Peptides (v.24, #7)

In vitro activities of native and designed peptide antibiotics against drug sensitive and resistant tumor cell lines by Sunkyu Kim; Sukwon S Kim; Yung-Jue Bang; Seong-Jin Kim; Byeong Jae Lee (945-953).
In order to develop peptide agents with reduced length and enhanced tumoricidal activity, we have designed gaegurin 6 (GGN6) derivatives through deletions and/or substitutions of amino acids. The deletion of hydrophobic amino terminal region completely abolished antitumor activity whereas the deletion of carboxy terminal region had little influence on antitumor activity. Antitumor activity of the PTP peptides did not correlate with antibacterial activity. PTP7, the most potent derivative, was found to have comparable antitumor activity to GGN6 in spite of reduced number of amino acids which is about half the size of gaegurin 6; furthermore, it showed little cytotoxicity on PBMCs and RBCs. GGN6 and PTP7 also showed equivalent cytotoxicity against drug sensitive (MCF-7) and multidrug-resistant cell lines (MCF-7/DOX). Plasma membrane blebbing and DNA fragmentation of peptide-treated tumor cells indicated that the peptides could induce apoptosis in tumor cells. These results suggest that GGN6 and its derivatives can be developed as new anticancer agents and may provide a new strategy for overcoming MDR which is a major problem in cancer therapy.
Keywords: MDR; Antimicrobial peptide; Gaegurin 6; Derivative; Antitumor activity; MCF-7/DOX;

Characterization of novel antimicrobial peptides from the skins of frogs of the Rana esculenta complex by Mohamed F Ali; Floyd C Knoop; Hubert Vaudry; J.Michael Conlon (955-961).
Rana esculenta is a hybridogenetic hybrid between Rana ridibunda and Rana lessonae and so is best considered as a complex of interbreeding species rather than a discrete single species. In this study, antimicrobial peptides were isolated from a pooled extract of the skins of specimens of the R. esculenta complex collected in the wild. In addition to several peptides belonging to the brevinin and esculentin families that have been previously isolated from skin secretions of a single specimen of R. esculenta, three newly described members of the brevinin-2 family (brevinin-2Ei, brevinin-2Ej, and brevinin-2Ek) and one member of the temporin family (temporin-1Ec) were purified and characterized. In addition, three structurally related peptides with no sequence similarity with antimicrobial peptides isolated from other species of ranid frogs, that potently and selectively inhibit the growth of the Gram-positive bacterium Escherichia coli (minimal inhibitory concentration (MIC<5 μM)), were identified. These peptides show limited amino acid sequence similarity to the homologous exon gene products that encode the N-terminal flanking peptides of preprocaerulein, preproxenopsin, and preprolevitide and so have been termed caerulein precursor-related fragments (CPRF-Ea, CPRF-Eb, and CPRF-Ec). The data suggest that there may be considerable polymorphism among specimens from different populations of the R. esculenta complex. It is proposed that the distribution and amino acid sequences of skin antimicrobial peptides may be useful markers for taxonomic classification of particular sub-populations and for an understanding of phylogenetic interrelationships.
Keywords: Antimicrobial peptide; Rana esculenta; Rana ridibunda; Rana lessonae; HPLC purification;

From the seeds of the Yunnan bean, we purified an antifungal peptide using affinity chromatography on Affi-gel blue gel, FPLC-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 75. The antifungal peptide was adsorbed on Affi-gel blue gel at pH 7.8 and Mono S at pH 4.5. It exhibited a molecular mass of 6.5 kDa in both gel filtration and sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Its N-terminal sequence closely resembled defensin-related peptides. The peptide exerted antifungal activity toward the fungal species Fusarium oxysporum and Mycosphaerella arachidicola, with an IC50 of 2 μM for the former fungus and 10 μM for the latter. It manifested a weaker mitogenic activity toward murine splenocytes than Concanavalin A. It also displayed antiproliferative activity on a murine leukemia (L1210), a hepatoma (HepG2), and a murine leukemia (M1) cell line. It inhibited human immunodeficiency virus-1 reverse transcriptase with an IC50 of 200 μM.
Keywords: Antifungal peptides; Isolation; Bean; Gymnocladus chinensis;

A novel antifungal peptide, with a molecular mass of 8 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis and in gel filtration on Superdex 75 and designated cucurmoschin, was isolated from the seeds of the black pumpkin. The peptide was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel. Cucurmoschin inhibited mycelial growth in the fungi Botrytis cinerea, Fusarium oxysporum and Mycosphaerella oxysporum. It inhibited translation in a cell-free rabbit reticulocyte lysate system with an IC50 of 1.2 μM. The N-terminal sequence of cucurmoschin was rich in arginine, glutamate and glycine residues.
Keywords: Antifungal peptide; Pumpkin; Seeds; Isolation;

A ribonuclease, with a molecular mass of 13 kDa and a ubiquitin-like N-terminal sequence, has been isolated from fruiting bodies of the mushroom Termitomyces globulus. The ribonuclease demonstrated ribonucleolytic activity toward poly A, poly C, poly G and poly U, with the activity toward poly A and poly C being much higher than that toward poly G and poly U. The ribonuclease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and CM-Sepharose. The enzyme required a temperature of 70 °C for expression of maximal activity. However, the enzyme expressed nearly the same optimal activity over a wide pH range of 5.0–8.0.
Keywords: Ribonuclease; Mushroom; Isolation;

Immunomodulation by peptide analogs of retroviral envelope protein by Shikhar Mehrotra; Kamla P Mishra; Virendra S Yadav; Madhushree Bhattacharya; Deepa Pandey; Wahajul Haq; Vijay K Singh (979-985).
The mechanism by which retroviral proteins exert their immunosuppressive influence has remained enigmatic. Early studies have demonstrated that retroviral infection suppresses cellular and humoral immune responses. A hydrophilic 26 amino acid region of the otherwise hydrophobic transmembrane envelope protein of murine and feline leukemia viruses, p15E, is conserved among the transmembrane envelope proteins of numerous animal retroviruses (e.g. murine, feline, bovine and simian) as well as in human T-cell leukemia virus, and to a lesser extent, in human immunodeficiency virus (HIV). We evaluated the immunomodulatory properties of various synthetic retroviral envelope peptides synthesized as overlapping fragments to this conserved sequence. We report that two small peptides inhibit human mixed lymphocyte reaction (MLR), interleukin-2 (IL-2) and tumor necrosis factor (TNF-α) production. These peptides did not affect human natural killer (NK) cell cytotoxicity in vitro, and nitric oxide (NO) production in mouse macrophage cells, RAW264.7. Our observations suggests immunomodulatory potential of two retroviral peptide analogs.
Keywords: Cytokines; Immunomodulators; Mixed lymphocyte reaction; Nitric oxide; Retroviral peptide;

A peptide from the extension of Lys-tRNA synthetase binds to transfer RNA and DNA by Kwabena P.A.B Yiadom; Rasha Hammamieh; Nkoli Ukpabi; Pearl Tsang; David C.H Yang (987-998).
Eukaryotic aminoacyl-tRNA synthetases have dispensable extensions appended at the amino- or carboxyl-terminus as compared to their bacterial counterparts. While a synthetic peptide corresponding to the basic amino-terminal extension in yeast Asp-tRNA synthetase binds to DNA, the extension in the intact protein evidently binds to tRNA and enhances the tRNA specificity of Asp-tRNA synthetase. On the other hand, the amino-terminal extension in human Asp-tRNA synthetase, both within the intact protein and as a synthetic peptide, binds to tRNA. Here, the tRNA binding of a synthetic peptide, hKRS(Arg25-Glu42), corresponding to the amino-terminal extension of human Lys-tRNA synthetase (hKRS) was analyzed. This basic peptide bound to tRNAPhe and the apparent-binding constant increased with increasing concentrations of Mg2+. The hKRS peptide also bound to DNA and polyphosphate; however, the apparent DNA-binding constants decreased at increasing concentrations of Mg2+. The ability of the hKRS peptide to adopt α-helical conformation was demonstrated by NMR and circular dichroism. A Lys-rich peptide derived from the elongation factor 1α was also examined and bound to DNA but not to tRNA.
Keywords: Elongation factor 1α; Nuclear magnetic resonance; tRNA; tRNA synthetase; Circular dichroism; RNA world; Nucleic acid binding peptides; Magnesium ion;

6746 SERA peptide analogues immunogenicity and protective efficacy against malaria is associated with short α helix formation: by Martha Patricia Alba; Luz Mary Salazar; Alvaro Puentes; Martha Pinto; Elizabeth Torres; Manuel Elkin Patarroyo (999-1006).
Erythrocyte high activity binding peptides (HABPs) have been identified for the Plasmodium falciparum serine repeat antigen (SERA). HABP 6746, located in this protein’s 50 kDa fragment had its critical binding residues replaced by amino acids having similar mass but different charge to change their immunologic properties. This peptide analogues were used to immunize Aotus monkeys that were challenged later on with a virulent P. falciparum strain to determine their protective efficacy. A shortening in α helix structure was found in the immunogenic and protective ones when their secondary structure was analyzed by NMR, to correlate their structure with their immunologic properties. These data, together with results from previous studies, suggest that this shortening in HABP helical configuration may lead to better fitting with immune system molecules, rendering them immunogenic and protective and therefore making them excellent candidates for consideration as components of a subunit based multicomponent synthetic vaccine against malaria.
Keywords: Malaria; Peptide; NMR; Synthetic vaccine;

Plasmodium falciparum normocyte binding protein (PfNBP-1) peptides bind specifically to human erythrocytes by John Jairo Valbuena; Ricardo Vera; Javier Garcı́a; Alvaro Puentes; Hernando Curtidor; Marisol Ocampo; Mauricio Urquiza; Zuly Rivera; Fanny Guzmán; Elizabeth Torres; Manuel Elkin Patarroyo (1007-1014).
Plasmodium falciparum normocyte binding protein-1 (PfNBP-1), a Plasmodium vivax RBP-1 orthologue is expressed in the apical merozoite area. PfNBP-1 binds directly to human erythrocyte membrane in a sialic acid-dependent but trypsin-resistant way. Erythrocyte binding assays were done with synthetic peptides covering the sequence reported as PfNBP-1. Two specific erythrocyte high activity binding peptides were found: 101 V F INDLDTYQY EY FY EWN Q 120 , peptide 26332, and 181 NTKETYLK EL N K KKMLQNKK 200 , peptide 26336. These two peptides’ binding was saturable and presenting nanomolar affinity constants. The critical binding residues (those residues underlined and highlighted in bold) were determined by competition assays with glycine-scan analogue peptides. These peptides were able to block merozoite in vitro invasion of erythrocytes.
Keywords: PfNBP-1; Plasmodium falciparum; Erythrocyte-binding;

P. falciparum: merozoite surface protein-8 peptides bind specifically to human erythrocytes by Alvaro Puentes; Javier Garcı́a; Marisol Ocampo; Luis Rodrı́guez; Ricardo Vera; Hernando Curtidor; Ramsés López; Jorge Suarez; John Valbuena; Magnolia Vanegas; Fanny Guzman; Diana Tovar; Manuel E Patarroyo (1015-1023).
This work determined Plasmodium falciparum merozoite surface protein-8 (MSP-8) regions specifically binding to membrane surface receptors on human erythrocytes. Five high activity binding peptides (HABPs), whose binding to erythrocytes became saturable and sensitive on being treated with neuraminidase and chymotrypsin were identified from the MSP-8 protein. Those amino acids directly involved in interaction with erythrocytes were also determined for each one of the HABPs. Some of them specifically recognized 28, 46, and 73 kDa erythrocyte membrane proteins. Some HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by up to 98%, suggesting the MSP-8 protein’s possible role in the invasion process.
Keywords: P. falciparum; MSP-8; High activity binding peptides; Critical residues; Invasion inhibition;

Monoclonal antibody G15-6A was generated by immunizing mice with Ascaris head extracts. It recognizes an antigen present in a single neuron, with a cell body in the dorsal rectal ganglion, that projects along the ventral cord to the nerve ring. Ascaris extracts were fractionated by HPLC and ammonium sulfate precipitation, and fractions assayed by dot-blotting with antibody G15-6A. A single immunoreactive polypeptide was purified; mass spectrometry showed a molecular weight of 11,542 Da. Partial N-terminal sequencing, followed by cloning of the transcript encoding the peptide, revealed a predicted peptide product comprising 109 amino acids, and a molecular mass of 11,863 Da. The N-terminus of the predicted peptide includes four more amino acids than are found in the isolated product.
Keywords: Neuropeptide; Monoclonal antibody; Dot-blot immunoassay; Ascaris suum; Nematode nervous system; RT-PCR;

Conformation of neuropeptide Y receptor antagonists: structural implications in receptor selectivity by Seetharama D.S Jois; Ambikaipakan Balasubramaniam (1035-1043).
Two NPY analogue peptides, BVD10 (Ile-Asn-Pro-Ile-Tyr-Arg-Leu-Arg-Tyr-OMe) and BVD15 (Ile-Asn-Pro-Ile-Tyr-Arg-Leu-Arg-Tyr-NH2) were characterized conformationally by NMR, CD and molecular dynamics simulations. The two peptides exhibit different secondary structure characteristics in trifluoroethanol. BVD10 exhibits a structure with two consecutive β-turns at Asn2-Pro3-Ile4-Tyr5 and Ile4-Tyr5-Arg6-Leu7. BVD15 exhibits a helical type of structure along with a β-turn at Asn2-Pro3-Ile4-Tyr5. Molecular modeling studies suggested that the C-terminus Tyr9 is oriented in different directions in the two peptides. The difference in the structures of peptides observed may contribute to the Y1 selectivity of BVD10 relative to BVD15.
Keywords: NMR; Beta-turn; Helical structure; NPY peptide; Trifluoroethanol;

We have reported previously that bradykinin (BK) induces potent and reproducible concentration-dependent contractions of the pig iris sphincter (PIS) muscle in vitro through the activation of BK B2 receptors. Here we attempted to investigate additional mechanisms by which BK induces contraction of the PIS in vitro. BK-mediated contraction of the PIS relied largely on the external Ca2+ influx by a mechanism sensitive to the L-, N- and P-type of Ca2+ channel selective blockers. Likewise, BK-induced contraction of the PIS was greatly inhibited by the CGRP-(8–37), NK2 or NK3 receptor antagonists (SR 48968, SR 142801), and to a lesser extent by the NK1 antagonist (FK 888). Capsaicin desensitization of PIS or capsazepine pre-incubation also significantly reduced BK-mediated contraction in the PIS. Furthermore, KT 5720 or GF 109203X (the protein kinase A and C inhibitors, respectively) also significantly inhibited BK-mediated contraction. Taken together, these results indicate that BK-mediated contraction of the PIS seems to be mediated primarily by the release of CGRP and tachykinins from sensory nerve fibers, and relies largely on extracellular Ca2+ influx via activation of L-, N- and P-type of Ca2+ channels. Finally, these responses are mediated by activation of both protein kinase A- and C-dependent mechanisms.
Keywords: Bradykinin; Pig iris sphincter; Calcium channel; Tachykinins; CGRP; Sensory fibers; Capsaicin; Protein kinases A and C;

Suppression of cytokine-induced expression of adrenomedullin and endothelin-1 by dexamethasone in T98G human glioblastoma cells by Kazuhiro Takahashi; Reiko Udono-Fujimori; Kazuhito Totsune; Osamu Murakami; Shigeki Shibahara (1053-1062).
There is accumulating evidence showing that glial cells and gliomas secrete some neuropeptides and vasoactive peptides, such as adrenomedullin and endothelin-1. We have previously shown that expression of these two peptides is induced by inflammatory cytokines in T98G human glioblastoma cells. Glucocorticoids are frequently used for the treatment of inflammatory diseases and glioblastomas. We therefore studied effects of dexamethasone on expression of adrenomedullin and endothelin-1 in T98G human glioblastoma cells. Dexamethasone dose-dependently increased adrenomedullin mRNA levels and immunoreactive-adrenomedullin levels in the medium in T98G cells, whereas it decreased immunoreactive-endothelin levels in the medium. A combination of three cytokines, interferon-γ (100 U/ml), tumor necrosis factor-α (20 ng/ml) and interleukin-1β (10 ng/ml) induced expression of adrenomedullin and endothelin-1 in T98G cells. Dexamethasone (10−8  mol/l) suppressed increases in expression of both adrenomedullin and endothelin-1 induced by these three cytokines. Thus, dexamethasone alone increased adrenomedullin expression whereas it suppressed the cytokine-induced expression of adrenomedullin in T98G cells. These findings raised the possibility that effects of dexamethasone on brain inflammation and glioblastomas may be partly mediated or modulated by its effects on expression of adrenomedullin and endothelin-1.
Keywords: Adrenomedullin; Endothelin; Glioblastoma; Glia; Dexamethasone; Cytokine;

It has been proved that there are the subfornical organ (SFO)-nucleus paraventricularis (NPV)-rostral ventrolateral medulla (RVL) angiotension II (AngII) pressor system and the central amygdaloid nucleus (AC)-lateral hypothalamus/perifornical region (LH/PF) emotional pressor system in the brain. Because the LH/PF contains abundant AngII ergic neurons projecting to the SFO, the purpose of the present study was to examine whether the (SFO-NPV-RVL) AngII pressor system takes part in the AC-pressor response via AngII ergic neurons in the LH/PF. The results showed that (1) l-glutamate microinjection into the AC or LH/PF induced pressor responses. (2) Both the AC- and LH/PF-pressor responses could be reversed by preinjection of [Sar1, Thr8]-angiotensin II (an antagonist of AngII) into either the SFO, NPV or RVL. Taken together with our previous findings that the projections of the CRF-ergic and SP-ergic neurons in the AC could activate the LH/PF, the above findings prove that: besides several known mechanisms of the brain AngII inducing pressor response, the (SFO-NPV-RVL) AngII pressor system also takes part in the AC-emotional pressor response via AngII ergic projections from the LH/PF to the SFO, which may be the neurophysiological basis of the brain AngII playing an important role in developing hypertension of the SHRs.
Keywords: Angiotensin II; Subfornical organ; Emotion; Central amygdaloid nucleus; Blood pressure;

Intrahippocampal infusion of the bombesin/gastrin-releasing peptide antagonist RC-3095 impairs inhibitory avoidance retention by Rafael Roesler; Carolina A. Meller; Márcia I. Kopschina; Diogo Onofre Souza; João Antônio Pêgas Henriques; Gilberto Schwartsmann (1069-1074).
Bombesin (BN)-like peptides regulate cell proliferation and cancer growth as well as neuroendocrine and neural functions. We evaluated the effects of the BN/gastrin-releasing peptide (GRP) antagonist RC-3095 on memory formation. Male Wistar rats were given a bilateral infusion of saline or RC-3095 (0.2, 1.0 or 5.0 μg) into the dorsal hippocampus either immediately or 2 h after training in an inhibitory avoidance (IA) task. Retention test trials were carried out 1.5 h (short-term memory) and 24 h (long-term memory) after training. RC-3095 impaired both short- and long-term retention only when given immediately after training. The results suggest that the hippocampal BN/GRP receptor system modulates IA memory formation.
Keywords: RC-3095; Bombesin receptor; Gastrin-releasing peptide; Anticancer drugs; Hippocampus; Neural plasticity; Behavior; Memory;

Kinetic-controlled hydrolysis of Leu-Val-Val-hemorphin-7 catalyzed by angiotensin-converting enzyme from rat brain by Makoto Hayakari; Kimihiko Satoh; Hiroshi Izumi; Toshihiro Kudoh; Junpei Asano; Takehiko Yamazaki; Shigeki Tsuchida (1075-1082).
Leu-Val-Val-hemorphin-7 (LVV-H7, LVVYPWTQRY), an opioid peptide, was found to be hydrolyzed sequentially by rat brain angiotensin-converting enzyme (ACE) in three steps through dipeptidyl carboxypeptidase activity. The kinetic constants evaluated were in order of: k 1 (0.19 min−1) ⪢  k 2 (0.0008 min−1) ≈  k 3 (0.0006 min−1) in 10 mM NaCl at pH 7.5 giving rise to LVV-H5 almost quantitatively. The decapeptide was noted to be hydrolyzed 164- and 346-fold more efficiently than angiotensin I (Ang I) in k cat and k cat/K m values, respectively, at their optimal conditions. The kinetic-controlled preferential action of the brain enzyme on LVV-H7 is suggestive of its multiple roles in vivo.
Keywords: Hemorphin; LVV-hemorphin-7; Opioid peptide; Angiotensin-converting enzyme;

The neuropeptide-degrading enzyme NL1 is expressed in specific neurons of mouse brain by Mélanie Carpentier; Mieczyslaw Marcinkiewicz; Guy Boileau; Luc DesGroseillers (1083-1091).
Metalloendopeptidases of the M13 family were shown to play critical roles in normal physiological processes such as pain control, hypertension and phosphate metabolism, and in pathological states such as Alzheimer’s disease. Recently, NL1, a novel member of the family, has been identified and shown to be expressed in several tissues both as a membrane-bound and a secreted protein. As a further step to understand the physiological role(s) of NL1 in mouse, we mapped NL1 mRNA expression pattern in embryos and in young animals at postnatal days p1 and p3, and in adult nervous tissue, using in situ hybridization at the cellular level. No expression could be detected in embryos and young animals. In contrast, NL1 expression was evident in adult brain, pituitary gland and spinal cord. In the central nervous system (CNS), NL1 mRNA was predominantly found in the ventro-posterior regions, which are mostly associated with vegetative functions. At the cellular level, NL1 mRNA was non-uniformly distributed within subpopulations of neurons. In the spinal cord, specific signal was observed in the gray matter. Then, in order to identify putative relevant substrates for NL1, we studied its enzymatic activity towards peptides known to be co-expressed in the NL1-positive domains. Our study showed that NL1 degrades several of these peptides in vitro, the most readily degraded peptides being Bradykinin and Substance P. These results suggest that NL1 is likely to play a critical role in the central nervous system.
Keywords: In situ hybridization; Neprilysin; Pituitary gland; Bradykinin; Substance P;

Antiviral and immunological benefits in HIV patients receiving intranasal peptide T (DAPTA) by Maria T. Polianova; Francis W. Ruscetti; Candace B. Pert; Rochelle E. Tractenberg; Gifford Leoung; Scott Strang; Michael R. Ruff (1093-1098).
d-Ala-Peptide T-amide (DAPTA), the first viral entry inhibitor, blocks chemokine (CCR5) receptors, not CD4. Early investigators could not “replicate” DAPTAs potent in vitro antiviral effect using the lab-adapted, X4, peptide T-insensitive strain, IIIB, delaying clinical virological studies. We now report that DAPTA, administered to eleven long-term infected (mean=17 years) patients with stable persistent plasma “virus” for up to 32 weeks did not change this level. Infectious virus could not be isolated from their plasma suggesting HIV RNA was devoid of replicative capacity. Progressively less actual virus (P<0.01) could be isolated from white blood cells (PBMCs). DAPTA flushed the monocyte reservoir to undetectable viral levels in most patients. Five of eleven had a mean CD4 increase of 33%. Immune benefits also included a four-fold increase in γ-interferon-secreting T-cells (antiviral cytotoxic T-cells) in the absence of drug-related toxicity. All five CD4 responders had increases in antiviral T cells and decreases in infected monocytes, an argument for initiating further studies promptly.
Keywords: Peptide T; AIDS; Entry inhibitor; CCR5; Antiviral; Clinical trial; Chemokine receptor; Viral reservoir; Cytotoxic T-cells;