on amphibian skin secretion peptides and their biosynthetic precursors.
Keywords: Amphibian; Venom; Mass spectrometry; Peptide; Cloning;
Effect of orphanin FQ/nociceptin (OFQ/N) and isoflurane on the prolactin secretory response in OFQ/N knockout mice by Kelly L. Zullig; Emily Murphree; Rainer K. Reinscheid; James Janik; Phyllis Callahan (1611-1614).
The prolactin secretory response to subcutaneous injection of orphanin FQ/nociceptin (OFQ/N) was measured in wild-type and OFQ/N knockout mice. These injections were given with and without isoflurane anesthesia, to determine if isoflurane would affect the prolactin secretory response. OFQ/N injection significantly increased prolactin levels in males and females, regardless of genotype, with a more robust response in females. Isoflurane pretreatment did not affect prolactin levels in controls or in animals injected with OFQ/N. This is the first report that exogenously administered OFQ/N stimulates prolactin secretion in mice and that brief isoflurane exposure does not significantly affect this response.