Peptides (v.28, #10)
Editorial Board (CO2).
Chronic caloric restriction induces forestomach hypertrophy with enhanced ghrelin levels during aging by Hyunwon Yang; Yun-Hee Youm; Chiaki Nakata; Vishwa Deep Dixit (1931-1936).
Caloric restriction (CR) is the only preventive intervention that has robust pro-longevity effects in experimental models. Various circulating hormones that regulate the state of negative energy balance may drive the multi-system beneficial effects of the CR phenomenon. Ghrelin, one such stomach-derived circulating peptide hormone stimulates food intake, promotes GH release and inhibits pro-inflammatory cytokines. We have recently demonstrated that ghrelin also reverses age-related thymic involution. Here, we report that chronic CR in aging mice results in reduction in body weight, and spleen size but remarkably, leads to a significant increase in the size and weight of stomach. The increased size of stomach was largely due to increased size of fundus (forestomach) and also smaller but statistically significant enlargement of antrum. The analysis of serial stomach sections revealed that chronic CR leads to a striking hypertrophy of lamina propria, stratum basale, stratum corneum and the stratified squamous epithelium of forestomach of the aged animals. We also report for the first time that chronic CR during aging significantly increases circulating ghrelin levels as well as total ghrelin production in the stomach and reverses age-related loss of ghrelin receptor expression in pituitary. Our data suggests that long-term CR-induced increased ghrelin production from hypertrophic stomach in mice may be an adaptive survival strategy in response to sustained negative energy balance that triggers heightened state of food seeking. Taken together, these data provide new insights into the underlying mechanism behind the salutary effects of chronic caloric restriction during aging process.
Keywords: Dietary restriction; Nutrition; Immune system; Ghrelin; GHS-R; Geriatrics; CR mimetic; Inflammation; Stomach; Fundus; Antrum; Forestomach; Aging; Thymus; Gastric bypass;
Obestatin and ghrelin in obese and in pregnant women by Elizabeth Fontenot; J.E. DeVente; E.R. Seidel (1937-1944).
We identified, through qPCR, receptor mRNA for a number of gut peptides in female human omental fat: the incretins, GIP and GLP-1, the orexigenic peptides PYY-Y1 and -Y2 and ghrelin, and the anorexigenic peptide obestatin. Four cohorts of women were examined: lean controls (BMI < 23), obese (BMI > 41), obese diabetic and term pregnant women. Human fat expressed receptor mRNAs for all six peptides. Pregnant women expressed roughly three times as much orphan GPR-39 receptor, a proposed obestatin receptor, than other women and less than half as much of the ghrelin receptor (GHSR-1a). An immunoblot probed with a GPR-39 selective antibody yielded a single band corresponding to the correct molecular weight (52 kDa) for the proposed obestatin receptor. Fluorescent immunohistochemistry of human fat employing the same antibody indicated the receptor protein was localized to the adipocyte cell membrane. The concentration of obestatin circulating in blood was measured in the same cohort of women and was significantly lower in obese and obese diabetic women compared to control.
Keywords: Ghrelin; Obestatin; Human adipose tissue; PYY; GLP-1; GIP; Pregnant; Diabetes; Obesity;
Structure–activity relationship of CART (cocaine- and amphetamine-regulated transcript) peptide fragments by Jana Maixnerová; Jan Hlaváček; Darja Blokešová; Wioleta Kowalczyk; Tomáš Elbert; Miloslav Šanda; Miroslava Blechová; Blanka Železná; Jiřina Slaninová; Lenka Maletínská (1945-1953).
CART (cocaine- and amphetamine-regulated transcript) peptides are neuropeptides abundant in the central nervous system and periphery found to be involved in the regulation of food intake behavior and other physiological processes. Recently, we reported specific binding of 125I-CART(61–102) to the rat adrenal pheochromocytoma cell line PC12, both intact cells and cell membranes. In this study, several fragments of CART(61–102) corresponding to its structural loops were synthesized and tested for their potency in binding experiments using PC12 intact cells and cell membranes and in feeding test with fasted mice. From all shorter peptides tested, only CART(74–86) and CART(62–86) containing disulfide bridges kept partial binding potency of the original molecule with K i in 10−5 and 10−4 M range. However, these fragments were not able to inhibit food intake after their central administration up to a dose of 4 nmol/mouse. The results showed that a compact structure containing three disulfide bridges is necessary for preservation of full biological activity of CART peptides.
Keywords: CART peptide; Fragments; Binding; PC12 cells; Food intake; Mice;
Antibodies induced by Plasmodium falciparum merozoite surface antigen-2-designed pseudopeptides possess neutralizing properties of the in vitro malarial infection by José Manuel Lozano; Francy J. Montoya-Fajardo; Johan Hoebeke; Gladys H. Cifuentes; Martha Forero; Manuel Elkin Patarroyo (1954-1965).
Pseudopeptide chemistry is gaining ground in the field of synthetic vaccine development. We have previously demonstrated the potential scope of introducing reduced amide peptide bond isosters in a site-directed design for obtaining structurally modified probes able to induce malaria infection-neutralizing antibodies derived from the MSP-1 antigen. This work reports the functional properties of polyclonal and monoclonal antibodies induced by site-directed designed MSP-2 N-terminus pseudopeptides and their capacity for antibody isotype switching in in vitro immunization. Structural properties of the native peptide and its pseudopeptide analogs are discussed within the context of these novel pseudopeptides’ induced monoclonal antibody functional and physical–chemical properties.
Keywords: Merozoite surface protein 2 (MSP-2); Isoster bond; In vitro immunization; Pseudopeptide; Malaria vaccine;
Involvement of cocaine–amphetamine regulated transcript in the differential feeding responses to nociceptin/orphanin FQ in dark agouti and Wistar Ottawa Karlsburg W rats by Roberta Filippetti; Ingrid Kloting; Maurizio Massi; Carlo Cifani; Carlo Polidori (1966-1973).
Wistar Ottawa Karlsburg W (WOKW) rats and their controls, dark agouti (DA), present different features: in particular, DA rats are lean, while the WOKW are obese and present symptoms of hypertension, dyslipidemia, hyperinsulinemia, and impaired glucose tolerance. The present study tested the hypothesis that these two strains would demonstrate different sensitivity to nociceptin/orphanin FQ (N/OFQ). N/OFQ was injected into the lateral brain ventricle (LBV) of sated DA and WOKW rats, and corticosterone levels in both strains were measured after LBV injection of N/OFQ. LBV N/OFQ injections dose-dependently produced a significant increase in food intake (4 h) in DA rats, but not in WOKW. However, corticosterone levels were increased by N/OFQ to a greater degree in WOKW than in DA rats. Gene sequencing and gene expression of ORL1 receptor and cocaine–amphetamine regulated transcript (Cart) peptide were evaluated to study the difference in N/OFQ-induced feeding behavior in the two strains. WOKW rats had a different amino acid sequence of Cart peptide and a significantly higher expression of Cart in the hypothalamus. The present data show that DA and WOKW rats demonstrate different sensitivity to N/OFQ, and suggest that Cart peptide might be the underlying mechanism of this difference.
Keywords: Nociceptin/orphanin FQ; Dark agouti rats; Wistar Ottawa Karlsburg W rats; Cocaine–amphetamine regulated transcript peptide;
The gastric effects of UFP-112, a new nociceptin/orphanin receptor agonist, in physiological and pathological conditions by M. Broccardo; R. Guerrini; G. Morini; C. Polidori; S. Agostini; C. Petrella; G. Improta (1974-1981).
Nociceptin/orphanin FQ (N/OFQ), the endogenous NOP receptor ligand, centrally modulates gastric motor and secretory functions and prevents ethanol-induced gastric lesions in rats. A recently synthesized N/OFQ analog, [(pF)Phe4Aib7Arg14Lys15]N/OFQ-NH2 (UFP-112), acts as a highly potent and selective peptide agonist for NOP receptors and produces longer-lasting in vitro and in vivo effects in mice than the natural ligand N/OFQ. In this study, we evaluated the effects of centrally (intracerebroventricularly/icv) and peripherally (intraperitoneally/ip) injected UFP-112 on gastric emptying and gastric acid secretion, and on the development of gastric mucosal lesions induced by 50% ethanol in the rat. When injected icv, it dose-dependently delayed gastric emptying of a phenol red meal (by up to 70%), decreased gastric secretion in water-loaded rats after 90 pylorus ligature, and reduced ethanol-induced gastric lesions (by up to 87%). In all three assays, UFP-112 was more effective than N/OFQ. The highly selective NOP receptor antagonist, UFP-101, decreased the efficacy of UFP-112, thus confirming that central NOP receptors mediate inhibitory control on these functional and pathological conditions in rats. Ip injected N/OFQ and UFP-112 induced non-dose-related gastric hypersecretory and antiulcer effects, which UFP-101 partially abolished. Ip N/OFQ appeared equiactive but about 30–100 times less potent than ip UFP-112 in stimulating gastric acid secretion and preventing lesion formation. When ip injected, both UFP-112 and N/OFQ left gastric emptying in rats unchanged, suggesting that peripheral NOP receptors have a role in mediating gastric hypersecretory and antiulcer effects but are not involved in regulating gastric motility. In addition, the inhibitory effects induced by this novel NOP receptor agonist lasted longer than those induced by N/OFQ. In conclusion, UFP-112 is a promising new pharmacological tool for studying the functional roles of the central and peripheral N/OFQ receptor system.
Keywords: UFP-112; Gastric emptying; Secretion and ulcers;
Changes of β-casomorphin content in human milk during lactation by Beata Jarmołowska; Katarzyna Sidor; Małgorzata Iwan; Krzysztof Bielikowicz; Maciej Kaczmarski; Elżbieta Kostyra; Henryk Kostyra (1982-1986).
Milk is the best, complete food important for the development and nourishment of a neonate. Except for nutrients, milk contains biologically active opioid peptides derived from β-casein, named β-casomorphins (BCMs), which can exert effects in the gastrointestinal tract as well as in the whole body of neonates. The content of β-casomorphins in human milk during maturation phases has not been studied so far. The aim of this study was to determine the content of β-casomorphin-5 and -7 in human milk in different phases of lactation. A significantly higher concentration of both β-casomorphins was found in colostrum than in mature milk. The concentration of β-casomorphin in milk collected in the second month of lactation was similar to the level obtained in the fourth month of lactation. The content of β-casomorphins in human milk was observed with the period of lactation. The level of opioid peptides may depend on the function of these peptides in neonate's body and may be associated with the maturation process.
Keywords: β-Casomorphins; Opioid peptides; Human milk; Lactation;
Beta-endorphin levels in longtailed and pigtailed macaques vary by abnormal behavior rating and sex by Carolyn M. Crockett; Gene P. Sackett; Curt A. Sandman; Aleksandra Chicz-DeMet; Kathleen L. Bentson (1987-1997).
Frequent or severe abnormal behavior may be associated with the release of endorphins that positively reinforce the behavior with an opiate euphoria or analgesia. One line of research exploring this association involves the superhormone, proopiomelanocortin (POMC). The products of POMC appear to be dysregulated in some human subjects who exhibit self-injurious behavior (SIB). Macaque monkeys have POMC very similar to humans, and some laboratory macaques display SIB or frequent stereotypies. We investigated associations between plasma levels of three immunoreactive POMC fragments with possible opioid action and abnormal behavior ratings in macaques. In 58 adult male and female macaques (24 Macaca fascicularis and 34 Macaca nemestrina), plasma levels of intact beta-endorphin (βE) and the N-terminal fragment (BEN) were significantly higher in animals with higher levels of abnormal behavior. The C-terminal fragment (BEC) was significantly higher in males but unrelated to ratings of abnormal behavior. Levels of ACTH, cortisol, and (βE-ACTH)/βE dysregulation index were unrelated to abnormal behavior. None of the POMC products differed significantly by subjects’ species, age, or weight. The finding that intact beta-endorphin is positively related to abnormal behavior in two species of macaque is consistent with some previous research on human subjects and nonprimates. The positive relation of the N-terminal fragment of βE to abnormal behavior is a new finding.
Keywords: Self-injurious behavior (SIB); Stereotypy; Abnormal behavior; Opioids; Beta-endorphin; Proopiomelanocortin (POMC); Adrenocorticotropic hormone (ACTH); Cortisol; Macaca fascicularis; Macaca nemestrina;
Rubiscolin-6, a δ opioid peptide derived from spinach Rubisco, has anxiolytic effect via activating σ1 and dopamine D1 receptors by Hajime Hirata; Soushi Sonoda; Shun Agui; Mariko Yoshida; Kousaku Ohinata; Masaaki Yoshikawa (1998-2003).
Rubiscolin-6 (Tyr-Pro-Leu-Asp-Leu-Phe) is a δ opioid peptide derived from the large subunit of spinach d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). We previously reported that rubiscolin-6 had an analgesic effect and stimulated memory consolidation. Here we show that intraperitoneally (i.p.) or orally administered rubiscolin-6 has an anxiolytic effect at a dose of 10 mg/kg or 100 mg/kg, respectively, in the elevated plus-maze test in mice. The anxiolytic effects of rubscolin-6 after i.p. (10 mg/kg) and oral (100 mg/kg) administration were blocked by a δ opioid receptor antagonist, naltrindole (1 mg/kg, s.c.), suggesting that the anxiolytic activity of rubiscolin-6 is mediated by δ opioid receptor. The anxiolytic effect of rubiscolin-6 (10 mg/kg, i.p.) was also blocked by a dopamine D1 antagonist, SCH23390 (30 μg/kg, i.p.), but not by a dopamine D2 antagonist, raclopride (15 μg/kg, i.p.). The anxiolytic effect of rubiscolin-6 (10 mg/kg, i.p.) was blocked by σ1 receptor antagonist, BMY14802 (0.5 mg/kg, i.p.) or BD1047 (10 mg/kg, i.p.). Taken together, the anxiolytic effect of rubiscolin-6 is mediated by σ1 and dopamine D1 receptors downstream of δ opioid receptor.
Keywords: Opioid peptide; Anxiolytic effect; Rubisco; Dopamine receptor; Sigma receptor;
NAP protects hippocampal neurons against multiple toxins by Ilona Zemlyak; Nathan Manley; Robert Sapolsky; lllana Gozes (2004-2008).
The femtomolar-acting protective peptide NAP (NAPVSIPQ), derived from activity-dependent neuroprotective protein (ADNP), is broadly neuroprotective in vivo and in vitro in cerebral cortical cultures and a variety of cell lines. In the present study, we have extended previous results and examined the protective potential of NAP in primary rat hippocampal cultures, using microtubule-associated protein 2 (MAP2) as a measure for neuroprotection. Results showed that NAP, at femtomolar concentrations, completely protected against oxygen-glucose deprivation, and cyanide poisoning. Furthermore, NAP partially protected against kainic acid excitotoxicity. In summary, we have significantly expanded previous findings in demonstrating here direct neuroprotective effects for NAP on vital hippocampal neurons that are key participants in cognitive function in vivo.
Keywords: NAP; Kainic acid; Oxygen-glucose deprivation; Sodium cyanide; Neuronal death;
MIF-1 and its peptidomimetic analogs attenuate haloperidol-induced vacuous chewing movements and modulate apomorphine-induced rotational behavior in 6-hydroxydopamine-lesioned rats by Jessica M. Castellano; Jody Batrynchuk; Kristine Dolbeare; Vaneeta Verma; Amandeep Mann; Kevin J. Skoblenick; Rodney L. Johnson; Ram K. Mishra (2009-2015).
Two melanocyte-stimulating hormone release inhibiting factor-1 (MIF-1) also known as l-prolyl-l-leucyl-glycinamide (PLG) peptidomimetic analogs, 3(R)-[[[2(S)-pyrrolidinyl]carbonyl]-amino]-3-(butyl)-2-oxo-1-pyrrolidineacetamide trifluoroacetate (A) and 3(R)-[[[2(S)-pyrrolidinyl]carbonyl]amino]-3-(benzyl)-2-oxo-1-pyrrolidineacetamide trifluoroacetate (B), were evaluated for their ability to modulate dopaminergic activity by measuring apomorphine-induced rotations in 6-hydroxydopamine (6-OHDA)-lesioned rats, and haloperidol (HP)-induced vacuous chewing movements (VCMs) in rats; animal models of Parkinson's disease (PD) and human tardive dyskinesia (TD), respectively. In the 6-OHDA model, both analogs were found to potentiate the contralateral rotational behavior induced by apomorphine dose-dependently and with approximately the same potency. Furthermore, each analog was able to significantly attenuate HP-induced VCMs with almost equal efficacy. The potency and efficacy of these analogs were significantly greater than their parent compound, PLG. These results suggest that both analogs can modulate dopaminergic activity in vivo, likely by the same mechanisms recruited by PLG previously reported.
Keywords: Dopamine; Melanocyte-stimulating hormone release inhibiting factor-1 (MIF-1); Parkinson's disease; Tardive dyskinesia; 6-Hydroxydopamine; Vacuous chewing movements;
The synthetic melanocortin (CKPV)2 exerts broad anti-inflammatory effects in human neutrophils by Franco Capsoni; Annamaria Ongari; Gualtiero Colombo; Flavia Turcatti; Anna Catania (2016-2022).
Natural melanocortin peptides exert broad effects on the host and they have remarkable therapeutic potential. However, successful use of melanocortins as therapeutic agents depends on the design of molecules that have more stable pharmacological profiles. The synthetic peptide (CKPV)2, based on the C-terminal sequence of α-melanocyte stimulating hormone (α-MSH), has anti-tumor necrosis factor-α (TNF-α) effects in vitro and in vivo and is a promising candidate to treat inflammation. Because neutrophil activity is a major target for anti-inflammatory therapies, we determined whether (CKPV)2 modulates human neutrophil functions in vitro. Incubation of freshly-separated human neutrophils with 10−12–10−6 M (CKPV)2 significantly inhibited activities relevant to the inflammatory reaction. Neutrophil migration toward the two chemoattractants interleukin 8 (IL-8) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) was significantly inhibited by (CKPV)2. (CKPV)2 also inhibited reactive oxygen intermediate (ROI) production induced by phorbol 12-myristate 13-acetate (PMA), but not that induced by fMLP. Because these effects of (CKPV)2 were abolished by the adenylyl cyclase inhibitor 2′,5′-dideoxyadenosine (ddAdo), they appear to be cAMP-dependent. Finally, the peptide reduced lipopolysaccharide (LPS)-stimulated expression of TNF-α, interleukin-1β (IL-1β), interleukin-8 (IL-8), and intercellular adhesion molecule 1 (ICAM-1), as well as TNF-α protein release in cell supernatants. The data indicate that (CKPV)2 modulates broad cAMP-dependent, anti-inflammatory pathways in human neutrophils.
Keywords: Melanocortin peptides; α-Melanocyte stimulating hormone (α-MSH); (CKPV)2; Human neutrophils; Chemotaxis; Respiratory burst; Lipopolysaccharide (LPS);
Apelin activates l-arginine/nitric oxide synthase/nitric oxide pathway in rat aortas by Yue Xia Jia; Zheng Fei Lu; Jing Zhang; Chun Shui Pan; Jing Hui Yang; Jing Zhao; Fang Yu; Xiao Hui Duan; Chao Shu Tang; Yong Fen Qi (2023-2029).
Apelin was recently found to be an inotropic polypeptide in isolated rat hearts, and intravenous injection of apelin can induce a transient decrease in blood pressure. To illustrate the mechanism of apelin-induced vasodilation, we observed the in vitro effects of apelin on the l-arginine (l-Arg)/nitric oxide (NO) pathway in the incubated, isolated rat aorta. Apelin stimulated vascular NO2 − product and NOS activation in a concentration- and time-dependent manner. Compared with no apelin treatment, incubation with apelin (10−9, 10−8, and 10−7 mol/L) increased NO2 − product by 33%, 46%, and 69% (all p < 0.01), respectively, and Ca2+-dependent constitutive NOS (cNOS) activity by 200%, 460%, and 550% (all p < 0.01), respectively. However, Ca2+-independent NOS (iNOS) activity was not significantly altered (p > 0.05). Apelin incubation (10−9, 10−8, and 10−7 mol/L) increased l-Arg uptake by 130%, 180%, and 240% (all p < 0.01), respectively. The mRNA level of cationic amino acid transporters, CAT-1 and CAT-2B, in rat aortic tissues treated with 10−7 mol/L apelin was increased by 110% and 128%, respectively (both p < 0.01). Incubation with 10−7 mol/L apelin elevated eNOS mRNA and protein levels, by 53% (p < 0.05) and 319% (p < 0.01), respectively. Collectively, these results demonstrate that apelin directly activated the vascular l-Arg/NOS/NO pathway, which could be one of the important mechanisms of apelin-regulated vascular function.
Keywords: Apelin; Nitric oxide; Nitric oxide synthase; Cationic amino acid transporters; l-Arginine;
Over-expression of neurotensin high-affinity receptor 1 (NTS1) in relation with its ligand neurotensin (NT) and nuclear ß-catenin in inflammatory bowel disease-related oncogenesis by Céline Bossard; Frédérique Souazé; Anne Jarry; Stéphane Bezieau; Jean-François Mosnier; Patricia Forgez; Christian L. Laboisse (2030-2035).
We investigated the expression of the neurotensin high-affinity receptor 1 (NTS1) during inflammatory bowel disease (IBD)-related colorectal oncogenesis, in colonic samples from 30 patients with IBD-related adenocarcinomas, dysplasias, and inflammatory mucosa (IM).The percentage of NTS1-positive epithelial cells progressively increased from the inflammatory condition to adenocarcinoma and was significantly higher in adenocarcinomas than in IM (p = 0.0169). In parallel, the percentage of neurotensin (NT)-positive epithelial cells increased during the IBD-related oncogenesis. Finally, as NTS1 is a ß-catenin inducible gene, we found that a number of preneoplastic lesions and adenocarcinomas co-expressed NTS1 and ß-catenin without NT expression. Therefore, this study suggests two pathways of NTS1 overexpression during IBD-related oncogenesis: one triggered by NT overexpression, and a second associated with an activation of the APC/β-catenin pathway, these two pathways being not mutually exclusive.
Keywords: Neurotensin high-affinity receptor 1 (NTS1); Neurotensin (NT); Inflammatory bowel disease; Colorectal oncogenesis; APC/β-catenin pathway;
Endothelin-1 reduces mesenteric microvascular hydraulic permeability via cyclic AMP and protein kinase A signal transduction by Terry J. Chong; Javid Sadjadi; Brian Curran; Gregory P. Victorino (2036-2041).
We have previously shown that endothelin-1 (ET-1) decreases microvascular hydraulic permeability. In this study, we tested the hypothesis that ET-1 exerts its permeability-decreasing effect through cAMP, cGMP, and protein kinase A (PKA) by determining the effect of ET-1 on venular fluid leak during inhibition of cAMP synthesis, inhibition of cGMP degredation, and inhibition of PKA. Rat mesenteric venules were cannulated to measure hydraulic permeability, L p (units × 10−7 cm/(s cmH2O)). L p was measured during continuous perfusion of 80 pM ET-1 and either (1) an inhibitor of cAMP synthesis (10 μM 2′,5′ddA), (2) an inhibitor of cGMP degradation (100 μM Zaprinast), or (3) an inhibitor of PKA (10 μM H-89). Inhibition of cAMP synthesis blocked the permeability decreasing effects of ET-1. The peak L p of the cAMP inhibitor alone and with ET-1 was 4.11 ± 0.53 and 3.86 ± 0.19, respectively (p = 0.36, n = 6). Inhibition of cGMP degradation did not block the permeability decreasing effects of ET-1. The peak L p during inhibition of cGMP degradation alone and with ET-1 was 2.26 ± 0.15 and 1.44 ± 0.09, respectively (p < 0.001, n = 6). Inhibition of PKA activation blocked the permeability decreasing effects of ET-1. The peak L p of the PKA inhibitor alone and with ET-1 was 2.70 ± 0.15 and 2.59 ± 0.15, respectively (p = 0.38, n = 6). The data support the notion that the signal transduction mechanism of ET-1 with regard to decreasing microvascular fluid leak involves cAMP production and PKA activation, but not cGMP degradation. Further understanding of intracellular mechanisms that control microvascular fluid leak could lead to the development of a pharmacologic therapy to control third space fluid loss in severely injured or septic patients.
Keywords: Endothelin-1; cAMP; cGMP; Protein kinase A; Microvascular permeability; Hydraulic permeability;
Jaburetox-2Ec: An insecticidal peptide derived from an isoform of urease from the plant Canavalia ensiformis by F. Mulinari; F. Stanisçuaski; L.R. Bertholdo-Vargas; M. Postal; O.B. Oliveira-Neto; D.J. Rigden; M.F. Grossi-de-Sá; C.R. Carlini (2042-2050).
Canatoxin, a urease isoform from Canavalia ensiformis seeds, shows insecticidal activity against different insect species. Its toxicity relies on an internal 10 kDa peptide (pepcanatox), released by hydrolysis of Canatoxin by cathepsins in the digestive system of susceptible insects. In the present work, based on the N-terminal sequence of pepcanatox, we have designed primers to amplify by PCR a 270-bp fragment corresponding to pepcanatox using JBURE-II cDNA (one of the urease isoforms cloned from C. ensiformis, with high identity to JBURE-I, the classical urease) as a template. This amplicon named jaburetox-2 was cloned into pET 101 vector to obtain heterologous expression in Escherichia coli of the recombinant protein in C-terminal fusion with V-5 epitope and 6-His tag. Jaburetox-2Ec was purified on Nickel-NTA resin and bioassayed in insect models. Dysdercus peruvianus larvae were fed on cotton seed meal diets containing 0.01% (w/w) Jaburetox-2Ec and, after 11 days, all individuals were dead. Jaburetox-2Ec was also tested against Spodoptera frugiperda larvae and caused 100% mortality. In contrast, high doses of Jaburetox-2Ec were innocuous when injected or ingested by mice and neonate rats. Modeling of Jaburetox-2Ec, in comparison with other peptide structures, revealed a prominent β-hairpin motif consistent with an insecticidal activity based on either neurotoxicity or cell permeation.
Keywords: Canatoxin; Insecticide; Spodoptera frugiperda; Dysdercus peruvianus; Molecular modeling; Heterologous expression;
Antibodies elicited by a virosomally formulated Plasmodium falciparum serine repeat antigen-5 derived peptide detect the processed 47 kDa fragment both in sporozoites and merozoites by Shinji L. Okitsu; Francesca Boato; Markus S. Mueller; Dong Bo Li; Denise Vogel; Nicole Westerfeld; Rinaldo Zurbriggen; John A. Robinson; Gerd Pluschke (2051-2060).
Serine repeat antigen-5 (SERA5) is a candidate antigen for inclusion into a malaria subunit vaccine. During merozoite release and reinvasion the 120 kDa SERA5 precursor protein (P120) is processed, and a complex consisting of an N-terminal 47 kDa (P47) and a C-terminal 18 kDa (P18) processing product associates with the surface of merozoites. This complex is thought to be involved in merozoite invasion of and/or egress from host erythrocytes. Here we describe the synthesis and immunogenic properties of virosomally formulated synthetic phosphatidylethanolamine (PE)–peptide conjugates, incorporating amino acid sequence stretches from the N-terminus of Plasmodium falciparum SERA5. Choosing an appropriate sequence was crucial for the development of a peptide that elicited high titers of parasite cross-reactive antibodies in mice. Monoclonal antibodies (mAbs) raised against the optimized peptide FB-23 incorporating amino acids 57–94 of SERA5 bound to both P120 and to P47. Western blotting analysis proved for the first time the presence of SERA5 P47 in sporozoites. In immunofluorescence assays, the mAbs stained SERA5 in all its predicted localizations. The virosomal formulation of peptide FB-23 is suitable for use in humans and represents a candidate component for a multi-valent malaria subunit vaccine targeting both sporozoites and blood stage parasites.
Keywords: Malaria vaccine; Virosomes; Synthetic peptides; Serine repeat antigen-5;
Evidence that the genes encoding the melittin-related peptides in the skins of the Japanese frogs Rana sakuraii and Rana tagoi are not orthologous to bee venom melittin genes: Developmental- and tissue-dependent gene expression by Hiroe Suzuki; J. Michael Conlon; Shawichi Iwamuro (2061-2068).
The antimicrobial melittin-related peptides (MRPs) isolated from skin extracts of the Japanese frogs, Rana sakuraii and Rana tagoi, show amino acid sequence similarity with melittin from the venom of honeybees but the evolutionary relationship between the amphibian and insect peptides is unknown. cDNA clones encoding the MRP precursor (preproMRP) were obtained from R. sakuraii and R. tagoi skin total RNA. The nucleotide and deduced amino acid sequences of the clones indicated that the preproMRPs are organized like typical amphibian antimicrobial peptide precursors, with a highly conserved signal peptide, a more variable intervening sequence, and a hypervariable mature peptide region. This organization is markedly different from that of prepromelittin, in which the melittin sequence is flanked by multiple Xaa-Pro and Xaa-Ala dipeptides. The data indicate, therefore, that the genes encoding frog skin MRPs are not orthologous to the genes encoding melittins from bee venom. In adult R. sakuraii specimens, preproMRP gene transcripts were detected in total RNA from skeletal muscle as well as skin but not from heart, stomach, small intestine, or liver. In R. tagoi, preproMRP mRNA was not detected in skin prior to the onset of metamorphosis, but its level increased markedly during metamorphosis reaching a maximum at the stages of metamorphic climax.
Keywords: Antimicrobial peptide; Rana tagoi; Rana sakuraii; Melittin-related peptide; Frog skin; Bee venom;
A new family of antimicrobial peptides from skin secretions of Rana pleuraden by Xu Wang; Yuzhu Song; Jianxu Li; Huan Liu; Xueqing Xu; Ren Lai; Keyun Zhang (2069-2074).
While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Guizhou region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the Yunnan frog, Rana pleuraden. Members of the new peptide family named pleurain-As are composed of 26 amino acids with a unique N-terminal sequence (SIIT) and a disulfide-bridged heptapeptide sequence (CRLYNTC). By BLAST search, pleurain-As had no significant similarity to any known peptides. Native and synthetic peptides showed antimicrobial activities against tested microorganisms including Gram-negative and Gram-positive bacteria and fungi. Twenty different cDNAs encoding pleurain-As were cloned from the skin cDNA library of R. pleuraden. The precursors of pleurain-As are composed of 69 amino acid residues including predicted signal peptides, acidic propieces, and cationic mature antimicrobial peptides. The preproregion of pleurain-A precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature pleurain-As are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor. Furthermore, pleurain-As could exert antimicrobial capability against Helicobacter pylori. This is the first report of naturally occurring peptides with anti-H. pylori activity from Rana amphibians.
Keywords: Amphibian; Antimicrobial peptides; Rana pleuraden; Helicobacter pylori;
Effect of aminoisobutyric acid (Aib) substitutions on the antimicrobial and cytolytic activities of the frog skin peptide, temporin-1DRa by J. Michael Conlon; Rokaya Al-Kharrge; Eman Ahmed; Haider Raza; Sehamuddin Galadari; Eric Condamine (2075-2080).
Temporin-1DRa (HFLGTLVNLAKKIL.NH2), first isolated from the skin of the California red-legged frog Rana draytonii, shows broad-spectrum antimicrobial activity but its therapeutic potential is limited by its toxicity against mammalian cells. The cytolytic properties of cationic α-helical peptides are determined by a complex interaction between cationicity, hydrophobicity, conformation, and amphipathicity. This study has investigated the cytolytic properties of conformationally constrained analogs of temporin-1DRa containing α-aminoisobutyric acid (Aib) substitutions. Cytolytic activity was determined against the bacteria Escherichia coli and Staphylococcus aureus, the opportunistic yeast pathogen, Candida albicans, human erythrocytes, HepG2 hepatoma-derived cells, and L929 fibroblasts. Aib substitutions at Gly4, Asn8, and Ala10 increased both % helicity, determined in methanol solution, and hydrophobicity resulting in increases in both antimicrobial potencies and toxicities against the mammalian cells. Substitution at Leu6 resulted in an appreciable decrease in cytolytic activity against all cells whereas the substitutions at His1, Phe2, Leu3, Thr5, and Val7 had only minor effects on activity. Substitutions at Leu9, Ile13, Leu14 produced analogs with decreased helicity and hydrophobicity that retained activity against microorganisms but showed appreciably lower cytolytic activities against mammalian cells. In particular, the fourfold increase in therapeutic index [ratio of LC50 against erythrocytes to minimum inhibitory concentration (MIC) against microorganisms] of [Aib13]temporin-1DRa identifies it as a compound with potential for development as a therapeutically valuable anti-infective agent.
Keywords: Temporin; Frog skin; Amphipathic α-helix; Cytolysis; Antimicrobial; α-Aminoisobutyric acid;
The concentration dependent biphasic effect of leptin on endogenous cholesterol synthesis in human monocytes by Zoltán Balogh; Gabriella Fóris; Béla Kosztáczky; György Paragh; Ildikó Seres; Emese Zsíros; Gabriella Kónya; György Paragh (2081-2083).
In human monocytes 100 ng/mL leptin increased both statin-inhibitable free radical and cholesterol production in vitro. In our recent study, we aimed to elucidate the concentration dependence of observed leptin-effect. Following leptin stimulation cholesterol synthesis was measured in the presence of inhibitors to determine affected signal pathways. Leptin at low (10–100 ng/mL) concentrations increased [14C]acetate incorporation, whereas at 250 ng/mL and higher concentrations it suppressed cholesterol synthesis. HMG CoA reductase, phosphatidyl-3-kinase (PI3K) and mitogen activated protein kinase (MAPK) were involved in mediating leptin effects at low concentrations, whereas the cholesterol synthesis suppression was abolished by inhibitors of protein kinase C (PKC) and PI3K.
α-Melanocyte stimulating hormone and ghrelin: Central interaction in feeding control by Pawel K. Olszewski; Eric M. Bomberg; Martha K. Grace; Allen S. Levine (2084-2089).
α-Melanocyte stimulating hormone (α-MSH) and ghrelin play significant yet opposite roles in the regulation of feeding: α-MSH inhibits, whereas ghrelin stimulates consumption. The two peptidergic systems may interact in the process of food intake control. A single report published thus far has shown that a synthetic agonist of the melanocortin receptors, MTII, injected in the hypothalamic paraventricular nucleus (PVN) decreases feeding generated by ghrelin. We found that very low doses of α-MSH and MTII administered ICV significantly reduced ghrelin-dependent hyperphagia. However, an endogenous molecule, α-MSH, infused in the PVN did not exert an inhibitory effect on ghrelin-induced consumption, whereas the effective dose of PVN MTII exceeded that necessary to decrease short-term deprivation-induced feeding. We conclude that it is likely that in feeding regulation α-MSH and ghrelin “interact” at the central nervous system level, but the involvement of the PVN in this interaction appears questionable.