Peptides (v.50, #C)

Regulation of corticotropin-releasing factor and urocortin 2/3 mRNA by leptin in hypothalamic N39 cells by Satoshi Yamagata; Kazunori Kageyama; Kanako Akimoto; Yutaka Watanuki; Toshihiro Suda; Makoto Daimon (1-7).
Corticotropin-releasing factor (CRF) activates the pituitary-adrenal axis during stress, and shows anorectic effects via CRF type 1 receptors in the hypothalamus. Both urocortin (Ucn) 2 and Ucn3 also act as anorectic neuropeptides via CRF type 2 receptors. Leptin, a product of the obesity gene secreted mainly from adipose tissue, reduces food intake and increases energy expenditure. A possible interaction between leptin and CRF/Ucns has been suggested, as leptin can regulate expression and activation of CRF and Ucns in the hypothalamus. This study aimed to explore the possible function of leptin in the hypothalamus, and its effects in regulating CRF and Ucns. The study identified mRNA expression of the leptin receptor (Ob-R) and its subtypes, CRF, and Ucn2/3 in mouse hypothalamic N39 cells. Leptin stimulated signal transducer and activators of transcription type 3 (STAT3) phosphorylation, directly increased the mRNA levels of both CRF and Ucn2/3 in hypothalamic cells, and increased Ob-Rb mRNA levels. A Janus kinase inhibitor inhibited the leptin-mediated increase in STAT3 phosphorylation, and then the increases in CRF and Ucn2/3 mRNA levels. Leptin may contribute to a stress response or anorectic effect via the regulation of CRF and Ucn2/3 in the hypothalamus.
Keywords: Leptin; Corticotropin-releasing factor; Urocortin; Hypothalamus; Stress;

Plasma visfatin, a possible prognostic marker in aneurysmal subarachnoid hemorrhage by Jun-Xing Wang; Yong Hou; Shan-Ping Ruan; Jie Wang; Xiao-Ming Hu (8-12).
Visfatin is linked to inflammation and associated with clinical outcomes of intracerebral hemorrhage. This study was designed to investigate whether visfatin might serve as a marker of severity and prognosis in aneurysmal subarachnoid hemorrhage. In this study, plasma visfatin levels of 172 consecutive patients and 172 sex and age-matched healthy subjects were determined using enzyme-linked immunosorbent assay. The recorded clinical outcomes included in-hospital mortality and 6-month mortality and unfavorable outcome (Glasgow Outcome Scale score of 1–3). Plasma visfatin level was substantially higher in patients than in healthy controls (92.1 ± 20.5 ng/mL vs. 12.4 ± 3.2 ng/mL; P  < 0.001), was significantly associated with the World Federation of Neurological Surgeons (WFNS) score (r  = 0.569, P  < 0.001) and Fisher score (r  = 0.657, P  < 0.001), was an independent predictor of in-hospital mortality [odds ratio (OR), 1.378; 95% confidence interval (CI), 1.036–1.866; P  = 0.002] and 6-month mortality (OR, 1.261; 95% CI, 1.018–1.745; P  = 0.004) and unfavorable outcome (OR, 1.207; 95% CI, 1.012–1.682; P  = 0.008) in multivariate logistic regression analysis and had high predictive value for in-hospital mortality [area under curve (AUC), 0.849; 95% CI, 0.787–0.899; P  < 0.001] and 6-month mortality (AUC, 0.868; 95% CI, 0.808–0.915; P  < 0.001) and unfavorable outcome (AUC, 0.859; 95% CI, 0.797–0.907; P  < 0.001) using receiver operating characteristic curves. AUCs of visfatin were similar to those of WFNS score and Fisher score (all P  > 0.05), but visfatin did not improve the predictive values of WFNS score and Fisher score (all P  > 0.05). Thus, visfatin may be associated with clinical severity of aneurysmal subarachnoid hemorrhage and also have prognostic value for clinical outcomes.
Keywords: Visfatin; Aneurysmal subarachnoid hemorrhage; Prognosis; Biomarker;

Alterations of irisin concentrations in saliva and serum of obese and normal-weight subjects, before and after 45 min of a Turkish bath or running by Suleyman Aydin; Suna Aydin; Tuncay Kuloglu; Musa Yilmaz; Mehmet Kalayci; İbrahim Sahin; Demet Cicek (13-18).
The purpose of this study was to ascertain (1) whether human saliva contains irisin and whether its level correlates with serum irisin concentration, (2) whether salivary glands, eccrine glands and sebaceous glands in human skin produce irisin, (3) how the changes in saliva and serum irisin concentrations after the Turkish bath at 47 ± 3 °C compare with the changes caused by moderate exercise in obese and normal weight subjects. Seven obese male subjects and seven normal weight subjects were enrolled for Turkish bath. Seven obese male subjects and seven normal weight subjects were also enrolled for moderate outdoor exercise, and thirteen male normal weight subjects neither exercised nor showered at the Turkish bath. From each participant, 1.5 ml of saliva and 5 ml blood were collected simultaneously before and after the moderate exercise and Turkish bath. Salivary glands and eccrine and sebaceous glands in the skin were screened immunohistochemically for irisin while serum and saliva irisin were measured with an ELISA. Submandibular glands, eccrine glands and sebaceous glands in the human skin showed strong irisin immunoreactivity. Human saliva contained irisin and its level was significantly higher than the serum levels in both obese and normal weight subjects. However, irisin concentrations were more markedly increased in both saliva and serum samples from subjects who had showered at a Turkish bath than in obese subjects who had exercised or in normal weight subjects. Human submandibular glands, eccrine sweat glands and sebaceous glands synthesize irisin.
Keywords: Irisin; Saliva; Serum; Obesity; Turkish bath; Exercise;

Antimicrobial activity of nisin against the swine pathogen Streptococcus suis and its synergistic interaction with antibiotics by Geneviève LeBel; Fanny Piché; Michel Frenette; Marcelo Gottschalk; Daniel Grenier (19-23).
Streptococcus suis serotype 2 is known to cause severe infections in pigs, including meningitis, endocarditis and pneumonia. Furthermore, this bacterium is considered an emerging zoonotic agent. Recently, increased antibiotic resistance in S. suis has been reported worldwide. The objective of this study was to evaluate the potential of nisin, a bacteriocin of the lantibiotic class, as an antibacterial agent against the pathogen S. suis serotype 2. In addition, the synergistic activity of nisin in combination with conventional antibiotics was assessed. Using a plate assay, the nisin-producing strain Lactococcus lactis ATCC 11454 proved to be capable of inhibiting the growth of S. suis (n  = 18) belonging to either sequence type (ST)1, ST25, or ST28. In a microdilution broth assay, the minimum inhibitory concentration (MIC) of purified nisin ranged between 1.25 and 5 μg/mL while the minimum bactericidal concentration (MBC) was between 5 and 10 μg/mL toward S. suis. The use of a capsule-deficient mutant of S. suis indicated that the presence of this polysaccharidic structure has no marked impact on susceptibility to nisin. Following treatment of S. suis with nisin, transmission electron microscopy observations revealed lysis of bacteria resulting from breakdown of the cell membrane. A time-killing curve showed a rapid bactericidal activity of nisin. Lastly, synergistic effects of nisin were observed in combination with several antibiotics, including penicillin, amoxicillin, tetracycline, streptomycin and ceftiofur. This study brought clear evidence supporting the potential of nisin for the prevention and treatment of S. suis infections in pigs.
Keywords: Streptococcus suis; Swine infection; Nisin; Antibiotic; Synergy;

Hematopoietic stem cell expansion caused by a synthetic fragment of leptin by Carol C. Dias; Amanda Nogueira-Pedro; Christiano M.V. Barbosa; Antonio C. Ribeiro-Filho; Frederick Wasinski; Ronaldo C. Araújo; Vani Xavier de Oliveira Jr; Antonio Miranda; Edgar J. Paredes-Gamero (24-27).
Leptin is a cytokine that regulates food intake, energy expenditure and hematopoiesis. Based on the tridimensional structure of the human leptin molecule, six fragments have been synthesized, (Ac-Lep23–47-NH2, [LEP1]; Ac-Lep48–71-NH2, [LEP2]; Ac-Lep72–88-NH2, [LEP3]; Ac-Lep92–115-NH2, [LEP4], Ac-[Ser117]-Lep116–140-NH2, [LEP5] and Ac-Lep141–164-NH2, [LEP6]), and their effects on hematopoiesis were evaluated. The mice were treated with 1 mg/kg LEP5 for 3 days. The mature and primitive hematopoietic populations were quantified. We observed that the mature populations from the bone marrow and spleen were not affected by LEP5. However, the peptide caused at least a two-fold increase in the number of hematopoietic stem cells, the most primitive population of the bone marrow. Additionally, the number of granulocyte/macrophage colony-forming units produced by bone marrow cells in methylcellulose also increased by 40% after treatment with LEP5, and the leptin receptor was activated. These results show that the leptin fragment LEP5 is a positive modulator of the in vivo expansion of hematopoietic stem cells.

Cationic lytic-type peptides have been studied for clinical application in various infections and cancers. This study aimed to determine the functions of our specially designed lytic peptide. To investigate the functional mechanism at the cell membrane level, we used giant unilayer vesicles (GUVs) mimicking cell membranes. In GUVs treated with FITC-labeled lytic peptide (lytic-FITC), fluorescence increased in a time-dependent manner. However, no inner fluorescence was detected in GUVs treated with lytic peptide and calcein. Next, distribution of lytic-FITC peptide on the cell membrane and in the cytoplasm was examined in a living human glioma U251 cell line. In the immunocytochemical study, some lytic peptide stains colocalized with early endosome antigen protein 1 (EEA-1). In cells treated with lytic peptide, the immunofluorescence intensity of lytic peptide increased in a concentration and treatment time-dependent manner. Cytotoxic activity of lytic peptide decreased after pretreatment with the endocytosis inhibitors cytochalasin D, chlorpromazine and amiloride. These findings suggest that lytic peptide exerts cytotoxic activity after cellular uptake via an endocytosis pathway. In conclusion, the influx mechanism of lytic peptide was shown to include not only disintegration and pore formation at the cell membrane, but also cell entry via endocytosis dependent and independent pathways.
Keywords: Cationic peptide; Lytic-type peptide; Anti-cancer peptide; Endocytosis;

Oxytocin receptor blockade reduces acquisition but not retrieval of taste aversion and blunts responsiveness of amygdala neurons to an aversive stimulus by Pawel K. Olszewski; Joseph R. Waas; Lydia L. Brooks; Florence Herisson; Allen S. Levine (36-41).
When gastrointestinal sickness induced by toxin injection is associated with exposure to novel food, the animal acquires a conditioned taste aversion (CTA). Malaise is accompanied by a surge in oxytocin release and in oxytocin neuronal activity; however, it is unclear whether oxytocin is a key facilitator of aversion or merely its marker. Herein we investigated whether blockade of the oxytocin receptor with the blood–brain barrier penetrant oxytocin receptor antagonist L-368,899 is detrimental for the acquisition and/or retrieval of lithium chloride (LiCl)-dependent CTA to a saccharin solution in mice. We also examined whether L-368,899 given prior to LiCl affects neuronal activity defined through c-Fos immunohistochemistry in select brain sites facilitating CTA acquisition. L-368,899 given prior to LiCl caused a 30% increase in saccharin solution intake in a two-bottle test, but when the antagonist was administered before the two-bottle test, it failed to diminish the retrieval of an existing CTA. LiCl administration increased c-Fos expression in the hypothalamic paraventricular and supraoptic nuclei, area postrema, nucleus of the solitary tract and basolateral and central (CNA) nuclei of the amygdala. L-368,899 injected before LiCl reduced the number of c-Fos positive CNA neurons and brought it down to levels similar to those observed in mice treated only with L-368,899. We conclude that oxytocin is one of the key components in acquisition of LiCl-induced CTA and the aversive response can be alleviated by the oxytocin receptor blockade. Oxytocin receptor antagonism blunts responsiveness of CNA to peripherally injected LiCl.
Keywords: Anorexia; Avoidance; Food intake;

Attenuation of systemic morphine-induced analgesia by central administration of ghrelin and related peptides in mice by Ping Zeng; Jia-xiang Chen; Bei Yang; Xing Zhi; Fa-xian Guo; Meng-li Sun; Jing-lei Wang; Jie Wei (42-49).
Ghrelin, an acylated 28-amino peptide secreted in the gastric endocrine cells, has been demonstrated to stimulate the release of growth hormone, increase food intake, and inhibit pro-inflammatory cascade, etc. Ghrelin mainly combines with its receptor (GHS-R1α) to play the role in physiological and pathological functions. It has been reported that ghrelin plays important roles in the control of pain through interaction with the opioid system in inflammatory pain and acute pain. However, very few studies show the effect of supraspinal ghrelin system on antinociception induced by intraperitoneal (i.p.) administration of morphine. In the present study, intracerebroventricular (i.c.v.) injection of ghrelin (0.1, 1, 10 and 100 nmol/L) produced inhibition of systemic morphine (6 mg/kg, i.p.) analgesia in the tail withdrawal test. Similarly, i.c.v. injection GHRP-6 and GHRP-2 which are the agonists of GHS-R1α, also decreased analgesia effect induced by morphine injected intraperitoneally in mice. Furthermore, these anti-opioid activities of ghrelin and related peptides were not blocked by pretreatment with the GHS-R1α selective antagonist [d-Lys3]-GHRP-6 (100 nmol/L, i.c.v.). These results demonstrated that central ghrelin and related peptides could inhibit the analgesia effect induced by intraperitoneal (i.p.) administration of morphine. The anti-opioid effects of ghrelin and related peptides do not interact with GHS-R1a. These findings may pave the way for a new strategy on investigating the interaction between ghrelin system and opioids on pain modulation.
Keywords: Ghrelin; GHRP-2; GHRP-6; [d-Lys3]-GHRP-6; Morphine; Tail withdrawal test;

C-type natriuretic peptide plasma levels are reduced in obese adolescents by S. Del Ry; M. Cabiati; V. Bianchi; S. Storti; C. Caselli; T. Prescimone; A. Clerico; G. Saggese; D. Giannessi; G. Federico (50-54).
The high prevalence of obesity in children may increase the magnitude of lifetime risk of cardiovascular disease (CD). At present, explicit data for recommending biomarkers as routine pre-clinical markers of CD in children are lacking. C-type natriuretic peptide (CNP) is assuming increasing importance in CD; in adults with heart failure, its plasma levels are related to clinical and functional disease severity. We have previously reported five different reference intervals for blood CNP as a function of age in healthy children; however, data on plasma CNP levels in obese children are still lacking. Aim of this study was to assess CNP levels in obese adolescents and verify whether they differ from healthy subjects. Plasma CNP was measured in 29 obese adolescents (age: 11.8 ± 0.4 years; BMI: 29.8 ± 0.82) by radioimmunoassay and compared with the reference values of healthy subjects. BNP was also measured. Both plasma CNP and BNP levels were significantly lower in the obese adolescents compared to the appropriate reference values (CNP: 3.4 ± 0.2 vs 13.6 ± 2.3 pg/ml, p  < 0.0001; BNP: 18.8 ± 2.6 vs 36.9 ± 5.5 pg/ml, p  = 0.003). There was no significant difference between CNP values in males and females. As reported in adults, we observed lower plasma CNP and BNP levels in obese children, suggesting a defective natriuretic peptide system in these patients. An altered regulation of production, clearance and function of natriuretic peptides, already operating in obese adolescents, may possibly contribute to the future development of CD. Thus, the availability of drugs promoting the action of natriuretic peptides may represent an attractive therapeutic option to prevent CD.
Keywords: C-type natriuretic peptide; Obesity; Adolescents; Natriuretic peptides;

Endogenous opiates and behavior: 2012 by Richard J. Bodnar (55-95).
This paper is the thirty-fifth consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2012 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); and immunological responses (Section 17).
Keywords: Opiate receptors; Beta-endorphin; Enkephalins; Dynorphin, Orphanin FQ/nociceptin; Endomorphins;

The median raphe nucleus participates in the depressive-like behavior induced by MCH: Differences with the dorsal raphe nucleus by Ximena López Hill; Claudia Pascovich; Jessika Urbanavicius; Pablo Torterolo; María Cecilia Scorza (96-99).
An emerging body of evidence involves the hypothalamic neuropeptide melanin-concentrating hormone (MCH) in the regulation of emotional states. We have reported a pro-depressive effect induced by MCH after its microinjection into the dorsal raphe nucleus (DR) evaluated in the forced swimming test (FST) in rats. Here we extended this study to the median raphe nucleus (MnR). Firstly, the presence of MCH-containing fibers in the rat MnR was analyzed by means of immunohistochemistry. Secondly, the behavioral effect induced by the microinjection of MCH into the MnR was assessed using the FST. Morphological results showed a large density of MCHergic fibers within the MnR. Behavioral results indicated that 100 ng of MCH (but not 50 ng) significantly increased the immobility time and decreased the swimming time, demonstrating a depressive-like effect. In contrast, climbing behavior was not significantly affected. Present findings revealed that the MnR neurons participate in the MCHergic control of affective-related behavioral responses. However, the behavioral patterns induced by MCH in the MnR and DR were different. This could be explained by anatomical and physiological differences between both nuclei.
Keywords: Neuropeptide; Mood disorders; Hypothalamus; Depression; Raphe nuclei;

Effect of atrial natriuretic peptide on reactive oxygen species-induced by hydrogen peroxide in THP-1 monocytes: Role in cell growth, migration and cytokine release by Paolo De Vito; Sandra Incerpi; Elisabetta Affabris; Zulema Percario; Monica Borgatti; Roberto Gambari; Jens Z. Pedersen; Paolo Luly (100-108).
Atrial natriuretic peptide (ANP), a cardiovascular hormone, elicits different biological actions in the immune system. The aim of the present study was to investigate in THP-1 monocytes the ANP effect on hydrogen peroxide (H2O2)-induced Reactive Oxygen Species (ROS), cell proliferation and migration. A significant increase of H2O2-dependent ROS production was induced by physiological concentration of ANP (10−10  M). The ANP action was partially affected by cell pretreatment with PD98059, an inhibitor of mitogen activated-protein kinases (MAPK) as well as by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K) and totally suppressed by diphenylene iodonium (DPI), an inhibitor of the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The hormone effect was mimicked by cANF and an ANP/NPR-C signaling pathway was studied using pertussis toxin (PTX). A significant increase of H2O2-induced cell migration was observed after ANP (10−10  M) treatment, conversely a decrease of THP-1 proliferation, due to cell death, was found. Both ANP actions were partially prevented by DPI. Moreover, H2O2-induced release of IL-9, TNF-α, MIP-1α and MIP-1β was not counteracted by DPI, whereas no effect was observed in any experimental condition for both IL-6 and IL-1β. Our results support the view that ANP can play a key role during the inflammatory process.
Keywords: Atrial natriuretic peptide; NPR-C; NADPH oxidase; Reactive oxygen species; Cytokines;

Melanin-concentrating hormone (MCH) is classically known for its role in regulating teleost fish skin color change for environmental adaptation. Recent evidence suggests that MCH also has appetite-stimulating properties. The gonadotropin-releasing hormone (GnRH) peptide family has dual roles in endocrine control of reproduction and energy status in fish. Atlantic cod (Gadus morhua) are a commercially important aquaculture species inhabiting the shores of Atlantic Canada. In this study, we examine MCH and GnRH transcript expression profiles during early development as well as in central and peripheral tissues and quantify juvenile Atlantic cod MCH and GnRH hypothalamic mRNA expressions following food deprivation. MCH and GnRH3 cDNAs are maternally deposited into cod eggs, while MCH has variable expression throughout early development. GnRH2 and GnRH3 mRNAs “turn-on” during mid-segmentation once the brain is fully developed. For both MCH and GnRH, highest expression appears during the exogenous feeding stages, perhaps supporting their functions as appetite regulators during early development. MCH and GnRH transcripts are found in brain regions related to appetite regulation (telencephalon/preoptic area, optic tectum/thalamus, hypothalamus), as well as the pituitary gland and the stomach, suggesting a peripheral function in food intake regulation. Atlantic cod MCH mRNA is upregulated during fasting, while GnRH2 and GnRH3 transcripts do not appear to be influenced by food deprivation. In conclusion, MCH might be involved in stimulating food intake in juvenile Atlantic cod, while GnRHs may play a more significant role in appetite regulation during early development.
Keywords: Atlantic cod (Gadus morhua); Melanin-concentrating hormone (MCH); Gonadotropin-releasing hormone (GnRH); Tissue distribution; Early ontogeny; Fasting;

The vascular inflammatory role of platelet activating factor acetylhydrolase (PAF-AH) is thought to be due to the formation of lysophosphatidyl choline and oxidized non-esterified fatty acids. This enzyme is considered a promising therapeutic target for the prevention of atherosclerosis and there is a need to expand the available chemical templates of PAF-AH inhibitors. This study demonstrated how natural PAF-AH inhibitory peptides were isolated and characterized from the red macroalga Palmaria palmata. The dried powdered alga was hydrolyzed using the food grade enzyme papain, and the resultant peptide containing fraction generated using RP-HPLC. Several oligopeptides were identified as potential PAF-AH inhibitors following bio-guided fractionation, and the amino acid sequences of these oligopeptides were confirmed by Q-TOF-MS and microwave-assisted solid phase de novo synthesis. The most promising PAF-AH inhibitory peptide had the amino acid sequence NIGK and a PAF-AH IC50 value of 2.32 mM. This peptide may constitute a valid drug template for PAF-AH inhibitors. Furthermore the P. palmata hydrolysate was nontoxic when assayed using the Zebrafish toxicity model at a concentration of 1 mg/ml.
Keywords: Atherosclerosis; Lp-PLA2; Enzyme inhibition; Darapladib; Platelet-activating factor acetylhydrolase; Bioactive peptides; Palmaria palmata;

Value of proBNP1–108 testing for the risk stratification of patients with systolic heart failure by Damien Gruson; Thibault Lepoutre; Sylvie A. Ahn; Michel F. Rousseau (125-128).
The study objectives were to determine the circulating levels of proBNP1–108, the precursor of B-type natriuretic peptide (BNP) and amino-terminal pro-BNP (NT-proBNP), in patients with systolic heart failure (HF) and to assess their prognosis value for cardiovascular (CV) death over a long-term follow-up. Seventy-three patients with systolic HF and 68 healthy volunteers were included. ProBNP1–108, BNP and NT-proBNP levels were measured with automated immunoassays and their predictive value for long-term survival was assessed through an 8 years follow-up. ProBNP1–108 levels were markedly increased in patients with systolic HF in comparison to healthy volunteers. In univariate proportional hazard model, survival was related to proBNP1–108, BNP, NT-proBNP, age, EF and glomerular filtration rate (eGFR). Kaplan–Meier survival curves according to proBNP tertiles diverged significantly, and the highest proBNP levels were related to patients with the highest risk of CV death. In a multivariate analysis including age, EF, proBNP1–108, BNP, NT-proBNP, and eGFR levels, NT-proBNP was the strongest predictor of long term CV death. Our study therefore demonstrated that high levels of proBNP1–108, measured with an assay with enhanced analytical specificity, are related to the long-term risk of cardiovascular death in systolic heart failure.
Keywords: ProBNP1–108; BNP; NT-proBNP; Heart failure; Prognosis;

Differential regulation of human cathelicidin LL-37 by free fatty acids and their analogs by Weiyu Jiang; Lakshmi T. Sunkara; Xiangfang Zeng; Zhuo Deng; Sarah M. Myers; Guolong Zhang (129-138).
LL-37 is the single cathelicidin host defense peptide in humans with direct antimicrobial and immunomodulatory activities. Specific regulation of LL-37 synthesis has emerged as a novel non-antibiotic approach to disease control and prevention. Short-chain fatty acids, and butyrate in particular, were found recently to be strong inducers of LL-37 gene expression without causing inflammation. Here, we further evaluated the LL-37-inducing efficiency of a broad range of saturated free fatty acids and their derivatives in human HT-29 colonic epithelial cells and U-937 monocytic cells by real-time RT-PCR. Surprisingly, we revealed that valerate, hexanoate, and heptanoate with 5–7 carbons are more potent than 4-carbon butyrate in promoting LL-37 gene expression in both cell types. Free fatty acids with longer than 7 or shorter than 4 carbons showed only a marginal effect on LL-37 expression. Studies with a series of fatty acid derivatives with modifications in the aliphatic chain or carboxylic acid group yielded several analogs such as benzyl butyrate, trans-cinnamyl butyrate, glyceryl tributyrate, and phenethyl butyrate with a comparable LL-37-inducing activity to sodium butyrate. On the other hand, although reactive, the anhydride derivatives of short- and medium-chain fatty acids are as potent as their corresponding free acid forms in LL-37 induction. Thus, these newly identified free fatty acids and their analogs with a strong capacity to augment LL-37 synthesis may hold promise as immune boosting dietary supplements for antimicrobial therapy.
Keywords: LL-37; Host defense peptides; Cathelicidins; Short-chain fatty acids; Butyrate; Hexanoate; Heptanoate;

Involvement of spinal release of α-neo-endorphin on the antinociceptive effect of TAPA by Hirokazu Mizoguchi; Takashi Kon-no; Hiroyuki Watanabe; Chizuko Watanabe; Akihiko Yonezawa; Takumi Sato; Tsukasa Sakurada; Shinobu Sakurada (139-144).
The antinociceptive effect of i.t.-administered Tyr-d-Arg-Phe-β-Ala (TAPA), an N-terminal tetrapeptide analog of dermorphin, was characterized in ddY mice. In the mouse tail-flick test, TAPA administered i.t. produced a potent antinociception. The antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with the κ-opioid receptor antagonist nor-binaltorphimine, as well as by the μ-opioid receptor antagonist β-funaltrexamine and the μ1-opioid receptor antagonist naloxonazine. TAPA-induced antinociception was also significantly suppressed by co-administration of the μ1-opioid receptor antagonist Tyr-d-Pro-Phe-Phe-NH2 (d-Pro2-endomorphin-2) but not by co-administration of the μ2-opioid receptor antagonists Tyr-d-Pro-Trp-Phe-NH2 (d-Pro2-endomorphin-1) and Tyr-d-Pro-Trp-Gly-NH2 (d-Pro2-Tyr-W-MIF-1). In CXBK mice whose μ1-opioid receptors were naturally reduced, the antinociceptive effect of TAPA was markedly suppressed compared to the parental strain C57BL/6ByJ mice. Moreover, the antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with antiserum against the endogenous κ-opioid peptide α-neo-endorphin but not antisera against other endogenous opioid peptides. In prodynorphin-deficient mice, the antinociceptive effect of TAPA was significantly reduced compared to wild-type mice. These results suggest that the spinal antinociception induced by TAPA is mediated in part through the release of α-neo-endorphin in the spinal cord via activation of spinal μ1-opioid receptors.
Keywords: TAPA; Antinociception; μ-Opioid receptor; α-Neo-endorphin; Release; Spinal cord;

Modulatory effects of NDP-MSH in the regenerating liver after partial hepatectomy in rats by Caterina Lonati; Andrea Carlin; Patrizia Leonardi; Franco Valenza; Silvano Bosari; Anna Catania; Stefano Gatti (145-152).
Melanocortins are endogenous peptides that exert protective actions on the host physiology. The broad modulatory effects of these molecules suggest that they might influence the mediator network induced during liver regeneration. The research aim was to determine if melanocortin treatment alters liver molecular changes induced by partial hepatectomy (PH). Rats under isoflurane anesthesia were subjected to standard 70% PH or sham surgery. Animals received a single i.v. injection of Nle4,DPhe7-α-melanocyte stimulating hormone (NDP-MSH) or saline 30 min before surgery. Sacrifice was performed at time intervals between 4 and 72 h. A preliminary screening based on TaqMan low-density array (TLDA) identified 71 transcripts altered by PH. Real-time PCR analysis revealed that NDP-MSH modulated the expression of a substantial proportion of these transcripts including several chemokines and their receptors. The critical signaling pathway interleukin-6 (IL-6)/signal transducer and activator of transcription (STAT)/suppressor of cytokine signaling (SOCS) was significantly enhanced by NDP-MSH. Further, peptide treatment considerably reduced the decline of IκBα protein caused by PH. Although the final organ regeneration was not substantially affected, NDP-MSH modulated expression of cell cycle mediators and exerted subtle influences on hepatocyte replication. Most of the changes brought about by NDP-MSH, a peptide approved for clinical use, should be salutary during liver regeneration.
Keywords: Melanocortin peptides; 70% partial hepatectomy (PH); Liver regeneration; Interleukin 6 (IL-6); Suppressor of cytokine signaling (SOCS);

An analog of the host-defense peptide hymenochirin-1B with potent broad-spectrum activity against multidrug-resistant bacteria and immunomodulatory properties by Milena Mechkarska; Manju Prajeep; Gordana D. Radosavljevic; Ivan P. Jovanovic; Amna Al Baloushi; Agnes Sonnevend; Miodrag L. Lukic; J. Michael Conlon (153-159).
Hymenochirin-1B (IKLSPETKDN10LKKVLKGAIK20GAIAVAKMV.NH2) is a cationic, amphipathic, α-helical, host-defense peptide, first isolated from skin secretions of the Congo clawed frog Hymenochirus boettgeri (Pipidae). Structure-activity relationships were investigated by synthesizing analogs in which the Pro5, Glu6 and Asp9 on the hydrophilic face of the α-helix are substituted by one or more l-lysine or d-lysine residues. Although replacement with l-lysine generates analogs with increased antimicrobial potency against a range of Gram-positive and Gram-negative bacteria (up to 8-fold), the peptides are more hemolytic. Increasing the cationicity of hymenochirin-1B while reducing the helicity by substitutions with d-lysine generates analogs that are between 2 and 8 fold more potent than the native peptide and are equally or less hemolytic. [E6k,D9k]hymenochirin-1B represents a candidate for drug development as it shows high potency against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and a range of Gram-negative bacteria, including multidrug-resistant strains of Acinetobacter baumannii and Stenotrophomonas maltophilia (MIC in the range 0.8–3.1 μM) and NDM-1 carbapenemase-producing clinical isolates of Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Citrobacter freundii (MIC in the range 3.1–6.25 μM), and low hemolytic activity (LC50  = 302 μM). [E6k,D9k]hymenochirin-1B, at a concentration of 2.5 μM, significantly (P  < 0.05) stimulates the production of the anti-inflammatory cytokines IL-4 and IL-10 by human peripheral blood mononuclear cells but is without significant effect on production of the pro-inflammatory cytokines TNF-α and IL-17.
Keywords: Antimicrobial peptide; Hymenochirin-1B; Structure-activity; MRSA; Carbapenem-resistant Enterobacteriaceae; Cytokine;