Peptides (v.45, #C)

A comparison of host-defense peptides in skin secretions of female Xenopus laevis  ×  Xenopus borealis and X. borealis  ×  X. laevis F1 hybrids by Milena Mechkarska; Manju Prajeep; Jérôme Leprince; Hubert Vaudry; Mohammed A. Meetani; Ben J. Evans; J. Michael Conlon (1-8).
Peptidomic analysis was used to compare the diversity of host-defense peptides in norepinephrine-stimulated skin secretions from laboratory-generated female F1 hybrids of Xenopus laevis and Xenopus borealis (Pipidae). Skin secretions of hybrids with maternal X. laevis (X LB ) contained 12 antimicrobial peptides (AMPs), comprising 8 from X. laevis and 4 from X. borealis. Magainin-B1, XPF-B1, PGLa-B1 CPF-B2, CPF-B3 and CPF-B4 from X. borealis and XPF-1, XPF-2, and CPF-6 from X. laevis were not detected and CPF-1 and CPF-7 were present in low concentration. The secretions contained caerulein and caerulein-B1 derived from both parents but lacked X. laevis xenopsin and X. borealis caerulein-B2. Skin secretions of hybrids with maternal X. borealis (X BL ) contained 14 AMPs comprising 6 from X. borealis and 8 from X. laevis. Magainin-B1, XPF-B1, PGLa-B1, CPF-B2, XPF-1, CPF-5, and CPF-7 were absent and CPF-B3, CPF-B4, CPF-1 and CPF-6 were present only in low concentration. Xenopsin and caerulein were identified in the secretions but caerulein-B2 was absent and caerulein-B1was present in low concentration. No peptides were identified in secretions of either X LB or X BL hybrids that were not present in the parental species. The data indicate that hybridization between X. laevis and X. borealis results in increased diversity of host-defense peptides in skin secretions but point to extensive AMP gene silencing compared with previously studied female X. laevis  ×  X. muelleri F1 hybrids and no novel peptide expression.
Keywords: Xenopus; F1 hybrid; Frog skin secretion; Antimicrobial peptide; Caerulein; Xenopsin; Allopolyploidization;

Role of oxytocin in energy metabolism by Valéria Ernestânia Chaves; Cristiane Queixa Tilelli; Nilton Almeida Brito; Márcia Nascimento Brito (9-14).
The basic mechanisms that lead obesity are not fully understood; however, several peptides undoubtedly play a role in regulating body weight. Obesity, a highly complex metabolic disorder, involves central mechanisms that control food intake and energy expenditure. Previous studies have shown that central or peripheral oxytocin administration induces anorexia. Recently, in an apparent discrepancy, rodents that were deficient in oxytocin or the oxytocin receptor were shown to develop late-onset obesity without changing their total food intake, which indicates the physiological importance of oxytocin to body metabolism. Oxytocin is synthesized not only within magnocellular and parvocellular neurons but also in several organs, including the ovary, uterus, placenta, testis, thymus, kidney, heart, blood vessels, and skin. The presence of oxytocin receptors in neurons, the myometrium and myoepithelial cells is well recognized; however, this receptor has also been identified in other tissues, including the pancreas and adipose tissue. The oxytocin receptor is a typical class I G protein-coupled receptor that is primarily linked to phospholipase C-β via Gq proteins but can also be coupled to other G proteins, leading to different functional effects. In this review, we summarize the present knowledge of the effects of oxytocin on controlling energy metabolism, focusing primarily on the role of oxytocin on appetite regulation, thermoregulation, and metabolic homeostasis.
Keywords: Oxytocin; Appetite control; Thermoregulation; Energy metabolism; Adipose tissue; Obesity;

Responsiveness of vomeronasal cells to a newt peptide pheromone, sodefrin as monitored by changes of intracellular calcium concentrations by Takeo Iwata; Tomoaki Nakada; Fumiyo Toyoda; Toshihiko Yada; Seiji Shioda; Sakae Kikuyama (15-21).
A peptide pheromone of the red-bellied male newt, sodefrin was tested for its ability to increase intracellular concentrations of Ca2+ ([Ca2+]i) in the dissociated vomeronasal (VN) cells of females by means of calcium imaging system. The pheromone elicited a marked elevation of [Ca2+]i in a small population of VN cells from sexually developed females. The population of cells exhibiting sodefrin-induced elevation of [Ca2+]i increased concentration-dependently. A pheromone of a different species was ineffective in this respect. The VN cells from non-reproductive females or from reproductive males scarcely responded to sodefrin in terms of elevating [Ca2+]i. In the cells from hypophysectomized and ovariectomized females, the sodefrin-inducible increase of [Ca2+]i never occurred. The cells from the operated newts supplemented with prolactin and estradiol exhibited [Ca2+]i responses to sodefrin with a high incidence. Thus, sex- and hormone-dependency as well as species-specificity of the responsiveness of the VN cells to sodefrin was evidenced at the cellular level. Subsequently, possibility of involvement of phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3) and/or PLC-diacylglycerol (DAG)-protein kinase C (PKC) pathways in increasing [Ca2+]i in VN cells in response to sodefrin was explored using pharmacological approaches. The results indicated that PLC is involved in generating the Ca2+ signal in all sodefrin-responsive VN cells, whereas IP3 in approximately 50% of the cells and DAG-PKC in the remaining cells. In the latter case, the increase of [Ca2+]i was postulated to be induced by the influx of Ca2+ through the L-type channel. The significance of the finding is discussed.
Keywords: Newt; Peptide pheromone; Sodefrin; Vomeronasal cell; Intracellular calcium concentration; Signal transduction pathway;

Influence of variants in the NPY gene on obesity and metabolic syndrome features in Spanish children by Josune Olza; Mercedes Gil-Campos; Rosaura Leis; Azahara I. Rupérez; Rafael Tojo; Ramón Cañete; Ángel Gil; Concepción M. Aguilera (22-27).
Variants in the neuropeptide Y (NPY) gene have been associated with obesity and its traits. The objective of the present study was to evaluate the association of single nucleotide polymorphisms (SNPs) in the NPY gene with obesity, metabolic syndrome features, and inflammatory and cardiovascular disease (CVD) risk biomarkers in Spanish children. We recruited 292 obese children and 242 normal-body mass index (BMI) children. Height, weight, BMI, waist circumference, clinical and metabolic markers, adipokines, and inflammatory (PCR, IL-6, IL-8 and TNF-α) and CVD risk biomarkers (MPO, MMP-9, sE-selectin, sVCAM, sICAM, and PAI-1) were analyzed. Seven SNPs in the NPY gene were genotyped. The results of our study indicate that anthropometric measurements, clinical and metabolic markers, adipokines (leptin and resistin), and inflammatory and CVD risk biomarkers were generally elevated in the obese group. The exceptions to this finding included cholesterol, HDL-c, and adiponectin, which were lower in the obese group, and glucose, LDL-c, and MMP-9, which did not differ between the groups. Both rs16147 and rs16131 were associated with the risk of obesity, and the latter was also associated with insulin resistance, triacylglycerols, leptin, and HDL-c. Thus, we confirm the association of rs16147 with obesity, and we demonstrate for the first time the association of rs16131 with obesity and its possible impact on the early onset of metabolic syndrome features, mainly triacylglycerols, in children.
Keywords: Obesity; NPY variants; Metabolic syndrome features; Children;

Three new antimicrobial peptides from the scorpion Pandinus imperator by Xian-Chun Zeng; Lingli Zhou; Wanxia Shi; Xuesong Luo; Lei Zhang; Yao Nie; Jinwei Wang; Shifen Wu; Bin Cao; Hanjun Cao (28-34).
Three novel cysteine-free venom peptides, which were referred to as Pantinin-1, Pantinin-2 and Pantinin-3, respectively, have been identified from the scorpion Pandinus imperator by cDNA cloning strategy. The precursor of each peptide consists of a signal peptide, a mature peptide with no disulfide bridges, and an acidic propeptide with a typical processing signal. Each of the three peptides is an α-helical, cationic and amphipathic molecule with 13 or 14 amino acid residues. Their amino acid sequences are homologous to those of some 13-mer antimicrobial peptides isolated from scorpions. Antimicrobial assay showed that all the three peptides possess relatively strong activities against Gram-positive bacteria and a fungus, but have very weak antimicrobial activities against Gram-negative bacteria. Toxicity assay showed that the three peptides exhibit very low or mild hemolytic activities against human red blood cells. It is interesting to see that Pantinin-3 is able to potently inhibit the growth of vancomycin-resistant Enterococcus (VRE) S13, a pathogen that can cause a number of human infections; this suggests that Pantinin-3 has great potential to be applied in the treatment of VRE infections. Our findings gain new insights into the structure/function relationships of the small linear cationic antimicrobial peptides from scorpions, and provide new templates for designing of antimicrobial agents targeting antibiotic-resistant pathogenic bacteria.
Keywords: Antimicrobial peptide; Scorpion venom; Pandinus imperator; Amphipathic peptide; Vancomycin-resistant Enterococcus; Antibiotic-resistant pathogen;

Plasma leptin level predicts hematoma growth and early neurological deterioration after acute intracerebral hemorrhage by Quan Du; Ding-Bo Yang; Yong-Feng Shen; Wen-Hua Yu; Zu-Yong Zhang; Qiang Zhu; Zhi-Hao Che; Qun-Jie Liu; Hao Wang; Xiao-Qiao Dong (35-39).
Higher plasma leptin levels have been associated with poor clinical outcomes after intracerebral hemorrhage. Nevertheless, their links with hematoma growth and early neurological deterioration are unknown. Therefore, we aimed to investigate the relationship between plasma leptin levels, hematoma growth, and early neurological deterioration in patients with acute intracerebral hemorrhage. We prospectively studied 102 consecutive patients with acute spontaneous basal ganglia hemorrhage presenting within 6 h from symptoms onset. Significant hematoma growth was defined as hematoma enlargement >33% at 24 h. Early neurological deterioration was defined as an increase of ≥4 points in National Institute of Health Stroke Scale score at 24 h from symptoms onset. We measured plasma leptin levels on admission using an enzyme-linked immunosorbent assay in a blinded fashion. In multivariate logistic regression analysis, plasma leptin level emerged as the independent predictor of hematoma growth (odds ratio, 1.182; 95% confidence interval, 1.061–2.598; P  = 0.008) and early neurological deterioration (odds ratio, 1.193; 95% confidence interval, 1.075–2.873; P  = 0.004). Using receiver operating characteristic curves, we calculated areas under the curve for hematoma growth (area under curve, 0.844; 95% confidence interval, 0.759–0.908) and early neurological deterioration (area under curve, 0.857; 95% confidence interval, 0.774–0.918). The predictive performance of leptin was similar to, but did not obviously improve that of hematoma volume. Thus, leptin may help in the prediction of hematoma growth and early neurological deterioration after intracerebral hemorrhage.
Keywords: Leptin; Intracerebral hemorrhage; Hematoma growth; Early neurological deterioration; Biomarker;

Mechanisms of glucagon degradation at alkaline pH by Nicholas Caputo; Jessica R. Castle; Colin P. Bergstrom; Julie M. Carroll; Parkash A. Bakhtiani; Melanie A. Jackson; Charles T. Roberts; Larry L. David; W. Kenneth Ward (40-47).
Glucagon is unstable and undergoes degradation and aggregation in aqueous solution. For this reason, its use in portable pumps for closed loop management of diabetes is limited to very short periods. In this study, we sought to identify the degradation mechanisms and the bioactivity of specific degradation products. We studied degradation in the alkaline range, a range at which aggregation is minimized. Native glucagon and analogs identical to glucagon degradation products were synthesized. To quantify biological activity in glucagon and in the degradation peptides, a protein kinase A-based bioassay was used. Aged, fresh, and modified peptides were analyzed by liquid chromatography with mass spectrometry (LCMS). Oxidation of glucagon at the Met residue was common but did not reduce bioactivity. Deamidation and isomerization were also common and were more prevalent at pH 10 than 9. The biological effects of deamidation and isomerization were unpredictable; deamidation at some sites did not reduce bioactivity. Deamidation of Gln 3, isomerization of Asp 9, and deamidation with isomerization at Asn 28 all caused marked potency loss. Studies with molecular-weight-cutoff membranes and LCMS revealed much greater fibrillation at pH 9 than 10. Further work is necessary to determine formulations of glucagon that minimize degradation and fibrillation.
Keywords: Glucagon; Degradation; Deamidation; Oxidation; Diabetes mellitus; Alkaline;

Cultured bovine adrenal chromaffin cells (BCCs) are employed to study first messenger-specific signaling by cytokines and neurotransmitters occurring in the adrenal medulla following immune-related stress responses. Here, we show that the cytokine TNF-alpha, and the neuropeptide transmitter PACAP, acting through the TNFR2 and PAC1 receptors, activate distinct signaling pathways, with correspondingly distinct transcriptomic signatures in chromaffin cells. We have carried out a comprehensive integrated transcriptome analysis of TNF-alpha and PACAP gene regulation in BCCs using two microarray platforms to maximize transcript identification. Microarray data were validated using qRT-PCR. More than 90% of the transcripts up-regulated either by TNF-alpha or PACAP were specific to a single first messenger. The final list of transcripts induced by each first messenger was subjected to multiple algorithms to identify promoter/enhancer response elements for trans-acting factors whose activation could account for gene expression by either TNF-alpha or PACAP. Distinct groups of transcription factors potentially controlling the expression of TNF-alpha or PACAP-responsive genes were found: most of the genes up-regulated by TNF-alpha contained transcription factor binding sites for members of the Rel transcription factor family, suggesting TNF-alpha-TNFR2 signaling occurs mainly through the NF-KB signaling pathway. Surprisingly, EGR1 was predicted to be the primary transcription factor controlling PACAP-modulated genes, suggesting PACAP signaling to the nucleus occurs predominantly through ERK, rather than CREB activation. Comparison of TNFR2-dependent versus TNFR1-dependent gene induction, and EGR1-mediated transcriptional activation, may provide a pharmacological avenue to the unique pathways activated by the first messengers TNF-alpha and PACAP in neuronal and endocrine cells.
Keywords: Affymetrix; Agilent; Chromaffin cell; Cyclic AMP; ERK; Gene induction; Microarray; NF-kB; PACAP; PAC1; Signal transduction; TNF-alpha; Transcriptional regulation; TNFR2;

High plasma copeptin level has been associated with clinical outcomes after acute illness. The present study was undertaken to investigate the plasma copeptin concentrations in preschool children with community-acquired pneumonia (CAP) and to analyze the correlations of copeptin with CAP-related complications and pleural effusion. Plasma copeptin concentrations of 100 healthy children and 165 preschool children with CAP were measured. 35 children (21.2%) presented with complicated CAP and 28 children (17.0%) presented with pleural effusion. The admission copeptin levels were significantly increased in all patients (49.7 ± 21.4 pmol/L), children with complicated CAP (73.0 ± 16.9 pmol/L), those with uncomplicated CAP (43.4 ± 17.8 pmol/L), those with pleural effusion (70.9 ± 17.4 pmol/L) and those without pleural effusion (45.3 ± 19.5 pmol/L) compared with healthy control individuals (9.0 ± 2.7 pmol/L, all P  < 0.001). Multivariate logistic regression analysis showed that plasma copeptin levels were independently related to CAP-related complications (odds ratio 1.214, 95% confidence interval 1.104–1.872, P  < 0.001) and pleural effusion (odds ratio 1.226, 95% confidence interval 1.109–1.917, P  < 0.001). A receiver operating characteristic curve analysis showed plasma copeptin level better predicted CAP-related complications (area under curve 0.876, 95% confidence interval 0.815–0.922) and pleural effusion (area under curve 0.831, 95% confidence interval 0.765–0.885). Thus, plasma copeptin level may represent a novel biomarker for predicting CAP-related complications in preschool children.
Keywords: Copeptin; Children; Community-acquired pneumonia; Complication; Pleural effusion; Biomarker;

Is oxytocin a therapeutic factor for ischemic heart disease? by Ali Mohammad Alizadeh; Parasto Mirzabeglo (66-72).
Preconditioning signaling pathways model for oxytocin. Our data suggest that these pathways involve an amplification loop leading to IP3/DAG and NO activation and subsequent PKC activation, and constant mitoKATP opening and mPTP closing. In fine, this could result in decrease of mitochondrial membrane potential and cardioprotection.Ischemic heart disease (IHD) is among the most important and top ranked causes of death in the world, and its preventive and interventional mechanisms are actively being investigated. Preconditioning may still be beneficial in some situations such as IHD. Development of cardioprotective agents to improve myocardial function, to decrease the incidence of arrhythmias, to delay the onset of necrosis, and to limit the total extent of infarction during IHD is of great clinical importance. In order to reduce morbidity, a new treatment modality must be developed, and oxytocin may indeed be one of the candidates. There is increasing experimental evidence indicating that oxytocin may have cardioprotective effects either by decreasing the extent of reperfusion injury or by pharmacologic preconditioning activity. This review shows that in the presence of oxytocin, the cardioprotective effects may be increased to some extent. The presented board of evidence focuses on the valuable effects of oxytocin on myocardial function and candidates it for future clinical studies in the realm of ischemic heart diseases.
Keywords: Oxytocin; Cardioprotection; Ischemic heart disease; Review;

Application of S-thanatin, an antimicrobial peptide derived from thanatin, in mouse model of Klebsiella pneumoniae infection by Guoqiu Wu; Pengpeng Wu; Xiulei Xue; Xuejiao Yan; Siru Liu; Chen Zhang; Zilong Shen; Tao Xi (73-77).
Thanatin was first discovered from the hemipteran insect Podisus maculiventris and showed a promising antimicrobial activity. Multidrug-resistant (MDR) clinical isolates of Klebsiella pneumoniae have developed resistance to current therapies. As an attempt to resolve this problem, the efficacy of thanatin and its analogues against clinical isolates of K. pneumoniae was studied in vitro and in vivo. S-thanatin showed an improved antimicrobial activity with the tested MIC values was 2–8-fold lower than those of other thanatin analogs. Antimicrobial assay indicated a high activity of S-thanatin against K. pneumoniae in vitro with MIC between 4 and 8 μg/ml. Its in vivo activity was evaluated using a K. pneumoniae-infected mice model. Adult male ICR mice were randomly grouped and given an intraperitoneal (i.p.) administration of 2 × 1010  colony-forming units of K. pneumoniae (CI 120204205). Afterwards, mouse groups were subjected to i.p. administration of saline or S-thanatin (5, 10, or 15 mg/kg). After an inspection of 72 h, the mice were finally sacrificed for analysis of in vivo bacterial growth and plasma endotoxin level. The results showed that S-thanatin administration apparently improved the survival rate and reduced the bacterial CFU from intra-abdominal fluid in mice. The plasma endotoxin level was improved as well. All above implied that S-thanatin, as an alternative, may provide a novel strategy for treating K. pneumoniae infection and other infections due to multidrug-resistant bacteria.
Keywords: S-thanatin; Antimicrobial peptide; Antimicrobial activity; Survival rates; Endotoxin level;

Antimicrobial activity of pleurocidin is retained in Plc-2, a C-terminal 12-amino acid fragment by Andre L.A. Souza; Paola Díaz-Dellavalle; Andrea Cabrera; Patricia Larrañaga; Marco Dalla-Rizza; Salvatore G. De-Simone (78-84).
An analysis of a series of five peptides composed of various portions of the pleurocidin (Plc) sequence identified a l2-amino acid fragment from the C-terminus of Plc, designated Plc-2, as the smallest fragment that retained a antimicrobial activity comparable to that of the parent compound. MIC tests in vitro with low-ionic-strength medium showed that Plc-2 has potent activity against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus but not against Enterococcus faecalis. The antifungal activity of the synthetic peptides against phytopathogenic fungi, such as Fusarium oxysporum, Colletotrichum sp., Aspergillus niger and Alternaria sp., also identified Plc-2 as a biologically active peptide. Microscopy studies of fluorescently stained fungi treated with Plc-2 demonstrated that cytoplasmic and nuclear membranes were compromised in all strains of phytopathogenic fungi tested. Together, these results identify Plc-2 as a potential antimicrobial agent with similar properties to its parent compound, pleurocidin. In addition, it demonstrated that the KHVGKAALTHYL residues are critical for the antimicrobial activity described for pleurocidin.
Keywords: Pleurocidin; Antimicrobial peptides; Antifungal activity; Cationic peptides; Pore-forming; Synthetic peptides; Small active sequence;

Comparative analysis of kisspeptin-immunoreactivity reveals genuine differences in the hypothalamic Kiss1 systems between rats and mice by Agnete Overgaard; Manuel Tena-Sempere; Isabelle Franceschini; Eloide Desroziers; Valerie Simonneaux; Jens D. Mikkelsen (85-90).
Kiss1 mRNA and its corresponding peptide products, kisspeptins, are expressed in two restricted brain areas of rodents, the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC). The concentration of mature kisspeptins may not directly correlate with Kiss1 mRNA levels, because mRNA translation and/or posttranslational modification, degradation, transportation and release of kisspeptins could be regulated independently of gene expression, and there may thus be differences in kisspeptin expression even in species with similar Kiss1 mRNA profiles. We measured and compared kisspeptin-immunoreactivity in both nuclei and both sexes of rats and mice and quantified kisspeptin-immunoreactive nerve fibers. We also determined Kiss1 mRNA levels and measured kisspeptin-immunoreactivity in colchicine pretreated rats. Overall, we find higher levels of kisspeptin-immunoreactivity in the mouse compared to the rat, independently of brain region and gender. In the female mouse AVPV high numbers of kisspeptin-immunoreactive neurons were present, while in the rat, the female AVPV displays a similar number of kisspeptin-immunoreactive neurons compared to the level of Kiss1 mRNA expressing cells, only after axonal transport inhibition. Interestingly, the density of kisspeptin innervation in the anterior periventricular area was higher in female compared to male in both species. Species differences in the ARC were evident, with the mouse ARC containing dense fibers, while the rat ARC contains clearly discernable cells. In addition, we show a marked sex difference in the ARC, with higher kisspeptin levels in females. These findings show that the translation of Kiss1 mRNA and/or the degradation/transportation/release of kisspeptins are different in mice and rats.
Keywords: Kisspeptin; Kiss1; AVPV; Arcuate nucleus; Kisspeptin antisera; Species differences;