Peptides (v.32, #7)

Stimulatory effect of intracerebroventricular administration of orexin A on food intake in the zebrafish, Danio rerio by Eri Yokobori; Kenji Kojima; Morio Azuma; Ki Sung Kang; Sho Maejima; Minoru Uchiyama; Kouhei Matsuda (1357-1362).
► The number of orexin A-containing neuronal cells increases in zebrafish fasted for 7 days. ► Orexin precursor mRNA levels also increase in fish fasted for 7 days. ► ICV administration of orexin A increases food consumption in zebrafish. ► Orexin A-induced orexigenic action is blocked by treatment with SB334867.Orexin is a potent orexigenic neuropeptide implicated in feeding regulation of mammals. However, except for the case of goldfish, the involvement of orexin in the feeding behavior of teleost fish has not well been studied. Therefore, we investigated the role of orexin on food intake using a zebrafish (Danio rerio) model. We examined the effect of feeding status on orexin-like immunoreactivity and the expression level of orexin transcript in the brain. The number of neuronal cells showing orexin-like immunoreactivity in the hypothalamic region, including the posterior tuberal nucleus, was significantly increased in fish fasted for 7 days. Orexin precursor mRNA levels in the brain obtained from fish fasted for 7 days were higher than those in fish that had been fed normally. We then investigated the effect of intracerebroventricular (ICV) administration of orexin A on food intake. Cumulative food intake was significantly increased by ICV administration of orexin A (at 0.3 and 3 pmol/g body weight, BW) during a 60-min observation period after treatment. The orexin A-induced orexigenic action (at 0.3 pmol/g BW) was blocked by treatment with an orexin receptor antagonist, SB334867, at 10 pmol/g BW. These results indicate that orexin A acts as feeding regulator in the zebrafish.
Keywords: Zebrafish; Orexin A; Food intake; Orexigenic action; SB334867; Orexin precursor mRNA;

Orange-spotted grouper (Epinephelus coioides) orexin: Molecular cloning, tissue expression, ontogeny, daily rhythm and regulation of NPY gene expression by Aifen Yan; Lingjuang Zhang; Zhiguo Tang; Yanhong Zhang; Chaobin Qin; Bo Li; Wensheng Li; Haoran Lin (1363-1370).
► Grouper orexin was identified by cDNA cloning. ► The prepro-orexin mRNA was widely present in brain and peripheral tissues. ► The prepro-orexin mRNA was first detected at the neural stage of grouper embryo. ► Grouper hypothalamic prepro-orexin mRNA significantly increased at the meal-time. ► Grouper orexin-A can stimulate NPY mRNA expression in vivo and in vitro.Orexin-A and -B, collectively called orexins, are hypothalamic neuropeptides involved in the regulation of food intake, sleep and energy balance. In this study, the full-length cDNA of prepro-orexin was isolated from the hypothalamus of orange-spotted grouper (Epinephelus coioides) using RT-PCR and RACE. The grouper prepro-orexin cDNA is 711 bp in length and encodes a 149-amino acid precursor protein that contains a 46-amino acid signal peptide, a 43-amino acid mature orexin-A peptide, a 27-amino acid mature orexin-B peptide and a 33-amino acid C terminus of unknown function. The tissue distribution and ontogeny of prepro-orexin were examined by quantitative real-time PCR. We found that the prepro-orexin mRNA is widely expressed in brain and peripheral tissues, with abundant expression in the hypothalamus. During the embryonic development, prepro-orexin mRNA was first detected in neurula stage embryos, and its expression gradually increased during the remainder of embryogenesis. Our analysis of grouper hypothalamic prepro-orexin expression showed that prepro-orexin mRNA levels were greater in the light phase than in the dark phase and increased significantly at meal-time. Intraperitoneal injection of orexin-A caused a dose-related increase in hypothalamus NPY mRNA expression level after 4 h. Orexin-A also increased NPY mRNA expression level from static hypothalamic fragments incubation. Our results imply that orexin may be involved in feeding in the orange-spotted grouper and orexin-A is a stimulator of NPY mRNA expression in vivo and in vitro.
Keywords: Orexin; Grouper; Daily rhythm; Regulation of NPY;

Developmental overfeeding alters hypothalamic neuropeptide mRNA levels and response to a high-fat diet in adult mice by Silvia Ferretti; Alice Fornari; Patrizia Pedrazzi; Massimo Pellegrini; Michele Zoli (1371-1383).
► We studied hypothalamic neuropeptide expression in developmentally overfed mice. ► Breast and/or adolescence overfed adult mice had reduced NPY mRNA levels. ► Adult high fat diet further reduced neuropeptide levels in developmentally overfed mice. ► Several peptidergic circuits are altered in developmentally overfed adult mice.It has been suggested that nutritional manipulations during the first weeks of life can alter the development of the hypothalamic circuits involved in energy homeostasis. We studied the expression of a large number of the hypothalamic neuropeptide mRNAs that control body weight in mice that were overfed during breastfeeding (mice grown in a small litter, SL) and/or during adolescence (adolescent mice fed a high-fat diet, AHF). We also investigated possible alterations in mRNA levels after 50 days of a high-fat diet (high-fat challenge, CHF) at 19 weeks of age. Both SL and AHF conditions caused overweight during the period of developmental overfeeding. During adulthood, all of the mouse groups fed a CHF significantly gained weight in comparison with mice fed a low-fat diet, but the mice that had undergone both breast and adolescent overfeeding (SL-AHF-CHF mice) gained significantly more weight than the control CHF mice. Of the ten neuropeptide mRNAs studied, only neuropeptide Y (NPY) expression was decreased in all of the groups of developmentally overfed adult mice, but CHF during adulthood by itself induced a decrease in NPY, agouti-related protein (AgRP) and orexin (Orx) mRNA levels. Moreover, in the developmentally overfed CHF mice NPY, AgRP, galanin (GAL) and galanin-like peptide (GalP) mRNA levels significantly decreased in comparison with the control CHF mice. These results show that, during adulthood, hypothalamic neuropeptide systems are altered (NPY) and/or abnormally respond to a high-fat diet (NPY, AgRP, GAL and GalP) in mice overfed during critical developmental periods.
Keywords: Hypothalamic neuropeptides; Metabolic imprinting; Breast overfeeding; Adolescent overfeeding; Overweight; Mice;

Hyperleptinemia in obese adolescents deregulates neuropeptides during weight loss by Ana R. Dâmaso; Aline de Piano; Priscila L. Sanches; Flávia Corgosinho; Lian Tock; Lila M. Oyama; Luciana Tock; Claudia M. Oller do Nascimento; Sérgio Tufik; Marco Túlio de Mello (1384-1391).
► Improvement in anthropometric and biochemical measures after interdisciplinary intervention. ► Increase of adiponectin and A/L and reduction in leptin and NPY/AgRP in hyperleptinemic. ► α-MSH and body fat mass (%) = independent predictors to explain leptin concentration. ► Hyperleptinemia deregulates orexigenic and anorexigenic neuropeptides during weight loss.Leptin has emerged over the past decade as a key hormone not only in energy balance regulation but also in neuroendocrine and inflammatory processes. The aim of the present study was to evaluate whether hyperleptinemia deregulates neuropeptides during weight loss. A total of 86 post-pubertal obese adolescents (with or without hyperleptinemia) participated in one year of interdisciplinary weight loss therapy (clinical, nutritional, psychological and exercise-related). Adipokine and neuropeptide concentrations were measured by ELISA, visceral fat was measured by ultrasound and body composition was measured by pletismography. The hyperleptinemic patients presented a lower alpha-MSH concentration and higher NPY/AgRP ratio while the adiponectin/leptin (A/L) ratio was lower compared with the non-hyperleptinemic group. After therapy, significant improvements in BM, BMI, body fat mass, visceral and subcutaneous fat, HOMA-IR, QUICKI, total cholesterol and triglycerides were observed in both groups. Indeed, we observed significant increases in adiponectin and A/L as well as reductions in leptin and NPY/AgRP ratio in the hyperleptinemic group. In the stepwise multiple linear regression analysis with leptin concentration as the dependent variable, α-MSH and body fat mass (%) were the independent predictors to explain leptin concentration. For the entire group, we found positive correlations between leptinemia and BMI and body fat mass (%) as well as a negative correlation with free fat mass (%) and alpha-MSH. Finally, we verified negative correlations between adiponectin/leptin ratio with total cholesterol and LDL-c, only in hyperleptinemic patients. In conclusion, the hyperleptinemia in obese adolescents deregulates neuropeptides during weight loss.
Keywords: Leptin; Obesity; Neuropeptides; Adolescent;

Effects of cell-type specific leptin receptor mutation on leptin transport across the BBB by Hung Hsuchou; Abba J. Kastin; Hong Tu; Emily N. Markadakis; Kirsten P. Stone; Yuping Wang; Steven B. Heymsfield; Streamson S. Chua; Silvana Obici; I. Jack Magrisso; Weihong Pan (1392-1399).
► Generation and characterization of ELKO and ALKO mice for the first time. ► Illustrating that membrane-bound mutant receptors without signaling functions can mediate leptin transport across the BBB. ► Leptin signaling may hinder its own transport across endothelial barrier. ► Astrocytic leptin receptors do not play a crucial role in basal level leptin transport. ► Implications for obesity, metabolic syndrome, and neuroinflammation.The functions of leptin receptors (LRs) are cell-type specific. At the blood–brain barrier, LRs mediate leptin transport that is essential for its CNS actions, and both endothelial and astrocytic LRs may be involved. To test this, we generated endothelia specific LR knockout (ELKO) and astrocyte specific LR knockout (ALKO) mice. ELKO mice were derived from a cross of Tie2-cre recombinase mice with LR-floxed mice, whereas ALKO mice were generated by a cross of GFAP-cre with LR-floxed mice, yielding mutant transmembrane LRs without signaling functions in endothelial cells and astrocytes, respectively. The ELKO mutation did not affect leptin half-life in blood or apparent influx rate to the brain and spinal cord, though there was an increase of brain parenchymal uptake of leptin after in situ brain perfusion. Similarly, the ALKO mutation did not affect blood–brain barrier permeation of leptin or its degradation in blood and brain. The results support our observation from cellular studies that membrane-bound truncated LRs are fully efficient in transporting leptin, and that basal levels of astrocytic LRs do not affect leptin transport across the endothelial monolayer. Nonetheless, the absence of leptin signaling at the BBB appears to enhance the availability of leptin to CNS parenchyma. The ELKO and ALKO mice provide new models to determine the dynamic regulation of leptin transport in metabolic and inflammatory disorders where cellular distribution of LRs is shifted.
Keywords: Leptin; BBB; Transport; Endothelial cells; Astrocytes; CNS effects;

Disulfide bond prolongs the half-life of therapeutic peptide-GLP-1 by Ying Li; Xin Li; Xuemin Zheng; Lida Tang; Weiren Xu; Min Gong (1400-1407).
► GLP-1 homodimeric analog (hdGLP1G10C) extended the biological half-life of GLP-1. ► hdGLP1G10C remained the biological activity of GLP-1. ► hdGLP1G10C showed better glucose tolerance and higher HbA1c reduction than GLP-1. ► hdGLP1G10C might be utilized as a long-lasting drug for type 2 diabetes.The multiple physiological characterization of glucagon-like peptide-1 (GLP-1) makes it a promising drug candidate for the therapy of type 2 diabetes. However, the half-life of GLP-1 is short in vivo due to rapid degradation by dipeptidyl peptidase-IV (DPP-IV) and renal clearance. This indicates that the stabilization of GLP-1 is critical for its utility in drug development. In this study, we developed a cluster of GLP-1 homodimeric analogs, which fused the mutated GLP-1 monomer by an intra-disulfide bridge. The stabilities of the GLP-1 homodimeric analogs were investigated and the physiological functions of the analogs were compared with those of wild-type GLP-1 in rats and human serum. Single dose glucose tolerance test was performed to investigate the administration frequency which satisfied the efficient glucose regulatory in rats. Multiple dose glucose tolerance tests were employed also to study the long-acting anti-diabetic activity of GLP-1 homodimeric analog. The results indicated that the GLP-1 homodimeric analog (hdGLP1G10C) remarkably raised the biological half-life of GLP-1; also HDGLP1G10C showed better glucose tolerance and higher HbA1c reduction than GLP-1 in rodents. Based upon the results in this study, it was suggested that hdGLP1G10C prolonged the stability of GLP-1 and retained the biological activity of GLP-1. The improved physiological characterization of hdGLP1G10C makes it as possible potent anti-diabetic drug in the treatment of type 2 diabetes mellitus.
Keywords: Long-acting GLP-1 analog; Disulfide bridge; Glucose tolerance test; HbA1c;

► GLP-1 analog (GLP715a) containing an inter-disulfide bond and a Trp-cage extended its half-life. ► The analog (GLP715a) remained the biological activity of GLP-1. ► GLP715a showed better glucose tolerance and higher HbA1c reduction than GLP-1. ► GLP715a might be utilized as a long-lasting drug for type 2 diabetes.The multiple physiological characterizations of glucagon-like peptide-1 (GLP-1) make it a promising drug candidate for the therapy of type 2 diabetes. However, the half-life of GLP-1 is short in vivo due to degradation by dipeptidyl peptidase-IV (DPP-IV) and renal clearance. Therefore, the stabilization of GLP-1 is critical for its utility in drug development. Based on our previous research, a GLP-1 analog that contained an intra-disulfide bond exhibited a prolonged biological half-life. In this study, we improved upon previous analogs with a novel GLP-1 analog that contained a tryptophan cage-like sequence for an improved binding affinity to the GLP-1 receptor. The binding capacities and the stabilities of GLP715a were investigated, and the physiological functions of the GLP715a were compared to those of the wild-type GLP-1 in animals. The results demonstrated that the new GLP-1 analog (GLP715a) increased its biological half-life to approximately 48 h in vivo; GLP715a also exhibited a higher binding affinity to the GLP-1 receptor than the wild-type GLP-1. The increased binding capacity of GLP715a to its receptor resulted in a quick response to glucose administration. The long-acting anti-diabetic property of GLP715a was revealed by its increased glucose tolerance, higher HbA1c reduction, more efficient glucose clearance and quicker insulin stimulation upon glucose administration compared to the wild-type GLP-1 in rodents. The improved physiological characterizations of GLP715a make it a possible potent anti-diabetic drug in the treatment of type 2 diabetes mellitus.
Keywords: Long-acting GLP-1 analogs; Disulfide bond; Tryptophan cage; Insulin stimulation; HbA1c;

Adrenomedullin protects against fructose-induced insulin resistance and myocardial hypertrophy in rats by Jing Zhang; Bao-Hong Zhang; Yan-Rong Yu; Chao-Shu Tang; Yong-Fen Qi (1415-1421).
► Endogenous ADM was increased in rats with insulin resistance induced by fructose. ► ADM has protective effect on fructose-induced insulin resistance. ► ADM inhibited cardiovascular hypertrophy associated with insulin resistance. ► ADM could act as a protective peptide against IR probably via its antioxidant effect.Adrenomedullin (ADM) has been recognized as a multipotent multifunctional peptide. To explore the pathophysiological roles of ADM in insulin resistance (IR), we studied the changes in ADM mRNA level in the myocardium and vessels and the effect of ADM supplementation on rats with IR induced by fructose feeding. Rats were fed 4% fructose in drinking water for 8 weeks, and ADM was administered subcutaneously in pure water through an Alzet Mini-osmotic Pump at 300 ng/kg/h for the last 4 weeks. Compared with controls, rats with IR showed increased levels of fasting blood sugar and serum insulin, by 95% and 67%, respectively (all P  < 0.01), and glycogen synthesis and glucose transport activity of the soleus decreased by 54% and 55% (all P  < 0.01). mRNA level and content of brain natriuretic peptide (BNP) in myocardial were all increased significantly. Fructose-fed rats showed increased immunoreactive-ADM content in plasma by 110% and in myocardia by 55% and increased mRNA level in myocardia and vessels (all P  < 0.01). ADM administration ameliorated the induced IR and myocardial hypertrophy. The glycogen synthesis and glucose transport activity of the soleus muscle increased by 41% (P  < 0.01) and 32% (P  < 0.05). ADM therapy attenuated myocardial and soleus lipid peroxidation injury and enhanced the antioxidant ability. Our results showed upregulation of endogenous ADM during fructose-induced IR and the protective effect of ADM on fructose-induced IR and concomitant cardiovascular hypertrophy probably by its antioxidant effect, which suggests that ADM could be an endogenous protective factor in IR.
Keywords: Adrenomedullin; Insulin resistance; Fructose-fed;

Caveolae are essential for angiotensin II type 1 receptor-mediated ANP secretion by Young-Bin Oh; Shan Gao; Jung Min Lim; Hyung Tae Kim; Byung-Hyun Park; Suhn Hee Kim (1422-1430).
► Methyl-β-cyclodextrin (MbCD) decreased a number of caveoli in rat atria. ► MbCD increased basal ANP secretion. ► MbCD attenuated stretch-induced ANP secretion. ► MbCD abolished angiotensin II-induced suppression and losartan-induced stimulation of ANP secretion. ► MbCD inhibited the process of proANP to ANP.Caveolae may act as mechanosensors and function as binding sites for calcium ions. The intracaveolar localization of atrial natriuretic peptide (ANP) derived from the direct interaction of atrial granules with caveolae has been demonstrated. The aim of this study was to define the effect of caveolae on ANP secretion induced by stretch and angiotensin II. The isolated perfused beating atria from Sprague-Dawley rats were used. To disrupt caveolae, 10 mM methyl-β-cyclodextrin (MbCD) was applied for 1 h and the number of caveoli were markedly decreased. MbCD increased basal ANP secretion and atrial diastolic pressure. The molecular profile of ANP in perfusate from control atria showed mainly one major peak corresponded to synthetic ANP whereas that from MbCD-treated atria showed two major immunoreactive peaks corresponded to synthetic rat ANP and proANP. High atrial stretch induced by elevating the height of outflow catheter from 5 cm H2O to 7.5 cm H2O increased atrial contractility and ANP secretion. The response of ANP secretion to high stretch was attenuated in MbCD-pretreated atria. Pretreatment with MbCD abolished angiotensin II-induced suppression and losartan-induced stimulation of ANP secretion. However, the effect of angiotenisin (1-7) on ANP secretion was not altered by MbCD treatment. The expression of angiotensin II type 1 receptor protein was reduced by MbCD treatment. These data suggest that caveolae are essential for angiotensin II type 1 receptor-mediated ANP secretion and relate to the processing of proANP.
Keywords: Caveolae; Caveolin; Atrial natriuretic peptide; Angiotensin II; Receptor; Atrial stretch; ANP profile;

Novel antihypertensive hexa- and heptapeptides with ACE-inhibiting properties: From the in vitro ACE assay to the spontaneously hypertensive rat by Pedro Ruiz-Giménez; José F. Marcos; Germán Torregrosa; Agustín Lahoz; Ricardo Fernández-Musoles; Salvador Valles; Enrique Alborch; Paloma Manzanares; Juan B. Salom (1431-1438).
► Synthetic heptapeptides were designed and screened as ACE inhibitors and antihypertensors. ► Heptapeptides inhibit in vitro ACE activity and ex vivo ACE-dependent vasoconstriction. ► Heptapeptides and lead hexapeptides lower blood pressure in hypertensive rats. ► Heptapeptides and hexapeptides do not modify normal blood pressure and are not cytotoxic.Bioactive ACE inhibiting peptides are gaining interest in hypertension treatment. We have designed and screened six synthetic heptapeptides (PACEI48 to PACEI53) based on two hexapeptide leads (PACEI32 and PACEI34) to improve ACE inhibitory properties and assess their antihypertensive effects. ACE activity was assayed in vitro and ex vivo. Selected peptides were administered to spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats. In vitro cytotoxicity was assessed with the MTT reduction test. The six heptapeptides at low micromolar concentration produced different degrees of in vitro inhibition of ACE activity using the synthetic substrate HHL or the natural substrate angiotensin I; and ex vivo inhibition of ACE-dependent, angiotensin I-induced vasoconstriction, but not angiotensin II-induced vasoconstriction. Oral administration of the hexapeptide PACEI32L, and the heptapeptides PACEI50L and PACEI52L, induced reductions in systolic blood pressure lasting up to 3 h in SHRs but not in WKY rats. Intravenous injection of PACEI32L and PACEI50L, but not PACEI52L, induced acute transient reductions in mean blood pressure of SHRs. d-Amino acid peptides showed five-fold less ACE inhibitory potency, no inhibitory effect on angiotensin I-induced vasoconstriction, and antihypertensive effect in SHRs after i.v. injection, but not after oral administration. The toxicity of peptides to reduce the viability of cultured cells was in the millimolar range. In conclusion, we have obtained novel rationally designed heptapeptides with improved ACE inhibitory properties when compared to lead hexapeptides. One selected hexapeptide and two heptapeptides show oral antihypertensive effects in SHRs and appear safe in cytotoxicity assays.
Keywords: Angiotensin converting enzyme; ACE inhibitor; Vasoconstriction; Hypertension; Spontaneously hypertensive rat; Antihypertensive peptide;

Pituitary adenylate cyclase-activating polypeptide plays an anti-inflammatory role in endotoxin-induced airway inflammation: In vivo study with gene-deleted mice by Krisztian Elekes; Katalin Sandor; Andras Moricz; Laszlo Kereskai; Agnes Kemeny; Eva Szoke; Aniko Perkecz; Dora Reglodi; Hitoshi Hashimoto; Erika Pinter; Janos Szolcsanyi; Zsuzsanna Helyes (1439-1446).
► In pituitary adenylate-cyclase polypeptide gene-deleted (PACAP−/−) mice, airway hyperreactivity is significantly higher 24 h after intranasal endotoxin administration and inflammatory histopathological changes are more severe both 6 h and 24 h later. ► Endotoxin-induced increase of myeloperoxidase (MPO) activity in the lung is almost double in PACAP−/− mice compared to the wild-types at 6 h. ► In contrast, there is no difference between the IL-1β concentrations of the lung of PACAP+/+ and PACAP−/− mice.The presence of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in capsaicin-sensitive peptidergic sensory nerves, inflammatory and immune cells suggest its involvement in inflammation. However, data on its role in different inflammatory processes are contradictory and there is little known about its functions in the airways. Therefore, our aim was to examine intranasal endotoxin-induced subacute airway inflammation in PACAP gene-deficient (PACAP−/−) and wild-type (PACAP+/+) mice. Airway responsiveness to inhaled carbachol was determined in unrestrained mice with whole body plethysmography 6 h and 24 h after LPS. Myeloperoxidase (MPO) activity referring to the number of accumulated neutrophils and macrophages was measured with spectrophotometry and interleukin-1β (IL-1β) concentration with ELISA from the lung homogenates. Histological evaluation and semiquantitative scoring were also performed. Bronchial responsiveness, as well as IL-1β concentration and MPO activity markedly increased at both timepoints. Perivascular edema dominated the histological picture at 6 h, while remarkable peribronchial granulocyte accumulation, macrophage infiltration and goblet cell hyperplasia were seen at 24 h. In PACAP−/− mice, airway hyperreactivity was significantly higher 24 h after LPS and inflammatory histopathological changes were more severe at both timepoints. MPO increase was almost double in PACAP−/− mice compared to the wild-types at 6 h. In contrast, there was no difference between the IL-1β concentrations of the PACAP+/+ and PACAP−/− mice. These results provide evidence for a protective role for PACAP in endotoxin-induced airway inflammation and hyperreactivity.
Keywords: Airway hyperresponsiveness; Whole body plethysmography; Enhanced pause (Penh); Myeloperoxidase activity; Interleukin-1β; Lipopolysaccharide;

Cigarette smoke-induced kinin B1 receptor promotes NADPH oxidase activity in cultured human alveolar epithelial cells by Sébastien Talbot; James Chi-Jen Lin; Karim Lahjouji; Jean-Philippe Roy; Jacques Sénécal; André Morin; Réjean Couture (1447-1456).
► Upregulation of kinin B1 receptor (B1R) and interleukin-1β. ► Enhanced superoxide anion production. ► Prevention of B1R induction and oxidative stress by NADPH oxidase inhibitors. ► Enhanced superoxide anion production by B1R activation.Pulmonary inflammation is an important pathological feature of tobacco smoke-related lung diseases. Kinin B1 receptor (B1R) is up-regulated in the rat trachea chronically exposed to cigarette-smoke. This study aimed at determining (1) whether exposure to total particulate matter of the cigarette smoke (TPM) can induce B1R in human alveolar epithelial A549 cells, (2) the mechanism of B1R induction, (3) the functionality of de novo synthesized B1R, and (4) the role of B1R in TPM-induced increase of superoxide anion (O2 ●–) level. Results show that A549 cells exposed to 10 μg/ml TPM increased O2 ●– level along with B1R (protein and mRNA) and IL-1β mRNA. In contrast, B2R and TNF-α mRNA were not affected by TPM. The increasing effect of TPM on O2 ●– level was not significantly affected by the B1R antagonist SSR240612. TPM-increased B1R mRNA was prevented by co-treatments with N-acetyl-l-cysteine (potent antioxidant), diphenyleneiodonium (NADPH oxidase inhibitor), IL-1Ra (interleukin-1R antagonist) and SN-50 (specific inhibitor of NF-kB activation) but not by pentoxifylline (TNF-α release inhibitor), indomethacin and niflumic acid (COX-1 and -2 inhibitors). Stimulation of B1R with a selective agonist (des-Arg9-BK, 10 μM; 30 min) increased O2 ●–production which was prevented by apocynin and diphenyleneiodonium (NADPH oxidase inhibitors). Data suggest that the increased expression of B1R by TPM in A549 cells is mediated by oxidative stress, IL-1β and NF-kB but not by cyclooxygenases or TNF-α. The amplification of O2 ●– levels via the activation of B1R-NADPH oxidase may exacerbate pulmonary inflammation and contribute to the chronicity of tobacco smoke-related lung diseases.
Keywords: Kinin B1 receptor; Interleukin-1 β; Cigarette smoke; Tobacco smoke; Inflammation; Oxidative stress;

► GRP receptors are massively overexpressed in the vascular bed of tumors. ► GRP receptor-positive vessels are also VEGF receptor-positive. ► Vascular targeting is proposed for ovarian, urothelial and renal cell cancers. ► GRP radiotracers can be used alone or preferably together with VEGF tracers.Gastrin-releasing peptide (GRP) and GRP receptors (GRPR) play a role in tumor angiogenesis. Recently, GRPR were found to be frequently expressed in the vasculature of a large variety of human cancers. Here, we characterize these GRPR by comparing the vascular GRPR expression and localization in a selection of human cancers with that of an established biological marker of neoangiogenesis, the vascular endothelial growth factor (VEGF) receptor. In vitro quantitative receptor autoradiography was performed in parallel for GRPR and VEGF receptors (VEGFR) in 32 human tumors of various origins, using 125I-Tyr-bombesin and 125I-VEGF165 as radioligands, respectively. Moreover, VEGFR-2 was evaluated immunohistochemically. All tumors expressed GRPR and VEGFR in their vascular system. VEGFR were expressed in the endothelium in the majority of the vessels. GRPR were expressed in a subpopulation of vessels, preferably in their muscular coat. The vessels expressing GRPR were all VEGFR-positive whereas the VEGFR-expressing vessels were not all GRPR-positive. GRPR expressing vessels were found immunohistochemically to co-express VEGFR-2. Remarkably, the density of vascular GRPR was much higher than that of VEGFR. The concomitant expression of GRPR with VEGFR appears to be a frequent phenomenon in many human cancers. The GRPR, localized and expressed in extremely high density in a subgroup of vessels, may function as target for antiangiogenic tumor therapy or angiodestructive targeted radiotherapy with radiolabeled bombesin analogs alone, or preferably together with VEGFR targeted therapy.
Keywords: Growth factors; Hormones; Gastrin-releasing peptide receptors; Vascular endothelial growth factor receptor; Neoangiogenesis; Tumoral vessels; Tumor targeting;

RYH: A minimal peptidic sequence obtained from beta-chain hemoglobin exhibiting an antimicrobial activity by Lucie Catiau; Johnatan Traisnel; Nour-Eddine Chihib; Guillaume Le Flem; Annick Blanpain; Oleg Melnyk; Didier Guillochon; Naïma Nedjar-Arroume (1463-1468).
► Peptides derived from β114-145 exhibit antibacterial activity. ► The most active peptide is the shortest one: β140-145. ► Minimal antimicrobial sequence from beta-chain: RYH. ► These active peptides were able to interact with cellular membrane. ► Minimum antimicrobial sequence: Y, R and one positively charged amino acid.Bovine hemoglobin is an animal protein described as source of bioactive peptides. Enzymatic hydrolysis of this protein results into some peptides exhibiting antimicrobial activity against Gram-positive and Gram-negative bacteria. In this study, a family of peptides from the beta chain (beta-114-145 derived peptides) obtained by peptic hydrolysis of bovine hemoglobin, was purified by reverse-phase HPLC and characterized by different analytical techniques (mass spectrometry, circular dichroism). The minimum inhibitory concentration was determined to show the antimicrobial activity of these peptides. Four bacterial strains were used: two Gram-negative (Escherichia coli and Salmonella Enteritidis) and two Gram-positive strains (Listeria innocua and Micrococcus luteus). The effect of these peptides on artificial membrane was also measured. Our findings showed that the peptide β114-145 and its peptic derivatives contain the RYH sequence. The most antimicrobial peptide is the RYH peptide which was the shortest one.
Keywords: Hemoglobin; Antimicrobial; MIC; Bacteriostatic effect; Liposome; Minimal sequence;

Wound healing activity of the human antimicrobial peptide LL37 by Reinaldo Ramos; João Pedro Silva; Ana Cristina Rodrigues; Raquel Costa; Luísa Guardão; Fernando Schmitt; Raquel Soares; Manuel Vilanova; Lucília Domingues; Miguel Gama (1469-1476).
► Recombinant LL37 inhibits activation of macrophages by LPS. ► Recombinant LL37 induces proliferation, migration and tubule-like structures formation by endothelial cells in vitro. ► Topical application of LL37 improves wound healing through vascularization and re-epithelialization.Antimicrobial peptides (AMPs) are part of the innate immune system and are generally defined as cationic, amphipathic peptides, with less than 50 amino acids, including multiple arginine and lysine residues. The human cathelicidin antimicrobial peptide LL37 can be found at different concentrations in many different cells, tissues and body fluids and has a broad spectrum of antimicrobial and immunomodulatory activities. The healing of wound is a complex process that involves different steps: hemostasis, inflammation, remodeling/granulation tissue formation and re-epithelialization. Inflammation and angiogenesis are two fundamental physiological conditions implicated in this process. We have recently developed a new method for the expression and purification of recombinant LL37. In this work, we show that the recombinant peptide P-LL37 with a N-terminus proline preserves its immunophysiological properties in vitro and in vivo. P-LL37 neutralized the activation of macrophages by lipopolysaccharide (LPS). Besides, the peptide induced proliferation, migration and tubule-like structures formation by endothelial cells. Wound healing experiments were performed in dexamethasone-treated mice to study the effect of LL37 on angiogenesis and wound regeneration. The topical application of synthetic and recombinant LL37 increased vascularization and re-epithelialization. Taken together, these results clearly demonstrate that LL37 may have a key role in wound regeneration through vascularization.
Keywords: Antimicrobial peptide; LL37; Angiogenesis; Wound healing;

Antimicrobial proline-rich peptides from the hemolymph of marine snail Rapana venosa by Pavlina Dolashka; Vesela Moshtanska; Valika Borisova; Aleksander Dolashki; Stefan Stevanovic; Tzvetan Dimanov; Wolfgang Voelter (1477-1483).
► Eleven antimicrobial peptides were isolated from the hemolymph of Rapana venosa snail. ► Seven from them exhibited antimicrobial activity against S. aureus.Hemolymph of Rapana venosa snails is a complex mixture of biochemically and pharmacologically active components such as peptides and proteins. Antimicrobial peptides are gaining attention as antimicrobial alternatives to chemical food preservatives and commonly used antibiotics. Therefore, for the first time we have explored the isolation, identification and characterisation of 11 novel antimicrobial peptides produced by the hemolymph of molluscs. The isolated peptides from the hemolymph applying ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC) have molecular weights between 3000 and 9500 Da, determined by mass spectrometric analysis. The N-terminal sequences of the peptides identified by Edman degradation matched no peptides in the MASCOT search database, indicating novel proline-rich peptides. UV spectra revealed that these substances possessed the characteristics of protein peptides with acidic isoelectric points. However, no Cotton effects were observed between 190 and 280 nm by circular dichroism spectroscopy. Four of the Pro-rich peptides also showed strong antimicrobial activities against tested microorganisms including Gram-positive and Gram-negative bacteria.
Keywords: Antimicrobial proline-rich peptides; Hemolymph of Rapana venosa; Gram-positive (Staphylococcus aureus) and a Gram-negative (Klebsiella pneumoniae) bacteria;

Determination of a new antibacterial peptide S-thanatin in rat plasma by an indirected-ELISA by Guoqiu Wu; Shenglan Wang; Xiyong Wang; Xiaofang Li; Xuepeng Deng; Zilong Shen; Tao Xi (1484-1487).
► The highest antiserum titer was 1:90,000 and the purity of antibody was >95%. ► The method does not require sample preconcentration and cleanup steps compared with typical instrumental methods. ► The sensitivity is higher than the previously reported instrumental methods. ► The method is expected to contribute to the therapeutic monitoring and further pharmacokinetic study of Ts.In this study, antimicrobial peptide S-thanatin (Ts) was chemically synthesized and linked to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) by carbodiimide reagent. Rabbits were immunized with Ts-KLH and polyclonal antibody against Ts was purified by fractional precipitation of ammonium sulfate, coupled with anion-exchange chromatography. The purified antibody specifically binding to Ts residues but not BSA molecules was observed by Western-Blot analysis. Ts-BSA was selected as immobilized antigen and reacted with the residual antibody after the excess of anti-Ts antibody was combined with Ts in the sample. The binding antibody was recognized by HRP-conjugated secondary antibody. Finally, the horseradish peroxidase in the complex could catalyze the TMB substrate, resulting in color development. The method was evaluated by analysis of linearity, precision and accuracy and successfully applied in determination of Ts in rat plasma. The data of the pharmacokinetic parameters were also obtained. The proposed ELISA has a great value in routine analysis of Ts for its therapeutic monitoring and pharmacokinetic studies.
Keywords: Antimicrobial peptide; S-thanatin; Indirected-ELISA; Pharmacokinetic;

Inhibitory effects and mechanisms of physiological conditions on the activity of enantiomeric forms of an α-helical antibacterial peptide against bacteria by Jinfeng Huang; Dianming Hao; Yu Chen; Yimin Xu; Juanjuan Tan; Yibing Huang; Fan Li; Yuxin Chen (1488-1495).
► In the presence of NaCl, CaCl2 or human serum albumin at physiological concentrations, inhibitory effects of physiological conditions on antimicrobial activity of enantiomeric peptides have been investigated. ► The electrostatic interaction between peptides and biomembrane was inhibited in the presence of salts. ► Salts especially CaCl2 weakened the ability of peptides to permeabilize the outer membrane of Gram-negative bacteria. ► Peptide showed different binding association abilities to HSA at different molar ratios, which is irrelevant to the peptide chirality and caused the reduction of peptide antimicrobial activity. ► d-peptide showed better performance than its l-enantiomer in all tested conditions owing to its resistance against proteolysis, hence, may be a promising candidate as therapeutic in clinical practices.Enantiomeric amphipathic α-helical antibacterial peptides were synthesized and their biophysical and biological properties under different physiological conditions were studied. In the absence of physiological factors, the l- and d-peptides exhibited similar antimicrobial activities against a broad spectrum of bacteria, even against clinical isolates with resistance to traditional antibiotics. However, in the presence of NaCl, CaCl2 or human serum albumin (HSA) at physiological concentrations, the enantiomers revealed bacterium-species dependent attenuations in antibacterial activity. In the presence of salts the electrostatic interaction between the peptides and the biomembrane was inhibited. Salts, especially CaCl2, weakened the ability of the peptides to permeabilize the outer membrane of Gram-negative bacteria, as determined by a 1-N-phenylnaphthylamine uptake assay. HSA exhibited variable inhibitory effects on the activity of the peptides when incubated with different bacterial strains. The peptides showed different binding association abilities to HSA at different molar ratios, regardless of their chirality, resulting in reduced peptide biological activity. The d-peptide performed better than its l-enantiomer in all conditions tested because of its resistance to proteolysis, and may therefore represent a promising candidate for development as a therapeutic agent.
Keywords: α-helical antimicrobial peptide; Enantiomer; Physiological conditions; Mechanism of action;

► Peptide with high antioxidant properties was isolated from the visceral waste of Magalaspis cordyla. ► Two steps of purification were executed using FPLC to obtain the active peptide. ► The sequence of purified peptide was determined as Ala–Cys–Phe–Leu (518.5 Da). ► Identified peptide proved better than natural antioxidant, α-tocopherol in PUFA protection.In the present study, a peptide having high antioxidant properties was isolated from horse mackerel viscera protein, Magalaspis cordyla. In vitro gastrointestinal digestion was employed to obtain potential protein hydrolysate and was subjected to consecutive chromatographic methods using fast protein liquid chromatography (FPLC) connected to diethyl amino ethyl (DEAE) anion exchange column and Sephadex G-25 gel filtration column. The activity of the fractions was tested against DPPH and hydroxyl radicals and the isolated peptide showed 89.2 and 59.1 percentage of scavenging. The amino acid sequence of purified peptide was determined using ESI-MS/MS as Ala–Cys–Phe–Leu (518.5 Da), it exhibited high activity against polyunsaturated fatty acid (PUFA) peroxidation than that of natural antioxidant, α-tocopherol.
Keywords: Horse mackerel; Antioxidant peptide; In vitro gastrointestinal; ESR spectrometer; Lipid peroxidation;

Peptidomic analysis of skin secretions demonstrates that the allopatric populations of Xenopus muelleri (Pipidae) are not conspecific by Milena Mechkarska; Eman Ahmed; Laurent Coquet; Jérôme Leprince; Thierry Jouenne; Hubert Vaudry; Jay D. King; J. Michael Conlon (1502-1508).
► Five antimicrobial peptides were isolated from skin secretions from Xenopus muelleri occupying a range in east Africa. ► Seven different antimicrobial peptides were isolated from skin secretions of X. muelleri occupying a non-contiguous range in west Africa. ► The western population of X. muelleri is more closely related to X. borealis than to X. muelleri from the eastern range consistent with a proposal to designate it as a separate species.Mueller's clawed frog Xenopus muelleri (Peters 1844) occupies two non-contiguous ranges in east and west Africa. The phylogenetic relationship between the two populations is unclear and it has been proposed that the western population represents a separate species. Peptidomic analysis of norepinephrine-stimulated skin secretions from X. muelleri from the eastern range resulted in the identification of five antimicrobial peptides structurally related to the magainins (magainin-M1 and -M2), xenopsin-precursor fragments (XPF-M1) and caerulein-precursor fragments (CPF-M1 and -M2) previously found in skin secretions of other Xenopus species. A cyclic peptide (WCPPMIPLCSRF.NH2) containing the RFamide motif was also isolated that shows limited structural similarity to the tigerinins, previously identified only in frogs of the Dicroglossidae family. The components identified in skin secretions from X. muelleri from the western range comprised one magainin (magainin-MW1), one XPF peptide (XPF-MW1), two peptides glycine-leucine amide (PGLa-MW1 and -MW2), and three CPF peptides (CPF-MW1, -MW2 and -MW3). Comparison of the primary structures of these peptides suggest that western population of X. muelleri is more closely related to X. borealis than to X. muelleri consistent with its proposed designation as a separate species. The CPF peptides showed potent, broad-spectrum activity against reference strains of bacteria (MIC 3–25 μM), but were hemolytic against human erythrocytes.
Keywords: Antimicrobial; Frog skin; Magainin; PGLa; Procaerulein; Proxenopsin; Tigerinin; Polyploidy;

The new kappa-KTx 2.5 from the scorpion Opisthacanthus cayaporum by Thalita Soares Camargos; Rita Restano-Cassulini; Lourival Domingos Possani; Steve Peigneur; Jan Tytgat; Carlos Alberto Schwartz; Erica Maria C Alves; Sonia Maria de Freitas; Elisabeth Ferroni Schwartz (1509-1517).
► We describe the κ-KTx2.5 and show it is a blocker of hKv1.1, and hKv1.4 channels. ► CD spectra showed a predominance of α-helices at different temperatures in water and water/TFE. ► κ-KTx2.5 had no activity on other ion channels, on bacterial growth and on smooth muscle tissue. ► A structural model of the kappa-KTx2.5-Kv1.2 complex is presented.The kappa-KTx family of peptides, which is the newest K+-channel blocker family from scorpion venom, is present in scorpions from the families Scorpionidae and Liochelidae. Differently from the other scorpion KTx families, the three-dimensional structure of the known kappa-KTxs toxins is formed by two parallel α-helices linked by two disulfide bridges. Here, the characterization of a new kappa-KTx peptide, designated kappa-KTx 2.5, derived from the Liochelidae scorpion Opisthacanthus cayaporum, is described. This peptide was purified by HPLC and found to be identical to OcyC8, a predicted mature sequence precursor (UniProtKB C5J89) previously described by our group. The peptide was chemically synthesized and the circular dichroism (CD) spectra of both, native and synthetic, conducted at different temperatures in water and water/trifluoroethanol (TFE), showed a predominance of α-helices. The kappa-KTx 2.5 is heat stable and was shown to be a blocker of K+-currents on hKv1.1, and hKv1.4, with higher affinity for Kv1.4 channels (IC50  = 71 μM). Similarly to the other kappa-KTxs, the blockade of K+-channels occurred at micromolar concentrations, leading to uncertainness about their proper molecular target, and consequently their pharmacologic effect. In order to test other targets, kappa-KTx2.5 was tested on other K+-channels, on Na+-channels, on bacterial growth and on smooth muscle tissue, a known assay to identify possible bradykinin-potentiating peptides, due to the presence of two contiguous prolines at the C-terminal sequence. It has no effect on the targets used except on hKv1.1, and hKv1.4 expressed in Chinese hamster ovary cells. Since the only plausible function found for kappa-KTx2.5 seems to be the blockade of K+-channels, a discussion regarding the analysis of structure–function relationships is included in this communication, based on sequence alignments of members of the kappa-KTx toxin family, and on computational simulation of a structural model of the kappa-KTx2.5-Kv1.2 complex.
Keywords: kappa-KTx; Liochelidae; Potassium channel blocker; Scorpion; Venom;

Virucidal activity of a scorpion venom peptide variant mucroporin-M1 against measles, SARS-CoV and influenza H5N1 viruses by Qiaoli Li; Zhenhuan Zhao; Dihan Zhou; Yaoqing Chen; Wei Hong; Luyang Cao; Jingyi Yang; Yan Zhang; Wei Shi; Zhijian Cao; Yingliang Wu; Huimin Yan; Wenxin Li (1518-1525).
► We investigated the antiviral activities of mucroporin and optimized mucroporin-M1. ► Mucroporin and mucroporin-M1 were scorpion venom-derived host defense peptides. ► Mucroporin-M1 but not mucroporin had antiviral activities against tested viruses. ► The tested viruses were measles, SARS-CoV and influenza H5N1 viruses. ► Mucroporin-M1 exerted its effects on viruses by directly binding to virus membranes.Outbreaks of SARS-CoV, influenza A (H5N1, H1N1) and measles viruses in recent years have raised serious concerns about the measures available to control emerging and re-emerging infectious viral diseases. Effective antiviral agents are lacking that specifically target RNA viruses such as measles, SARS-CoV and influenza H5N1 viruses, and available vaccinations have demonstrated variable efficacy. Therefore, the development of novel antiviral agents is needed to close the vaccination gap and silence outbreaks. We previously indentified mucroporin, a cationic host defense peptide from scorpion venom, which can effectively inhibit standard bacteria. The optimized mucroporin-M1 can inhibit gram-positive bacteria at low concentrations and antibiotic-resistant pathogens. In this investigation, we further tested mucroporin and the optimized mucroporin-M1 for their antiviral activity. Surprisingly, we found that the antiviral activities of mucroporin-M1 against measles, SARS-CoV and influenza H5N1 viruses were notably increased with an EC50 of 7.15 μg/ml (3.52 μM) and a CC50 of 70.46 μg/ml (34.70 μM) against measles virus, an EC50 of 14.46 μg/ml (7.12 μM) against SARS-CoV and an EC50 of 2.10 μg/ml (1.03 μM) against H5N1, while the original peptide mucroporin showed no antiviral activity against any of these three viruses. The inhibition model could be via a direct interaction with the virus envelope, thereby decreasing the infectivity of virus. This report provides evidence that host defense peptides from scorpion venom can be modified for antiviral activity by rational design and represents a practical approach for developing broad-spectrum antiviral agents, especially against RNA viruses.
Keywords: Mucroporin-M1; Scorpion venom; Measles; SARS-CoV; H5N1; Antiviral;

Antinociceptive properties of nasal delivery of Neurotoxin-loaded nanoparticles coated with polysorbate-80 by Yeping Ruan; Li Yao; Bingbing Zhang; Shuijuan Zhang; Jianyou Guo (1526-1529).
Display Omitted► Neurotoxin-loaded nanoparticles generate a significant improvement of drug levels in the brain. ► Neurotoxin-loaded nanoparticles overcoated with polysorbate-80 generate a significant improvement of drug levels in the brain. ► The nasal delivery of Neurotoxin generates a significant improvement of drug levels in the brain.Neurotoxin-1 (NT) is an analgesic peptide which is endowed an exceptional specificity of action that blocks transmission of the nerve impulse. The aim of this study was to evaluate the potential application of nanoparticles technology as drug carrier system for the nasal delivery of NT. Mice were administered intranasally (i.n.) with NT (NT-P-NP), free NT solution (F-NT) and intravenously (i.v.) with NT (IV-NT) respectively. The NT levels in animal brain and antinociceptive activity of NT were analyzed. The result on brain transport showed that nanoparticles could exert enhanced delivery of NT into the brain significantly after i.n. administration. The results of antinociceptive activity showed that NT-P-NP increased immobility in the open-field test, both phases of formalin test were significantly inhibited by the NT-P-NP and NT-P-NP significantly inhibited the reaction time to thermal stimuli at 60 and 90 min. Both NT-P-NP and IV-NT were able to inhibit constrictions in acetic acid-induced writhing reaction. These data suggest that NT-loaded nanoparticles coated with polysorbate-80 could generate a significant improvement of drug levels in the brain. Intranasal administration of Neurotoxin-1 entrapped in nanoparticles coated with polysorbate-80 is an attractive alternative to intravenous administration.
Keywords: Neurotoxin-1; Nanoparticles; Polysorbate-80; Antinociceptive properties; Nasal delivery;

Antinociceptive effects of spinally administered nociceptin/orphanin FQ and its N-terminal fragments on capsaicin-induced nociception by Soh Katsuyama; Hirokazu Mizoguchi; Takaaki Komatsu; Chikai Sakurada; Minoru Tsuzuki; Shinobu Sakurada; Tsukasa Sakurada (1530-1535).
► Intrathecal injection of N/OFQ caused a dose-dependent antinociceptive effect. ► The N-terminal fragments of N/OFQ were antinociceptive with a potency lower than N/OFQ. ► Antinociceptive effect of N/OFQ (1–13) and (1–11) lasted longer than that of N/OFQ. ► N/OFQ (1–13)-induced antinociception is mediated through spinal NOP receptors.Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand for the N/OFQ peptide (NOP) receptors, has been shown to be metabolized into some fragments. We examined to determine whether intrathecal (i.t.) N/OFQ (1–13), (1–11) and (1–7) have antinociceptive activity in the pain-related behavior after intraplantar injection of capsaicin. The i.t. administration of N/OFQ (0.3–1.2 nmol) produced an appreciable and dose-dependent inhibition of capsaicin-induced paw-licking/biting response. The N-terminal fragments of N/OFQ, (1–13) and (1–11), were antinociceptive with a potency lower than N/OFQ. Calculated ID50 values (nmol, i.t.) were 0.83 for N/OFQ, 2.5 for N/OFQ (1–13) and 4.75 for N/OFQ (1–11), respectively. The time-course effect revealed that the antinociceptive effects of these N-terminal fragments lasted longer than those of N/OFQ. Removal of amino acids down to N/OFQ (1–7) led to be less potent than N/OFQ and its fragments, (1–13) and (1–11). Antinociception induced by N/OFQ or N/OFQ (1–13) was reversed significantly by i.t. co-injection of [Nphe1]N/OFQ (1–13)NH2, a peptidergic antagonist for NOP receptors, whereas i.t. injection of the antagonist did not interfere with the action of N/OFQ (1–11) and (1–7). Pretreatment with the opioid receptor antagonist naloxone hydrochloride did not affect the antinociception induced by N/OFQ and its N-terminal fragments. These results suggest that N-terminal fragments of N/OFQ are active metabolites and may modulate the antinociceptive effect of N/OFQ in the spinal cord. The results also indicate that N/OFQ (1–13) still possess antinociceptive activity through NOP receptors.
Keywords: Nociceptin/orphanin FQ (N/OFQ); N-Terminal fragments of N/OFQ; Capsaicin-induced nociception; Antinociception; N/OFQ peptide (NOP) receptor; Spinal cord;

► Very few insect (neuro)peptides are still be bioactive after passage through the gut. ► Lipidization improves the efficacy of some orally ingested pharmaca/peptides. ► Injected corazonin-in-oil melanized the cuticle of an albino Locusta mutant. ► Orally ingested corazonin-in-oil melanizes the cuticle of nymphs and adults. ► Peptide in vegetable oil may serve as a simple but efficient slow release formula.Upon realizing that for drug delivery in the body, lipidization is a technique used in the pharmaceutical industry, we took in consideration that corazonin melanizes the cuticle of albino Locusta migratoria only when injected in an emulsion in oil, not when applied in a watery solution. In this study, we investigate the possibility for oral uptake of corazonin dispersed in oil, and validated the activity by a melanization assay. Not only was it active, it also induced red cuticular coloration in some animals, and it was also unexpectedly lethal for nymphs, but not for adults. These results necessitate the revision of the potential of (some) peptides for insect control. Also, they suggest practical recommendations for the application of other peptides in physiological assays where oil could be used as a simple slow release formula.

Shared and separate functions of the RAMP-based adrenomedullin receptors by Kenji Kuwasako; Kazuo Kitamura; Sayaka Nagata; Tomomi Hikosaka; Yoshio Takei; Johji Kato (1540-1550).
► AM is a novel hypotensive peptide that exerts a variety of strongly protective effects against multiorgan damage. ► Many of the effects of AM are mediated via the RAMP2-based AM1 receptor. ► The evidence now suggests that the RAMP3-based AM2 receptor also has distinct functions in vivo. ► AM2 is an AM-related peptide whose biological actions are similar to those of AM. ► AM2 strongly interacts with the AM1 and AM2 receptors and may also bind to a unique receptor.Adrenomedullin (AM) is a novel hypotensive peptide that exerts a variety of strongly protective effects against multiorgan damage. AM-specific receptors were first identified as heterodimers composed of calcitonin-receptor-like receptor (CLR), a G protein coupled receptor, and one of two receptor activity-modifying proteins (RAMP2 or RAMP3), which are accessory proteins containing a single transmembrane domain. RAMPs are required for the surface delivery of CLR and the determination of its phenotype. CLR/RAMP2 (AM1 receptor) is more highly AM-specific than CLR/RAMP3 (AM2 receptor). Although there have been no reports showing differences in intracellular signaling via the two AM receptors, in vitro studies have shed light on their distinct trafficking and functionality. In addition, the tissue distributions of RAMP2 and RAMP3 differ, and their gene expression is differentially altered under pathophysiological conditions, which is suggestive of the separate roles played byAM1 and AM2 receptors in vivo. Both AM and the AM1 receptor, but not the AM2 receptor, are crucial for the development of the fetal cardiovascular system and are able to effectively protect against various vascular diseases. However, AM2 receptors reportedly play an important role in maintaining a normal body weight in old age and may be involved in immune function. In this review article, we focus on the shared and separate functions of the AM receptor subtypes and also discuss the potential for related drug discovery. In addition, we mention their possible function as receptors for AM2 (or intermedin), an AM-related peptide whose biological functions are similar to those of AM.
Keywords: Adrenomedullin; Adrenomedullin 2; Calcitonin-receptor-like receptor; Receptor activity-modifying protein; Genetically engineered mouse; Cardiovascular diseases;

► The renin-angiotensin system is now evolving as an endocrine, paracrine and intracrine system. ► The renin/ACE/Ang II/AT1 receptor axis is the most recognized cascade of the entire system. ► The prorenin/(Pro)renin receptor/MAP kinases ERK 1/2 axis is activated in diseased states. ► The ACE2/Ang (1–7) Mas receptor axis opposes the effects induced by Ang II via AT1 receptors.Although renin, the rate-limiting enzyme of the renin-angiotensin system (RAS), was first discovered by Robert Tigerstedt and Bergman more than a century ago, the research on the RAS still remains stronger than ever. The RAS, once considered to be an endocrine system, is now widely recognized as dual (circulating and local/tissue) or multiple hormonal systems (endocrine, paracrine and intracrine). In addition to the classical renin/angiotensin I-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II receptor (AT1/AT2) axis, the prorenin/(Pro)renin receptor (PRR)/MAP kinase axis, the ACE2/Ang (1–7)/Mas receptor axis, and the Ang IV/AT4/insulin-regulated aminopeptidase (IRAP) axis have recently been discovered. Furthermore, the roles of the evolving RAS have been extended far beyond blood pressure control, aldosterone synthesis, and body fluid and electrolyte homeostasis. Indeed, novel actions and underlying signaling mechanisms for each member of the RAS in physiology and diseases are continuously uncovered. However, many challenges still remain in the RAS research field despite of more than one century's research effort. It is expected that the research on the expanded RAS will continue to play a prominent role in cardiovascular, renal and hypertension research. The purpose of this article is to review the progress recently being made in the RAS research, with special emphasis on the local RAS in the kidney and the newly discovered prorenin/PRR/MAP kinase axis, the ACE2/Ang (1–7)/Mas receptor axis, the Ang IV/AT4/IRAP axis, and intracrine/intracellular Ang II. The improved knowledge of the expanded RAS will help us better understand how the classical renin/ACE/Ang II/AT1 receptor axis, extracellular and/or intracellular origin, interacts with other novel RAS axes to regulate blood pressure and cardiovascular and kidney function in both physiological and diseased states.
Keywords: Angiotensin converting enzyme 2 (ACE2); Angiotensin II (Ang II); AT1 receptor signaling; Insulin-regulated aminopeptidase (IRAP); Mas receptor; (Pro)renin receptor (PRR);