Peptides (v.32, #9)

Circulating kisspeptin levels exhibit sexual dimorphism in adults, are increased in obese prepubertal girls and do not suffer modifications in girls with idiopathic central precocious puberty by Jimena Pita; Vicente Barrios; Teresa Gavela-Pérez; Gabriel Á. Martos-Moreno; María T. Muñoz-Calvo; Jesús Pozo; Adela Rovira; Jesús Argente; Leandro Soriano-Guillén (1781-1786).
► Kisspeptin levels do not change throughout puberty in males. ► Kisspeptin are higher in adult females than in prepubertal and pubertal females. ► Kisspeptin is elevated in prepubertal obese girls but not in idiopathic CPP girls. ► Kisspeptin does not seem to be a good marker for pubertal disorders. ► Adipose tissue may contribute to circulating kisspeptin synthesis.The system KISS1–KISS1R is one of the main regulators of the hypothalamic-pituitary-gonadal axis and constitutes a link between metabolism and reproduction through its interaction with leptin. The aim of this study was to clarify the possible utility of kisspeptin as a pubertal marker and/or the possible influence of nutritional status in kisspeptin levels. To this end, we have studied kisspeptin plasma levels throughout sexual development and in prepubertal obese girls and girls affected by idiopathic central precocious puberty (CPP). Plasma kisspeptin concentrations were analyzed by RIA. An increase in kisspeptin levels was observed in adult females compared to healthy prepubertal and pubertal girls (p  < 0.001) and to adult males (p  < 0.001). Additionally, kisspeptin was increased in prepubertal obese girls compared to healthy prepubertal girls (p  < 0.01) and girls with idiopathic CPP (p  < 0.05). As revealed by the regression analysis, in prepubertal healthy and obese girls and girls with idiopathic CCP, the parameters that influenced kisspeptin levels were BMI (R 2  = 0.10, p  < 0.05) and leptin levels (R 2  = 0.14, p  < 0.01). In conclusion, kisspeptin levels do not seem to be a good pubertal marker. The results obtained in prepubertal and idiopathic CCP girls point to a relationship between leptin, BMI and kisspeptin at least in this group, and suggest a possible role for adipose tissue in the modulation kisspeptin synthesis.
Keywords: Kisspeptin; Leptin; Obesity; Central precocious puberty;

B-type natriuretic peptide and anthropometric measures in a Brazilian elderly population with a high prevalence of Trypanosoma cruzi infection by Alline Maria Beleigoli; Maria Fernanda Lima-Costa; Maria de Fátima Haueisen Diniz; Antônio Luiz Ribeiro (1787-1792).
► Brain natriuretic peptide (BNP) has hemodynamic and lipolytic actions. ► Trypanosoma cruzi has the heart and the adipose tissue as important target organs. ► BNP is inversely related to measures of body mass and body composition in the elderly population of the Bambuí Cohort Study of Aging. ► Despite T.cruzi actions in the adipose tissue, the relation between BNP and anthropometric measures is not different between the infected and non-infected groups.B-type natriuretic peptide (BNP) is a diagnostic and prognostic tool in heart failure and also in Chagas disease, which is caused by the protozoan Trypanosoma cruzi and has cardiomyopathy as a main feature. BNP lipolytic actions and T. cruzi infection in the adipose tissue have been recently described. We aim to investigate the relationship between BNP and anthropometric measures and whether it is influenced by T. cruzi infection. We measured BNP, body mass index (BMI), waist circumference (WC), triceps skin-fold thickness (TSF) and performed serological, biochemical and electrocardiographic exams in 1398 subjects (37.5% infected with T. cruzi) in a community-dwelling elderly population in Bambui city, Brazil. Linear multivariate regression analysis was performed to investigate determinants of BNP levels. BNP levels were significantly (p  < 0.05) higher in T. cruzi-infected subjects than in the non-infected group (median = 121 and 64 pg/mL, respectively). BMI, WC and TSF in infected subjects were significantly lower than those in non-infected subjects (24.3 vs. 25.5 kg/m2; 89.2 vs. 92.4 cm; and 14.5 vs. 16.0 mm, respectively). There was an inverse relationship between BNP levels and BMI (b  = −0.018), WC (b  = −0.005) and TSF (b  = −0.193) levels. Infected and non-infected groups showed similar inverse relationships between BNP and BMI (b  = −0.021 and b  = −0.015, respectively). In conclusion, there was an inverse relationship between BNP levels and the anthropometric measures. Despite the actions in the adipose tissue, T. cruzi infection did not modify the associations between BNP and BMI, suggesting that body mass does not modify the accuracy of BNP in Chagas disease.
Keywords: B-type natriuretic peptide; Body mass index; Anthropometric measures; Chagas disease; Metabolism; Adipose tissue;

Up-regulation of apelin in brain tissue of patients with epilepsy and an epileptic rat model by Xiaogang Zhang; Xi Peng; Min Fang; Chunlei Zhou; Fenghua Zhao; Ying Zhang; Yali Xu; Qiong Zhu; Jing Luo; Guojun Chen; Xuefeng Wang (1793-1799).
► We firstly found in the present study were that: apelin was significantly elevated in the epileptogenic temporal neocortex of TLE patients as compared with non-epileptic controls. ► Increased expression of apelin was also found in the hippocampus and adjacent cortex of rat model induced by lithium–pilocarpine. ► The most of apelin positive cells were colocalization with neurons but not glial cells both in human and rat model. ► Our results demonstrated that the increased expression of apelin in the brain may be involved in human TLE.Prolonged epileptic seizures or SE can cause neuronal cell death. However, the exact role of neuroprotectant against brain injury during epileptic seizure needs to be further elucidated. The aim of this study was to investigate the expression of the apelin, a novel neuroprotective peptide, in brain tissues of the patients with temporal lobe epilepsy (TLE) and experimental rats using immunohistochemistry, immunofluorescence and Western blotting analysis and to discuss the possible role of apelin in TLE. Thirty temporal neocortical tissue samples from the patients with drug-refractory TLE underwent surgical therapy and nine histologically normal temporal lobes tissues as controls were used in our study. Fifty-six Sprague–Dawley rats were randomly divided into seven groups, including one control group and six groups with epilepsy induced by lithium–pilocarpine. Hippocampus and adjacent cortex were taken from the controls and epileptic rats at 1, 3, 7, 14, 30, and 60 days after onset of seizures. Apelin was mainly expressed in the neurons of TLE patients and controls, and was significantly increased in TLE patients compared with the controls. Apelin was also expressed in the neurons of experimental and control rats, it was gradually increased in the experimental rat post-seizure and reached a stable high level in chronic epileptic phase. Our results demonstrated that the increased expression of apelin in the brain may be involved in human TLE.
Keywords: Apelin; Epilepsy; Status epilepticus; Neuroprotection; Human; Rat;

Sequence variations of Env signal peptide alleles in different clinical stages of HIV infection by Joaquim Xavier da Silva; Octávio Luiz Franco; Mikael Araújo Guimarães Lemos; Marcos Vinícius Pereira Gondim; Francisco Prosdocimi; Enrique Roberto Argañaraz (1800-1806).
► Correlation between Env-signal peptide and the HIV infectivity. ► Differentiation between Env-sp N-termini of early- and late-stage virus specimens. ► Identification of Env-sp sequence alterations related to viral adaptive functions.The human immunodeficiency virus has been shown to increase its infectivity throughout the course of infection. This virus selection property has been associated with genome mutations and recombinations among virus variants, causing amino acid residue alterations in important viral proteins. In order to explore the contribution of Env signal peptide (Env-sp) to Env glycoprotein expression and its possible relationship to increased virus infectivity observed at late stages of infection, we characterized Env-sp sequences derived from twelve patients at “early” and “late” stages of HIV infection without antiretroviral therapy use. In spite of the remarkable overall similarity between both stages, we observed the deletion of a sequence of neutral and basic residues at the Env-sp amino terminus in virus from early stage specimens and the insertion of basic residues in the hydrophobic region on late-stage viral isolates. The Env-sp sequence alterations may have viral adaptive functions during HIV infection.
Keywords: HIV-1; AIDS; Env; Signal peptide sequences;

Antibacterial and anti-biofilm effects of cathelicidin peptides against pathogens isolated from cystic fibrosis patients by A. Pompilio; M. Scocchi; S. Pomponio; F. Guida; A. Di Primio; E. Fiscarelli; R. Gennaro; G. Di Bonaventura (1807-1814).
► The in vitro activity of 6 cathelicidin-derived peptides against S. aureus, P. aeruginosa, and S. maltophilia CF strains were evaluated in comparison with tobramycin. ► SMAP-29, BMAP-28, and BMAP-27 showed relevant antibacterial activity, and in some cases higher than tobramycin. ► SMAP-29, BMAP-27, and BMAP-28 at sub-MICs significantly reduce biofilm formation, although at a lower extent than tobramycin. ► Bactericidal SMAP-29, BMAP-28, and BMAP-27 concentrations significantly reduce preformed P. aeruginosa and S. maltophilia biofilms, although at a lower extent than tobramycin. ► Some cathelicidin-derived peptides may be useful in early prophylactic and therapeutic treatment of CF lung disease.Six different cathelicidin-derived peptides were compared to tobramycin for antibacterial and anti-biofilm effects against S. aureus, P. aeruginosa, and S. maltophilia strains isolated from cystic fibrosis patients. Overall, SMAP-29, BMAP-28, and BMAP-27 showed relevant antibacterial activity (MIC50 4–8 μg/ml), and in some cases higher than tobramycin. In contrast, indolicidin, LL-37, and Bac7(1–35) showed no significant antimicrobial activity (MIC50  > 32 μg/ml). Killing kinetics experiments showed that in contrast to tobramycin the active cathelicidin peptides exert a rapid bactericidal activity regardless of the species tested. All three peptides significantly reduced biofilm formation by S. maltophilia and P. aeruginosa strains at 1/2× MIC, although at a lower extent than tobramycin. In addition, BMAP-28, as well as tobramycin, was also active against S. aureus biofilm formation. Preformed biofilms were significantly affected by bactericidal SMAP-29, BMAP-27 and BMAP-28 concentrations, although at a lesser extent than tobramycin. Overall, our results indicate the potential of some cathelicidin-derived peptides for the development of novel therapeutic agents for cystic fibrosis lung disease.
Keywords: Antimicrobial peptide; Cathelicidin; Cystic fibrosis; Antibiotic; Multidrug-resistance;

Plantaricin A synthesized by Lactobacillus plantarum induces in vitro proliferation and migration of human keratinocytes and increases the expression of TGF-β1, FGF7, VEGF-A and IL-8 genes by Daniela Pinto; Barbara Marzani; Fabio Minervini; Maria Calasso; Giammaria Giuliani; Marco Gobbetti; Maria De Angelis (1815-1824).
► Plantaricin A improved the proliferation and migration of human keratinocytes. ► Plantarcin A affected the expression of genes involved in wound repair. ► The use of plantarcin A for dermatological purposes should be considered.This work showed the effect of pheromone plantaricin A (PlnA) on the proliferation and migration of the human keratinocytes NCTC 2544. PlnA was chemically synthesized and used as pure peptide or biologically synthesized during co-cultivation of Lactobacillus plantarum DC400 and Lactobacillus sanfranciscensis DPPMA174. The cell-free supernatant (CFS) was used as the crude preparation containing PlnA. The inductive effect of PlnA on the proliferation of NCTC 2544 cells was higher than that found for hyaluronic acid, a well known skin protective compound. As shown by scratch assay and image analyses, PlnA enhanced the migration of NCTC 2544 cells. Compared to the basal serum free medium (control), the highest inductive effect was found using 10 μg/ml of chemically synthesized PlnA. Similar results (P  > 0.05) were found for CFS. In agreement, the percentage of the starting scratch area was decreased after treatment (24 h) with PlnA. The expression of transforming growth factor-β1 (TGF-β1), keratinocyte growth factor 7 (FGF7), vascular endothelial growth factor (VEGF-A), and interleukin-8 (IL-8) genes was affected by PlnA. Compared to control, TGF-β1 gene was under expressed in the first 4 h of treatments and up-regulated after 8–24 h. On the contrary, FGF7 gene was strongly up-regulated in the first 4 h of treatments. Compared to control, VEGF-A and IL-8 genes were always up-regulated during the 4–24 h from scratching. Since capable of promoting the proliferation and migration of the human keratinocytes and of stimulating IL-8 cytokine, the use of PlnA for dermatological purposes should be considered.
Keywords: Plantaricin A; Lactobacillus plantarum; Transforming growth factor-β1; Keratinocyte growth factor 7; Vascular endothelial growth factor (VEGF-A); Interleukin-8;

Adrenomedullin production is increased in colorectal adenocarcinomas; its relation to matrix metalloproteinase-9 by Tomomi Hikosaka; Toshihiro Tsuruda; Sayaka Nagata; Kenji Kuwasako; Kazuyo Tsuchiya; Shinri Hoshiko; Haruhiko Inatsu; Kazuo Chijiiwa; Kazuo Kitamura (1825-1831).
► Gene expression and peptide concentration of adrenomedullin (AM) were highly expressed in colorectal cancer tissues. ► The level of preproAM mRNA was positively correlated with MMP-9, but not with VEGF-A. ► Immunoreactivity of AM was present in cancer cells, while MMP-9 localized in stroma cells surrounding the tumors.Adrenomedullin (AM) is highly expressed in various cancer cell lines, suggesting a possible association with cancer growth. In the present study, we examined the expression and/or concentration of AM, its related peptide, adrenomedullin2/intermedin (AM2/IMD) and their receptors in human colorectal cancer and the surrounding normal tissue. In addition, we assessed the correlation between the expression of AM and AM2/IMD with that of vascular endothelial growth factor (VEGF)-A and matrix metalloproteinase (MMP)-9. Using a specific immunoradiometric assay, we found that AM concentrations were 2–11-fold higher in colorectal cancer tissues than in the surrounding normal tissues. Moreover, real-time quantitative RT-PCR showed that the expression levels of preproAM (+548%), preproAM2/IMD (+2674%), calcitonin receptor-like receptor (CLR) (+518%), receptor activity modifying protein (RAMP)2 (+281%), RAMP3 (+178%), VEGF-A (+277%) and MMP-9 (+864%) mRNAs were significantly higher in cancer tissues than in the surrounding normal tissues, and there was a positive correlation between the gene expressions of MMP-9 and preproAM (r  = 0.352; p  = 0.005), but not with preproAM2/IMD (r  = 0.041, p  = 0.406). Both AM and AM2/IMD immunoreactivity were detected mainly within cancer cells, whereas MMP-9 immunoreactivity was mostly seen in the surrounding stroma. These findings suggest that AM produced in colorectal tumors acts in concert with MMP-9 in the stroma to contribute to the pathogenesis of colorectal cancer.
Keywords: Adrenomedullin; AM2/IMD; MMP-9; VEGF; Metastasis; Stroma;

Identifying the regulatory element for human angiotensin-converting enzyme 2 (ACE2) expression in human cardiofibroblasts by Tang-Ching Kuan; Tzu-Hui Yang; Cheng-Hao Wen; Mu-Yuan Chen; I-Liang Lee; Chih-Sheng Lin (1832-1839).
► Identification of regulatory sequence for the expression of human angiotensin-converting enzyme 2, ace2, in human cardiofibroblasts (HCFs). ► Putative binding site for angiotensin II-stimulated transcriptional activation of ace2 is homologous to Ikaros binding domain. ► Pro-inflammatory cytokines TGF-β1 and TNF-α do not affect the ACE2 expression in HCFs.Angiotensin-converting enzyme 2 (ACE2) has been proposed as a potential target for cardioprotection in regulating cardiovascular functions, owing to its key role in the formation of the vasoprotective peptides angiotensin-(1–7) from angiotensin II (Ang II). The regulatory mechanism of ace2 expression, however, remains to be explored. In this study, we investigated the regulatory element within the upstream of ace2. The human ace2 promoter region, from position −2069 to +20, was cloned and a series of upstream deletion mutants were constructed and cloned into a luciferase reporter vector. The reporter luciferase activity was analyzed by transient transfection of the constructs into human cardiofibroblasts (HCFs) and an activating domain was identified in the −516/−481 region. Deletion or reversal of this domain within ace2 resulted in a significant decrease in promoter activity. The nuclear proteins isolated from the HCFs formed a DNA–protein complex with double stranded oligonucleotides of the −516/−481 domain, as detected by electrophoretic mobility shift assay. Site-directed mutagenesis of this region identified a putative protein binding domain and a potential binding site, ATTTGGA, homologous to that of an Ikaros binding domain. This regulatory element was responsible for Ang II stimulation via the Ang II–Ang II type-1 receptor (AT1R) signaling pathway, but was not responsible for pro-inflammatory cytokines TGF-β1 and TNF-α. Our results suggest that the nucleotide sequences −516/−481 of human ace2 may be a binding domain for an as yet unidentified regulatory factor(s) that regulates ace2 expression and is associated with Ang II stimulation.
Keywords: Angiotensin-converting enzyme 2; Regulatory element; Angiotensin II; Human cardiofibroblasts;

An integrin-binding N-terminal peptide region of TIMP-2 retains potent angio-inhibitory and anti-tumorigenic activity in vivo by Dong-Wan Seo; W. Carl Saxinger; Liliana Guedez; Anna Rita Cantelmo; Adriana Albini; William G. Stetler-Stevenson (1840-1848).
► The α3β1 binding domain is localized to the N-terminal domain of the TIMP-2. ► This integrin binding domain encompasses residues Ile43–Ala66, or B–C loop. ► Synthetic peptides containing sequences from this region suppress endothelial growth. ► Peptides from this region are anti-angiogenic and anti-tumorigenic in vivo. ► TIMP-2 binds to the endothelial cell surface through two distinct loops.Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits angiogenesis by several mechanisms involving either MMP inhibition or direct endothelial cell binding. The primary aim of this study was to identify the TIMP-2 region involved in binding to the previously identified receptor integrin α3β1, and to determine whether synthetic peptides derived from this region retained angio-inhibitory and tumor suppressor activity. We demonstrated that the N-terminal domain of TIMP-2 (N-TIMP-2) binds to α3β1 and inhibits vascular endothelial growth factor-stimulated endothelial cell growth in vitro, suggesting that both the α3β1-binding domain and the growth suppressor activity of TIMP-2 localize to the N-terminal domain. Using a peptide array approach we identify a 24 amino acid region of TIMP-2 primary sequence, consisting of residues Ile43–Ala66, which shows α3β1-binding activity. Subsequently we demonstrate that synthetic peptides from this region compete for TIMP-2 binding to α3β1 and suppress endothelial growth in vitro. We define a minimal peptide sequence (peptide 8-9) that possesses both angio-inhibitory and, using a murine xenograft model of Kaposi's sarcoma, anti-tumorigenic activity in vivo. Thus, both the α3β1-binding and the angio-inhibitory activities co-localize to a solvent exposed, flexible region in the TIMP-2 primary sequence that is unique in amino acid sequence compared with other members of the TIMP family. Furthermore, comparison of the TIMP-2 and TIMP-1 protein 3-D structures in this region also identified unique structural differences. Our findings demonstrate that the integrin binding, tumor growth suppressor and in vivo angio-inhibitory activities of TIMP-2 are intimately associated within a unique sequence/structural loop (B–C loop).
Keywords: Angiogenesis; Angiogenesis inhibitors; Tissue inhibitor of metalloproteinases-2 (TIMP-2); Peptide inhibitors; Kaposi's sarcoma;

The bradykinin B1 receptor antagonist R-954 inhibits Ehrlich tumor growth in rodents by Patricia Dias Fernandes; Niele de Matos Gomes; Pierre Sirois (1849-1854).
► The effects of the bradykinin B1 antagonist R-954 were evaluated on the inflammatory reaction associated with the progression of Ehrlich ascitic tumor in mice and rats. ► R-954 reduced the inflammatory mediators, tumor growth as well as total cell numbers in medulla, blood, and ascitic fluid which follow the tumor inoculation. ► R-954 also inhibited the increase of solid tumor inoculated in rat paw.The present study investigated the effects of a new bradykinin B1 receptor antagonist, R-954, on the development of Ehrlich ascitic tumor (EAT) induced by the intraperitoneal inoculation of EAT cells in mice and the formation of a solid tumor by the subcutaneous injection of the cells in rat paw. The development of the tumor was associated with an increase in mouse total cell counts in bone marrow (10.8-fold), ascitic fluid (14.6-fold), and blood (12.6-fold). R-954 (2 mg/kg, s.c.) significantly reduced the ascitic fluid volume (63.7%) and the mouse weight gain (30.5%) after 10 consecutive days of treatment. The B1 antagonist as well as the anti-neoplasic drug vincristine also significantly inhibited the increase in total cell count in bone marrow, ascitic fluid, and blood. R-954 reduced significantly the total protein extravasation (57.3%), the production of nitric oxide (56%), PGE2 production (82%), and TNFα release (85.7%) in mice peritoneal cavity whereas vincristine reduced the release of these inflammatory mediators by 84–94%. The increase in paw edema after intraplantar injection of EAT cells was reduced by approximately 52% by either R-954 or vincristine treatment. In conclusion, this study presents for the first time the antitumoral activity of a new bradykinin B1 receptor antagonist on ascitic and solid tumors induced by Ehrlich cell inoculation in mice and rats.
Keywords: Bradykinin B1 antagonist; R-954; Cancer; Bradykinin; Ehrlich ascitic tumor;

Induction of LYVE-1/stabilin-2-positive liver sinusoidal endothelial-like cells from embryoid bodies by modulation of adrenomedullin-RAMP2 signaling by Takuma Arai; Takayuki Sakurai; Akiko Kamiyoshi; Yuka Ichikawa-Shindo; Nobuyoshi Iinuma; Yasuhiro Iesato; Teruhide Koyama; Takahiro Yoshizawa; Ryuichi Uetake; Akihiro Yamauchi; Lei Yang; Hisaka Kawate; Shinichiro Ogawa; Akira Kobayashi; Shinichi Miyagawa; Takayuki Shindo (1855-1865).
► Co-administration of AM and SB431542 enhanced differentiation of LYVE-1/stabilin-2-positive endothelial cells in EBs-culture system. ► These cells showed robust endocytosis and upregulated expression of LSEC-specific markers and possess fenestrae-like structure. ► In RAMP2-null liver, LYVE-1 was downregulated in LSEC, and the sinusoidal structure was disrupted. ► Our findings highlight the importance of AM–RAMP2 signaling for development of LSEC.Embryonic stem cells (ESCs) are a useful source for various cell lineages. So far, however, progress toward reconstitution of mature liver morphology and function has been limited. We have shown that knockout mice deficient in adrenomedullin (AM), a multifunctional endogenous peptide, or its receptor-activity modifying protein (RAMP2) die in utero due to poor vascular development and hemorrhage within the liver. In this study, using embryoid bodies (EBs)-culture system, we successfully induced liver sinusoidal endothelial-like cells by modulation of AM–RAMP2. In an EB differentiation system, we found that co-administration of AM and SB431542, an inhibitor of transforming growth factor β (TGFβ) receptor type 1, markedly enhanced differentiation of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)/stabilin-2-positive endothelial cells. These cells showed robust endocytosis of acetylated low-density lipoprotein (Ac-LDL) and upregulated expression of liver sinusoidal endothelial cells (LSECs)-specific markers, including factor 8 (F8), Fc-γ receptor 2b (Fcgr2b), and mannose receptor C type 1 (Mrc1), and also possessed fenestrae-like structure, a key morphological feature of LSECs. In RAMP2-null liver, by contrast, LYVE-1 was downregulated in LSECs, and the sinusoidal structure was disrupted. Our findings highlight the importance of AM–RAMP2 signaling for development of LSECs.
Keywords: Adrenomedullin (AM); Receptor activity-modifying protein (RAMP); Lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1); Stabilin-2; Liver sinusoidal endothelial cells (LSEC); Embryonic stem cells;

Effects of vaspin, chemerin and omentin-1 on feeding behavior and hypothalamic peptide gene expression in the rat by Luigi Brunetti; Chiara Di Nisio; Lucia Recinella; Annalisa Chiavaroli; Sheila Leone; Claudio Ferrante; Giustino Orlando; Michele Vacca (1866-1871).
► 24 h after adipokines administration, feeding and gene expression were analyzed. ► Vaspin decreased food intake, while chemerin and omentin-1 had no effect. ► Vaspin decreased NPY gene expression and increased POMC gene expression. ► Chemerin increased both AgRP and POMC gene expression. ► Omentin-1 did not modify gene expression of AgRP, NPY, orexin-A, CART, CRH, POMC.Visceral adipose tissue-derived serpin (vaspin) improves glucose tolerance and insulin sensitivity in diet-induced obese mice. Chemerin may increase insulin sensitivity in adipose tissue and seems to be associated with several key aspects of metabolic syndrome. Decreased levels of omentin-1 are associated with increasing obesity and insulin resistance. Our study aimed to investigate the effects of vaspin, chemerin and omentin-1 acute administration on feeding and hypothalamic gene expression of peptides which play a key role in feeding regulation. 35 rats were injected into the arcuate nucleus (ARC) of the hypothalamus with either saline (n  = 8), vaspin (1 μg/kg; n  = 9), chemerin (8 μg/kg; n  = 9), or omentin-1 (8 μg/kg; n  = 9). Food intake in the following 24 h was recorded, thereafter rats were sacrificed. Total RNA was extracted from hypothalami and reverse transcribed to evaluate hypothalamic gene expression of agouti-related peptide (AgRP), neuropeptide Y (NPY), orexin-A, cocaine- and amphetamine-regulated transcript (CART), corticotrophin releasing hormone (CRH) and proopiomelanocortin (POMC), by real-time reverse transcription polymerase chain reaction. Compared to vehicle, vaspin injection significantly decreased feeding, while chemerin and omentin-1 had no effect in the tested dose. Vaspin treatment significantly decreased NPY and increased POMC gene expression. Chemerin treatment led to a significant increase of both AgRP and POMC gene expression. Omentin-1 treatment did not modify gene expression of the investigated peptides. Therefore, vaspin is an adipokine triggering anorectic pathways in the hypothalamus, where reduction of NPY and increase of POMC mRNA levels mediate feeding inhibition. Chemerin and omentin-1 have no effect on feeding in the tested dose.
Keywords: Chemerin; Feeding; Neuropeptide Y; Omentin-1; Proopiomelanocortin; Vaspin;

Inhibitory effect of corticotropin-releasing factor on food intake in the bullfrog, Aquarana catesbeiana by Noriaki Morimoto; Kazumasa Hashimoto; Reiko Okada; Hiroshi Mochida; Minoru Uchiyama; Sakae Kikuyama; Kouhei Matsuda (1872-1875).
► CRF precursor mRNA levels increase in adult bullfrog fasted for 7 days. ► ICV administration of CRF decreases food consumption in adult bullfrog. ► CRF-induced anorexigenic action is blocked by treatment with alpha-helical CRF(9–41).Corticotropin-releasing factor (CRF) and CRF-related peptides exert hypophysiotropic and anorexigenic effects in mammals and teleost fish. In anuran amphibians, CRF acts as a potent stimulator of thyrotropin release from the pituitary. According to our recent study, CRF also acts as an anorexigenic factor for the cessation of food intake in the metamorphosing bullfrog larvae. However, the anorexigenic action of CRF has not been confirmed in adult bullfrogs. In this context, we examined the effect of feeding status on the expression level of the CRF transcript in the hypothalamus of the adult bullfrog. Levels of CRF mRNA in the hypothalami from bullfrogs fasted for 7 days were lower than in those from the bullfrogs that had been fed normally. Subsequently, we developed a method for measuring food intake in adult bullfrogs, and then investigated the effect of CRF on their food consumption in these animals. Intracerebroventricular (ICV) administration of CRF at 1 and 10 pmol/g body weight (BW) induced a significant decrease of food intake during 60 min. The CRF-induced anorexigenic action was blocked by treatment with a CRF receptor 1/CRF receptor 2 antagonist, α-helical CRF(9–41), at 100 pmol/g BW. These results provide direct evidence for the inhibitory effect of CRF on food intake, and suggest the involvement of CRF in the regulation of feeding through a CRF receptor-signaling pathway in the adult bullfrog.
Keywords: Bullfrog; CRF; Food intake; Anorexigenic action; α-Helical CRF(9–41); Intracerebroventricular injection;

Molecular identification of ghrelin receptor (GHS-R1a) and its functional role in the gastrointestinal tract of the guinea-pig by Takio Kitazawa; Tatsuro Nakamura; Atsuki Saeki; Hiroki Teraoka; Takeo Hiraga; Hiroyuki Kaiya (1876-1886).
Display Omitted► Growth hormone secretagogue receptor1a (GHS-R1a) was identified in guinea-pig brain cDNA and compared with that of other animals. ► Guinea-pig GHS-R1a expressed in HEK 293 cells responded to ghrelin, GHRP-6 and hexarelin in a concentration-dependent manner. ► GHS-R1a mRNA expression was high in the pituitary and hypothalamus, but very low in the gastrointestinal tract. ► Ghrelin was ineffective in producing responses in the stomach and colon but caused slight contraction in the small intestine. ► Functional roles of ghrelin and peripheral GHS-R1a for regulation of gastrointestinal motility are weak in guinea-pigs compared with other animal species.Ghrelin stimulates gastric motility in vivo in the guinea-pig through activation of growth hormone secretagogue receptor (GHS-R). In this study, we identified GHS-R1a in the guinea-pig, and examined its distribution and cellular function and compared them with those in the rat. Effects of ghrelin in different regions of gastrointestinal tract were also examined. GHS-R1a was identified in guinea-pig brain cDNA. Amino acid identities of guinea-pig GHS-R1a were 93% to horses and 85% to dogs. Expression levels of GHS-R1a mRNA were high in the pituitary and hypothalamus, moderate in the thalamus, cerebral cortex, pons, medulla oblongata and olfactory bulb, and low in the cerebellum and peripheral tissues including gastrointestinal tract. Comparison of GHS-R1a expression patterns showed that those in the brain were similar but the expression level in the gastrointestinal tract was higher in rats than in guinea-pigs. Guinea-pig GHS-R1a expressed in HEK 293 cells responded to rat ghrelin and GHS-R agonists. Rat ghrelin was ineffective in inducing mechanical changes in the stomach and colon but caused a slight contraction in the small intestine. 1,1-Dimethyl-4-phenylpiperazinium and electrical field stimulation (EFS) caused cholinergic contraction in the intestine, and these contractions were not affected by ghrelin. Ghrelin did not change spontaneous and EFS-evoked [3H]-efflux from [3H]-choline-loaded ileal strips. In summary, guinea-pig GHS-R1a was identified and its functions in isolated gastrointestinal strips were characterized. The distribution of GHS-R1a in peripheral tissues was different from that in rats, suggesting that the functional role of ghrelin in the guinea-pig is different from that in other animal species.
Keywords: Growth hormone secretagogue receptor 1a; Guinea-pig; Rat; Tissue distribution; Gastrointestinal tract;

Biological properties of prolactin-releasing peptide analogs with a modified aromatic ring of a C-terminal phenylalanine amide by Lenka Maletínská; Andrea Špolcová; Jana Maixnerová; Miroslava Blechová; Blanka Železná (1887-1892).
► PrRP20 analogs with changes at C-terminal Phe amides were characterized. ► PrRP20 analogs showed high affinities for the GPR10 receptor and cell signaling. ► PrRP20 analogs showed highly significant and long-lasting anorexigenic effect.Prolactin-releasing peptide (PrRP)-induced secretion of prolactin is not currently considered a primary function of PrRP, but the development of late-onset obesity in both PrRP and PrRP receptor knock-out mice indicates the unique anorexigenic properties of PrRP. In our recent study, we showed comparable potencies of peptides PrRP31 and PrRP20 in binding, intracellular signaling and prolactin release in pituitary RC-4B/C cells, and anorexigenic effect after central administration in fasted mice. In the present study, eight analogs of PrRP20 with C-terminal Phe amide modified with a bulky side chain or a halogenated aromatic ring revealed high binding potency, activation of mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK1/2) and cAMP response element-binding protein (CREB) and prolactin release in RC-4B/C cells. In particular, [PheNO2 31]PrRP20, [1-Nal31]PrRP20, [2-Nal31]PrRP20 and [Tyr31]PrRP20 showed not only in vitro effects comparable or higher than those of PrRP20, but also a very significant and long-lasting anorexigenic effect after central administration in fasted mice. The design of potent and long-lasting PrRP analogs with selective anorexigenic properties promises to contribute to the study of food intake disorders.
Keywords: PrRP20 analogs; RC-4B/C pituitary cells; Binding; MAPK/ERK1/2; CREB; Prolactin release; Food intake;

Central administration of neuronostatin induces antinociception in mice by Ai-min Yang; Wan-wen Ge; Song-song Lu; Shao-bin Yang; Shu-fang Su; Ze-yun Mi; Qiang Chen (1893-1901).
► I.c.v. administration of neuronostatin produced a dose- and time-related antinociceptive effect in the tail immersion assay in mice. ► Neuronostatin produces its full analgesic effect by activating μ-and κ-opioid receptors. ► The analgesic effects of neuronostatin are dependent upon the central melanocortin system ► C-terminal amidation modification of neuronostatin is essential to exert its antinociceptive effect. ► Neuronostatin (6 nmol, i.c.v.) increased c-Fos protein expression in the periaqueductal gray (PAG) and the nucleus raphe magnus (NRM).Neuronostatin, a recently discovered endogenous bioactive peptide, was encoded by pro-mRNA of somatostatin that contributes to modulation of nociception. However, nociceptive effect of neuronostatin is still not fully known. The aim of this study was to evaluate effect of neuronostatin on nociception and elucidate its possible mechanism of action. Intracerebroventricular (i.c.v.) administration of neuronostatin (0.3, 3, 6, 12 nmol/mouse) produced a dose- and time-related antinociceptive effect in the tail immersion assay in mice, an acute pain model. The antinociceptive effect of neuronostatin was significantly antagonized by naloxone, and was strongly inhibited by co-injection with β-funaltrexamine or nor-binaltorphimine, but not by naltrindole. Also, melanocortin 3/4 receptor antagonist, SHU9119, completely blocked the effect of neuronostatin. These data indicated the involvement of both μ- and κ-opioid receptors and central melanocortin system in the analgesic response induced by neuronostatin. In addition, neuronostatin (6 nmol, i.c.v.) increased c-Fos protein expression in the periaqueductal gray (PAG) and the nucleus raphe magnus (NRM) that have a pivotal role in regulating descending pain pathways. Taken together, this study is the first to reveal that neuronostatin produces antinociceptive effect via opioid and central melanocortin systems, which is associated with an increase in neuronal activity the PAG and NRM.
Keywords: Neuronostatin; Antinociception; Opioid receptors; Central melanocortin system; c-Fos; Mice;

Synthetic peptides from two Pf sporozoite invasion-associated proteins specifically interact with HeLa and HepG2 cells by Gabriela Arévalo-Pinzón; Hernando Curtidor; Marina Muñoz; Manuel A. Patarroyo; Manuel E. Patarroyo (1902-1908).
► SIAP-1 and -2 peptides specifically interact with HeLa and HepG2 cell receptors. ► Proteoglycans mediate the specific binding of SIAP-1 and -2 HABPs. ► Antibodies against SIAP-1 and -2 peptides recognize the native proteins in P. falciparum sporozoites.Two recently described molecules have been associated with sporozoite traversal ability and hepatocyte entry: sporozoite invasion-associated proteins (SIAP)-1 and -2. The HeLa and HepG2 cell binding ability of synthetic peptides spanning the whole SIAP-1 and -2 sequences has been studied in the search for identifying these proteins’ functionally active specific regions. Twelve HepG-2 and seventeen HeLa cell high-activity binding peptides (HABPs) have been identified in SIAP-1, 8 of them having high specific binding affinity for both cell lines. Four HepG2 HABPs and two HeLa HABPs have been identified in SIAP-2, one of them interacting with both HeLa and HepG2 cells. SIAP-1 and SIAP-2 HABPs bound specifically and saturably to heparin sulfate and chondroitin sulfate-type membrane receptors on host cells. Circular dichroism assays have shown high α-helix content in SIAP-1 and SIAP-2 HABP secondary structure. Immunofluorescence analysis has revealed that specific peptides against SIAP proteins are highly immunogenic in mice and that anti-SIAP-1 and -2 antibodies recognize the native protein in Plasmodium falciparum sporozoites. Polymorphism studies have shown that a most SIAP-1 and -2 HABPs are conserved among P. falciparum strains. Our results have suggested that SIAP-1 and -2 participate in host–pathogen interactions during cell-traversal and hepatocyte invasion and highlighted the relevance of the ongoing identification and study of potentially new molecules when designing a fully protective antimalarial vaccine.
Keywords: Cell-traversal; High-activity binding peptide; Antimalarial vaccine; Plasmodium falciparum; Sporozoite invasion;

► A previously designed antimicrobial peptide from Argopecten purpuratus hemocytes was expressed in E. coli using a chimaeric gene. ► Identity of the resulting recombinant peptide was confirmed by mass spectrometry analyses ► Peptide aliquots showed a very effective antifungal capacity against four important pathogenic plant fungi. ► Microscopy analyses of antifungal challenges revealed a decreased formation of reproductive structures caused by the peptide. ► Results lead to schedule further assays to propose applicability of this AMP in plant antifungal programs.Antimicrobial peptides (AMP) have been widely described in several organisms from different kingdoms. We recently designed and evaluated a synthetic version of an AMP isolated and characterized from Argopecten purpuratus hemocytes. This study describes the generation of a chimaeric gene encoding for Ap-S, the use of this construct to transform E. coli strain BL21, and the evaluation of the purified recombinant Ap-S (rApS) as an antifungal agent. The proposed gene coding for rAp-S consists of 93 nucleotides arranged downstream from the IPTG-inducible T7 promoter. The best synthesis conditions were obtained after E. coli cultivation at 26 °C for 3 h, which allowed for the production of an rAp-S-enriched fraction containing the peptide at 249 μM. Mass spectrometry analysis of the purified rAp-S (3085.80 Da) showed the addition of a glycine residue on its N-terminal end derived from vector design and peptide purification. The purified rApS fraction was assayed for antifungal activity by direct addition of purified rApS elution to potato dextrose agar media at a final concentration of 81 nM. These assays showed important growth inhibitions of both biotrophic (Fusarium oxysporum, Trichoderma harzianum) and necrotrophic (Botrytis cinerea, Alternaria spp.) fungi in that the hyphae structures and spore count were affected in all cases. The strategy of cloning and expressing rAp-S in E. coli, the high yield obtained and its successful use for controlling pathogenic fungi suggest that this molecule could be applied to agricultural crops using various management strategies.
Keywords: Antifungal peptide; Recombinant E. coli expression; Fungal plant pathogen; cDNA design; Chimaeric gene sequence;

Isolation and characterization of peptide antibiotics LI-F04 and polymyxin B6 produced by Paenibacillus polymyxa strain JSa-9 by Yang Deng; Zhaoxin Lu; Hua Bi; Fengxia Lu; Chong Zhang; Xiaomei Bie (1917-1923).
► Polymyxin B6 and two linear LI-F04 peptides are found in cell pellets of Paenibacillus polymyxa JSa-9. ► We firstly identify structure-linear LI-F antibiotic from a natural bacterial isolate. ► These antibiotics from strain JSa-9 have a potential application in biological controls. Paenibacillus polymyxa JSa-9 had been found to produce five cyclic LI-F type antibiotics which were released into culture medium in accordance with our previous report. In this study, another three kinds of antagonistic compounds were extracted from P. polymyxa JSa-9 cell pellets and (or) spores by methanol. Using high performance liquid chromatography (HPLC) method, two antagonistic fractions were separated and collected from the methanol extract. One showed inhibition against Escherichia coli and Staphylococcus aureus, while the other was active against Aspergillus niger and S. aureus. By means of electrospray ionization mass spectroscopy (ESI-MS), infrared spectroscopy (IR), and amino acid analysis, two kinds of compounds from fraction B with molecular masses of 901 and 915 Da were characterized as the linear lipopeptide analogs of antibiotics LI-F04a and LI-F04b, respectively. Another antimicrobial substance from fraction A could be attributed to polymyxin B6.
Keywords: Paenibacillus polymyxa; LI-F04; Polymyxin B6; Identification; Antimicrobial activity;

Display Omitted► A model was proposed to predict the biological activity of Hymenoptera peptides. ► The model was built based on structural properties of peptides from the literature. ► PCA with PLS regression was performed with chemometry of each peptide. Six groups related to the functional role of these peptides were observed.When searching for prospective novel peptides, it is difficult to determine the biological activity of a peptide based only on its sequence. The “trial and error” approach is generally laborious, expensive and time consuming due to the large number of different experimental setups required to cover a reasonable number of biological assays. To simulate a virtual model for Hymenoptera insects, 166 peptides were selected from the venoms and hemolymphs of wasps, bees and ants and applied to a mathematical model of multivariate analysis, with nine different chemometric components: GRAVY, aliphaticity index, number of disulfide bonds, total residues, net charge, pI value, Boman index, percentage of alpha helix, and flexibility prediction. Principal component analysis (PCA) with non-linear iterative projections by alternating least-squares (NIPALS) algorithm was performed, without including any information about the biological activity of the peptides. This analysis permitted the grouping of peptides in a way that strongly correlated to the biological function of the peptides. Six different groupings were observed, which seemed to correspond to the following groups: chemotactic peptides, mastoparans, tachykinins, kinins, antibiotic peptides, and a group of long peptides with one or two disulfide bonds and with biological activities that are not yet clearly defined. The partial overlap between the mastoparans group and the chemotactic peptides, tachykinins, kinins and antibiotic peptides in the PCA score plot may be used to explain the frequent reports in the literature about the multifunctionality of some of these peptides. The mathematical model used in the present investigation can be used to predict the biological activities of novel peptides in this system, and it may also be easily applied to other biological systems.
Keywords: Polycationic peptides; Insect venoms; PCA; System biology; Toxins;

Cellular uptake of transportan 10 and its analogs in live cells: Selectivity and structure–activity relationship studies by Jingjing Song; Ming Kai; Wei Zhang; Jindao Zhang; Liwei Liu; Bangzhi Zhang; Xin Liu; Rui Wang (1934-1941).
► TP10 and its analogs showed uptake selectivity to cancer cells. ► The increase of charge can enhance the translocation ability of TP10. ► Higher membrane disturbance leads to larger translocation ability. ► The MD simulations can explain the difference in translocation ability.Transportan 10 (TP10) is an amphipathic cell-penetrating peptide with high translocation ability. In order to obtain more details of structure–activity relationship of TP10, we evaluated the effects of structure and charge on its translocation ability. Our results demonstrated that disrupting the helical structure or Arg substitution could remarkably decrease the cellular uptake of TP10. However, increasing the number of positive charge was an effective strategy to enhance translocation ability of TP10. Furthermore, the molecular dynamics simulation supported the results derived from experiments, suggesting that higher membrane disturbance leads to higher cellular uptake of peptides. In addition, our study also demonstrated TP10 and its analogs preferentially entered cancer cells rather than normal cells. The uptake selectivity toward cancer cells makes TP10 and its analogs as potent CPPs for drug delivery.
Keywords: Cell penetrating peptide; Structure–activity relationship; Uptake selectivity; Molecular dynamics simulations;

Lipopolysaccharide increases gastric and circulating NUCB2/nesfatin-1 concentrations in rats by Andreas Stengel; Miriam Goebel-Stengel; Janusz Jawien; Peter Kobelt; Yvette Taché; Nils W.G. Lambrecht (1942-1947).
► LPS reduces plasma acyl ghrelin and desacyl ghrelin levels. ► In contrast, circulating nesfatin-1 is increased. ► Increased nesfatin-1 levels under these conditions are likely to be stomach derived. ► These data highlight the differential regulation of ghrelin and nesfatin-1. ► Decreased ghrelin and increased nesfatin-1 likely contribute to the LPS-induced anorexia.Bacterial lipopolysaccharide (LPS) is an established animal model to study the innate immune response to Gram-negative bacteria mimicking symptoms of infection including reduction of food intake. LPS decreases acyl ghrelin associated with decreased concentrations of circulating ghrelin-O-acyltransferase (GOAT) likely contributing to the anorexigenic effect. We also recently described the prominent expression of the novel anorexigenic hormone, nucleobindin2 (NUCB2)/nesfatin-1 in gastric X/A-like cells co-localized with ghrelin in different pools of vesicles. To investigate whether LPS would affect gastric and circulating NUCB2/nesfatin-1 concentration, ad libitum fed rats were equipped with an intravenous (iv) catheter. LPS was injected intraperitoneally (ip, 100 μg/kg) and blood was withdrawn before and at 2, 5, 7 and 24 h post injection and processed for NUCB2/nesfatin-1 radioimmunoassay. Gastric corpus was collected to measure NUCB2 mRNA expression by RT-qPCR and NUCB2/nesfatin-1 protein concentration by Western blot. Injection of LPS increased plasma NUCB2/nesfatin-1 concentrations by 43%, 78% and 62% compared to vehicle at 2 h, 5 h and 7 h post injection respectively (p  < 0.05) and returned to baseline at 24 h. The plasma NUCB2/nesfatin-1 increase at 2 h was associated with increased corpus NUCB2 mRNA expression (p  < 0.01), whereas NUCB2 mRNA was not detectable in white blood cells. Likewise, gastric NUCB2 protein concentration was increased by 62% after LPS compared to vehicle (p  < 0.01). These data show that gastric NUCB2 production and release are increased in response to LPS. These changes are opposite to those of ghrelin in response to LPS supporting a differential gastric regulation of NUCB2/nesfatin-1 and ghrelin expression derived from the same cell by immune challenge.
Keywords: Endotoxin; Ghrelin; Nucleobindin2; Stomach; X/A-like cell;

Intracerebroventricular administration of neuronostatin induces depression-like effect in forced swim test of mice by Ai-min Yang; Yue-ke Ji; Shu-fang Su; Shao-bin Yang; Song-song Lu; Ze-yun Mi; Qing-zhen Yang; Qiang Chen (1948-1952).
► Intracerebroventricular (i.c.v.) administration of neuronostatin induces depression-like effect in the forced swim in mice. ► Neuronostatin had no effect on locomotor activity in habituated mice. ► C-terminal amidation modification of neuronostatin is essential to exert its depressive effect. ► The depressive effect induced by neuronostatin was mediated by the central melanocortin system and GABAA receptor.Neuronostatin is a recently discovered endogenous bioactive peptide that is encoded by pro-mRNA of somatostatin. In the present study, we investigated the effect of neuronostatin on mood regulation in the forced swim test of mice. Our results showed intracerebroventricular (i.c.v.) administration of neuronostatin produced an increase in the immobility time, suggesting that neuronostatin induced depression-like effect. In order to rule out the possibility that neuronostatin had increased immobility time by a non-specific reduction in general activity, the effect of neuronostatin on locomotor activity was examined. Neuronostatin had no influence on locomotor activity in mice. In addition, the depression-like effect of neuronostatin was completely reversed by melanocortin 3/4 receptor antagonist SHU9119 or GABAA receptor antagonist bicuculline, but not by opioid receptor antagonist naloxone. These data suggested that the depression-like effect induced by i.c.v. administered neuronostatin was dependent upon the central melanocortin system and GABAA receptor. In conclusion, the results of this study report that neuronostatin induces depression-like effect. These findings reveal that neuronostatin is a new neuropeptide with an important role in regulating depressive behavior.

Discovery and development of a synthetic peptide derived from lactoferrin for clinical use by Carlo P.J.M. Brouwer; Mahfuzur Rahman; Mick M. Welling (1953-1963).
► Antimicrobial peptides eliminate infections with drug-resistant pathogens. ► Radiolabeling of peptides enables the scintigraphic detection of infections. ► Antimicrobial peptides are well tolerated without toxic side effects. ► Prophylaxis in patients receiving organ or stem cell transplantation. ► Possible application in coating transplants and biosensing.There is an urgent need to develop new antimicrobial drugs especially for combating the rise of infections caused by multi-resistant pathogens such as MRSA and VRSA. The problem of antibiotic resistant micro-organisms is expected to increase disproportionally and controlling of infections is becoming difficult because of the rapid spread of those micro-organisms. Primary therapy with classical antibiotics is becoming more ineffective. Combinational therapy of antibiotics with antimicrobial peptides (AMP's) has been suggested as an alternative approach to improve treatment outcome. Their unique mechanism of action and safety profile makes AMP's appealing candidates for simultaneous or sequential use in different cases of infections. In this review, for antimicrobial treatment the application of synthetic antimicrobial peptide hLF(1-11), derived from the first 11 amino acids of human lactoferrin is evaluated in both pre-clinical and clinical settings. Present information indicates that this derivate from lactoferrin is well tolerated in pre-clinical tests and clinical trials and thus hLF(1-11) is an interesting candidate for further exploration in various clinical indications of obscure infections, including meningitis. Another approach of using AMP's is their use in prevention of infections e.g. as coating for dental or bone implants or in biosensing applications or useful as infection specific radiopharmaceutical.
Keywords: Antimicrobial peptide; Lactoferrin; Synthetic derivate; hLF(1-11); Infection; Clinical trial;

Regulation of C-type natriuretic peptide expression by Donald F. Sellitti; Nancy Koles; Maria C. Mendonça (1964-1971).
► Introduction to the family of natriuretic peptides, their receptors, and functions. ► Functional significance of CNP in bone, neural, and cardiovascular tissue. ► Hormonal and pathophysiological determinants of CNP expression. ► Structure of the CNP gene, emphasizing regulatory sequences in promoter. ► Putative CNP transcription factors.C-type natriuretic peptide (CNP) is a member of the small family of natriuretic peptides that also includes atrial natriuretic peptide (ANP) and brain, or B-type natriuretic peptide (BNP). Unlike them, it performs its major functions in an autocrine or paracrine manner. Those functions, mediated through binding to the membrane guanylyl cyclase natriuretic peptide receptor B (NPR-B), or by signaling through the non-enzyme natriuretic peptide receptor C (NPR-C), include the regulation of endochondral ossification, reproduction, nervous system development, and the maintenance of cardiovascular health. To date, the regulation of CNP gene expression has not received the attention that has been paid to regulation of the ANP and BNP genes. CNP expression in vitro is regulated by TGF-β and receptor tyrosine kinase growth factors in a cell/tissue-specific and sometimes species-specific manner. Expression of CNP in vivo is altered in diseased organs and tissues, including atherosclerotic vessels, and the myocardium of failing hearts. Analysis of the human CNP gene has led to the identification of a number of regulatory sites in the proximal promoter, including a GC-rich region approximately 50 base pairs downstream of the Tata box, and shown to be a binding site for several putative regulatory proteins, including transforming growth factor clone 22 domain 1 (TSC22D1) and a serine threonine kinase (STK16). The purpose of this review is to summarize the current literature on the regulation of CNP expression, emphasizing in particular the putative regulatory elements in the CNP gene and the potential DNA-binding proteins that associate with them.
Keywords: C-type natriuretic peptide; Gene expression; Regulation;

Tachykinins and the hypothalamo–pituitary–gonadal axis: An update by Mercedes Lasaga; Luciano Debeljuk (1972-1978).
► Substance P and Neurokinin B play a role in neuroendocrine control of reproduction. ► Strong evidence indicates that arcuate NKB/dynorphin/kisspeptin neurons regulate pulsatile secretion of GnRH. ► A better understanding of the mechanisms involved in the arcuate NK3 receptor signaling could contribute to improve treatment of reproductive disorders.Tachykinins play a critical role in neuroendocrine regulation of reproduction. The best known members of the family are substance P (SP), neurokinin A and neurokinin B. Tachykinins mediate their biological actions through three G protein-coupled receptors, named NK1, NK2, and NK3. SP was suggested to play an important role in the ovulatory process in mammals and humans. Recent findings suggest a role of tachykinins in the aging of the hypothalamo–pituitary–gonadal axis. A high presence of SP was found in the sheep pars tuberalis and evidence indicates that it may have some role in the control of prolactin secretion. The presence of SP was confirmed in Leydig cells of the rat testes of animals submitted to constant light or treated with estrogens. Tachykinins were found to increase the motility of human spermatozoa. Tachykinins were also found to be present in the mouse ovary and more specifically, in the granulose cells. It is possible that tachykinins may play an important role in the ovarian function. NKB has been implicated in the steroid feedback control of GnRH release. Human mutations in the gene encoding this peptide or its receptor (TACR3) lead to a defect in the control of GnRH. A specific subset of neurons in the arcuate nucleus of the hypothalamus, colocalized three neuropeptides, kisspeptin, NKB and dynorphin. This subpopulation of neurons mediates the gonadal hormone feedback control of GnRH secretion. NKB/NK3 signaling plays a role in puberty onset and fertility in humans. This minireview summarizes the recent data about the action of tachykinins on the hypothalamo–pituitary–gonadal axis.
Keywords: Tachykinins; SP; NKB; Hypothalamus; Pituitary; Gonads;