Peptides (v.88, #C)

Cardiovascular effects of exogenous adrenomedullin and CGRP in Ramp and Calcrl deficient mice by J.B. Pawlak; S.E. Wetzel-Strong; M.K. Dunn; K.M. Caron (1-7).
Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) are potent vasodilator peptides and serve as ligands for the G-protein coupled receptor (GPCR) calcitonin receptor-like receptor (CLR/Calcrl). Three GPCR accessory proteins called receptor activity-modifying proteins (RAMPs) modify the ligand binding affinity of the receptor such that the CLR/RAMP1 heterodimer preferably binds CGRP, while CLR/RAMP2 and CLR/RAMP3 have a stronger affinity for AM. Here we determine the contribution of each of the three RAMPs to blood pressure control in response to exogenous AM and CGRP by measuring the blood pressure of mice with genetic reduction or deletion of the receptor components. Thus, the cardiovascular response of Ramp1−/− , Ramp2 +/−, Ramp3 −/−, Ramp1 −/−/Ramp3 −/− double-knockout (dKO), and Calcrl +/− mice to AM and CGRP were compared to wildtype mice. While under anesthesia, Ramp1−/− male mice had significantly higher basal blood pressure than wildtype males; a difference which was not present in female mice. Additionally, anesthetized Ramp1 −/−, Ramp3 −/−, and Calcrl +/− male mice exhibited significantly higher basal blood pressure than females of the same genotype. The hypotensive response to intravenously injected AM was greatly attenuated in Ramp1 −/− mice, and to a lesser extent in Ramp3 −/− and Calcrl +/− mice. However, Ramp1 −/−/Ramp3 −/− dKO mice retained some hypotensive response to AM. These results suggest that the hypotensive effect of AM is primarily mediated through the CLR/RAMP1 heterodimer, but that AM signaling via CLR/RAMP2 and CLR/RAMP3 also contributes to some hypotensive action. On the other hand, CGRP’s hypotensive activity seems to be predominantly through the CLR/RAMP1 heterodimer. With this knowledge, therapeutic AM or CGRP peptides could be designed to cause less hypotension while maintaining canonical receptor-RAMP mediated signaling.
Keywords: Adrenomedullin; CGRP; RAMP; Calcrl; Hypotension; Blood pressure;

Recombinant Lampetra japonica RGD peptide (rLj-RGD3) is a soluble toxin protein with three RGD (Arg-Gly-Asp) motifs and a molecular weight of 13.5 kDa. The aim of this study was to investigate the effects and mechanisms of rLj-RGD3 on tumor growth and survival in pancreatic carcinoma Panc-1 cell-bearing mice. A Panc-1 human pancreatic carcinoma-bearing nude mouse model was successfully generated, and the animals were treated with different doses of rLj-RGD3 for 3 weeks. The volume and weight of the subcutaneous tumors, the survival of the nude mice, histopathological changes, the intratumoral MVD, the number of apoptotic Panc-1 cells, and apoptosis-related proteins and gene expressions were determined. rLj-RGD3 significantly decreased the tumor volumes and weights, and the maximum tumor volume and weight IR values were 53.2% (p < 0.001) and 55.9% (p < 0.001), respectively. The life expectancy of Panc-1-bearing nude mice treated with rLj-RGD3 was increased by 56.3% (p < 0.001). Meanwhile, rLj-RGD3 promoted the expression of Bax, caspase-3, and caspase-9 and inhibited Bcl-2 and VEGF expression. In addition, rLj-RGD3 did not change FAK, PI3K and Akt expression, but p-FAK, p-PI3K and p-Akt, levels were down-regulated. These results show that rLj-RGD3 induced potent anti-tumor activity in vivo and suppressed the growth of transplanted Panc-1 cells in a nude mouse model, implying that rLj-RGD3 may serve as a potent clinical therapeutic agent for human pancreatic carcinoma.
Keywords: rLj-RGD3; Pancreatic carcinoma; Panc-1 cells; Anti-tumor; Mice;

In vivo digestomics of milk proteins in human milk and infant formula using a suckling rat pup model by Yasuaki Wada; Brett S. Phinney; Darren Weber; Bo Lönnerdal (18-31).
Human milk is the optimal mode of infant feeding for the first several months of life, and infant formulas serve as an alternative when breast-feeding is not possible. Milk proteins have a balanced amino acid composition and some of them provide beneficial bioactivities in their intact forms. They also encrypt a variety of bioactive peptides, possibly contributing to infant health and growth. However, there is limited knowledge of how milk proteins are digested in the gastrointestinal tract and bioactive peptides are released in infants. A peptidomic analysis was conducted to identify peptides released from milk proteins in human milk and infant formula, using a suckling rat pup model. Among the major milk proteins targeted, α-lactalbumin and β-casein in human milk, and β-lactoglobulin and β-casein in infant formula were the main sources of peptides, and these peptides covered large parts of the parental proteins’ sequences. Release of peptides was concentrated to specific regions, such as residues 70–92 of β-casein in human milk, residues 39–55 of β-lactoglobulin in infant formula, and residues 57–96 and 145–161 of β-CN in infant formula, where resistance to gastrointestinal digestion was suggested. In the context of bioactive peptides, release of fragments containing known bioactive peptides was confirmed, such as β-CN-derived opioid and antihypertensive peptides. It is therefore likely that these fragments are of biological significance in neonatal health and development.
Keywords: Human milk; Infant formula; Milk proteins; Bioactive peptides; Suckling rat pups;

Phoenixin is negatively associated with anxiety in obese men by Tobias Hofmann; Elena Weibert; Anne Ahnis; Ulf Elbelt; Matthias Rose; Burghard F. Klapp; Andreas Stengel (32-36).
Phoenixin was recently identified in the rat hypothalamus and initially implicated in reproductive functions. A subsequent study described an anxiolytic effect of the peptide. The aim of the study was to investigate a possible association of circulating phoenixin with anxiety in humans. We therefore enrolled 68 inpatients with a broad spectrum of psychometrically measured anxiety (GAD-7). We investigated men since a menstrual cycle dependency of phoenixin has been assumed. Obese subjects were enrolled since they often report psychological comorbidities. In addition, we also assessed depressiveness (PHQ-9) and perceived stress (PSQ-20). Plasma phoenixin levels were measured using a commercial ELISA. First, we validated the ELISA kit performing a spike-and-recovery experiment showing a variance of 6.7 ± 8.8% compared to the expected concentrations over the whole range of concentrations assessed, while a lower variation of 1.6 ± 0.8% was observed in the linear range of the assay (0.07–2.1 ng/ml). We detected phoenixin in the circulation of obese men at levels of 0.68 ± 0.50 ng/ml. These levels showed a negative association with anxiety scores (r  = −0.259, p  = 0.043), while no additional associations with other psychometric parameters were observed. In summary, phoenixin is present in the human circulation and negatively associated with anxiety in obese men, a population often to report comorbid anxiety.
Keywords: Emotion; Gut-brain axis; Psychobiology; Psychoneuroendocrine; Psychosomatic;

Chronic blockade of the AT2 receptor with PD123319 impairs insulin signaling in C57BL/6 mice by M.C. Muñoz; V. Burghi; J.G. Miquet; I.A. Cervino; D.T. Quiroga; L. Mazziotta; F.P. Dominici (37-45).
The renin-angiotensin system modulates insulin action. Angiotensin type 1 receptor exerts a deleterious effects while the angiotensin type 2 receptor (AT2R) appears to have beneficial effects providing protection against insulin resistance and type 2 diabetes. Although recent reports indicate that agonism of AT2R ameliorates diabetes and insulin resistance, the phenotype of AT2R-knockout mice seems to be controversial relating this aspect. Thus, in this study we have explored the role of AT2R in the control of insulin action. To that end, C57Bl/6 mice were administered the synthetic AT2R antagonist PD123319 for 21 days (10 mg/kg/day ip); vehicle treated animals were used as control. Glucose tolerance, metabolic parameters, in vivo insulin signaling in main insulin-target tissues as well as levels of adiponectin, TNF-α, MCP-1 and IL-6 in adipose tissue were assessed. AT2R blockade with PD123319 induced a marginal effect on glucose homeostasis but an important reduction in the insulin-induced phosphorylation of the insulin receptor and Akt in both liver and adipose tissue. Insulin signaling in skeletal muscle remained unaltered after treatment with PD123319, which could explain the minimal effect on glucose homeostasis induced by PD123319. Our current results reinforce the notion that the AT2R has a physiological role in the conservation of insulin action.
Keywords: Angiotensin II; Angiotensin type 2 receptor; Glucose tolerance; Insulin; Renin-angiotensin system;

A novel GLP-1 analog, a dimer of GLP-1 via covalent linkage by a lysine, prolongs the action of GLP-1 in the treatment of type 2 diabetes by Yingying Pan; Siwei Shi; Xun Lao; Jinlong Zhang; Shiming Tan; Zirong Wu; Jing Huang (46-54).
GLP-1 is an incretin hormone that can effectively lower blood glucose, however, the short time of biological activity and the side effect limit its therapeutic application. Many methods have been tried to optimize GLP-1 to extend its in vivo half-time, reduce its side effect and enhance its activity. Here we have chosen the idea to dimerize GLP-1 with a C-terminal lysine to form a new GLP-1 analog, DLG3312. We have explored the structure and the biological property of DLG3312, and the results indicated that DLG3312 not only remained the ability to activate the GLP-1R, but also strongly stimulated Min6 cell to secrete insulin. The in vivo bioactivities have been tested on two kinds of animal models, the STZ induced T2DM mice and the db/db mice, respectively. DLG3312 showed potent anti-diabetic ability in glucose tolerance assay and single-dose administration of DLG3312 could lower blood glucose for at least 10 hours. Long-term treatment with DLG3312 can reduce fasted blood glucose, decrease water consumption and food intake and significantly reduce the HbA1c level by 1.80% and 2.37% on STZ induced T2DM mice and the db/db mice, respectively. We also compared DLG3312 with liraglutide to investigate its integrated control of the type 2 diabetes. The results indicated that DLG3312 almost has the same effect as liraglutide but with a much simpler preparation process. In conclusion, we, by using C-terminal lysine as a linker, have synthesized a novel GLP-1 analog, DLG3312. With simplified preparation and improved physiological characterizations, DLG3312 could be considered as a promising candidate for the type 2 diabetes therapy.
Keywords: GLP-1 analog; Dimerization; Type 2 diabetes; Animal experiment; Long-term treatment;

Increased plasma orexin-A levels in patients with insomnia disorder are not associated with prepro-orexin or orexin receptor gene polymorphisms by Shi Tang; Weiwei Huang; Shanshan Lu; Lili Lu; Guohua Li; Xu Chen; Xiaomin Liu; Xin Lv; Zhangning Zhao; Ruisheng Duan; Yifeng Du; Jiyou Tang (55-61).
Orexins, also known as hypocretins, play a regulatory role in the sleep-wake cycle by activating orexin receptors. Previous animal studies have shown that sleep deprivation can elevate orexinergic peptide levels. However, the relationship between insomnia disorder and orexin-A levels in humans has not been explored. In the current study, we examined plasma orexin-A levels in patients with insomnia disorder and in normal sleepers. We also studied the possible mechanisms underlying changes in orexin-A levels between the study groups; this included investigations of prepro-orexin and orexin receptor gene polymorphisms as well as exploration of other variables. We measured plasma orexin-A levels in 228 patients with insomnia disorder and 282 normal sleepers. The results indicated that the patients with insomnia disorder had significantly higher orexin-A levels than normal sleepers (63.42 ± 37.56 vs. 54.84 ± 23.95 pg/ml). A positive relationship was detected between orexin-A level and age in patients with insomnia disorder. Orexin-A levels were elevated in relation to course of insomnia, as well as in relation to increased Insomnia Severity Index score. None of the evaluated prepro-orexin gene single nucleotide polymorphisms were informative between the two study populations. After sequencing all orexin receptor exons, one variation (rs2271933) in the OX1R gene and one variation (rs2653349) in the OX2R gene were found. However, no significant differences were found in either genotypic or allelic frequency distributions between the two study groups. It is suggested that the increased plasma orexin-A levels in patients with insomnia disorder are associated with the course and severity of insomnia, but not with prepro-orexin and orexin receptor gene polymorphisms.
Keywords: Insomnia; Orexin-A; Prepro-orexin; Orexin receptors; Gene variations;

Increased FNDC5/Irisin expression in human hepatocellular carcinoma by Melania Gaggini; Manuela Cabiati; Serena Del Turco; Teresa Navarra; Paolo De Simone; Franco Filipponi; Silvia Del Ry; Amalia Gastaldelli; Giuseppina Basta (62-66).
The fibronectin type III domain containing 5 (FNDC5)/Irisin, a novel energy-regulating hormone, is associated with lipid and carbohydrate metabolism. It is produced in low amounts by normal hepatic tissue, while in human hepatocellular carcinoma (HCC), in which aberrant de novo lipogenesis (DNL) occurs, the hepatic expression of FNDC5/Irisin is still unknown. The gene expression of FNDC5/Irisin, associated to key regulators of DNL, inflammation and cancer progression was evaluated in liver tissue of 18 patients with HCC undergoing liver transplantation and of 18 deceased donors.Hepatic mRNA expression of FNDC5/Irisin and stearoyl-CoA desaturase (SCD-1), main enzymatic regulator of DNL, were significantly higher in HCC patients than in donors (p < 0.0001 and p = 0.015, respectively). The hepatic mRNA expression of the neurogenic locus notch homolog protein 1 (NOTCH1) tended to be higher in HCC patients than in donors (p = 0.06). Only in HCC patients, hepatic FNDC5/Irisin strongly correlated with the transcription factor sterol regulatory element–binding factor 1, SCD-1, NOTCH1, tumor necrosis factor-α and Interleukin-6 mRNA expression. Further, in HCC patients, FNDC5/Irisin mRNA tended to correlate to plasma lipid profile namely triglycerides, palmitic/linoleic acid and polyunsaturated fatty acid/saturated fatty acid ratios.In conclusion, HCC-liver tissue over-expressed FNDC5/Irisin in association with gene expression of mediators involved in lipogenesis, inflammation and cancer, suggesting a possible protective role of the hormone from the liver damage.
Keywords: Hepatocellular carcinoma; Fibronectin type III domain containing 5; Irisin; De novo lipogenesis; Cancer;

Prostanoids counterbalance the synergism between endothelin-1 and angiotensin II in mesenteric veins of trained rats by Agnaldo Bruno Chies; Priscilla Bianca de Oliveira; Patrícia de Souza Rossignoli; Rafaela de Fátima Ferreira Baptista; Roger William de Lábio; Spencer Luiz Marques Payão (67-73).
Exercise-induced adaptations of the modulating mechanisms that influence the angiotensin (Ang II) responses assume different features depending on the venous bed. In femoral veins, exercise mobilizes vasodilator prostanoids to cooperate with NO in order to maintain reduced Ang II responses. On the other hand, exercise’s influence on the Ang II responses in veins that drain blood from the mesenteric region has been poorly described. Therefore, the present study aimed to identify the effects of a single bout of exercise, as well as exercise training, on the Ang II responses in mesenteric veins. The present study also aimed to investigate the involvement of prostanoids, NO and ET-1 in eventual exercise-induced modifications in these veins. To this end, mesenteric veins taken from resting-sedentary, exercised-sedentary, resting-trained and exercised-trained animals were studied in organ baths. In addition, the mRNA expression of prepro-endothelin-1 (ppET-1), as well as that of the ETA and ETB receptors, were quantified by real-time PCR in these veins. The results show that, either in absence or in presence of L-NAME, the Ang II responses were not different between groups. In the presence of indomethacin, higher Ang II responses were observed in the resting-trained animals than in the resting-sedentary animals. This difference, however, disappeared when L-NAME, BQ-123 or BQ-788 were added during incubation. In addition, no differences in ppET-1, ETA or ETB mRNA expression were observed between groups. Furthermore, in the presence of PD123,319, the Ang II responses in the exercised-sedentary animals were higher than those in the resting-sedentary animals. In conclusion, exercise training mobilizes endothelin-1 (ET-1) to reinforce the Ang II-induced responses mainly through ETA activation. On the other hand, vasodilator prostanoids are mobilized to act in parallel with NO in order to counterbalance the Ang II responses that have been potentiated by ET-1 in these trained animals.
Keywords: Angiotensin II; Endothelin-1; Exercise; Nitric oxide; Prostanoids; Vein;

Glucagon increases insulin levels by stimulating insulin secretion without effect on insulin clearance in mice by Gina Song; Giovanni Pacini; Bo Ahrén; David Z. D’Argenio (74-79).
Circulating insulin is dependent on a balance between insulin appearance through secretion and insulin clearance. However, to what extent changes in insulin clearance contribute to the increased insulin levels after glucagon administration is not known. This study therefore assessed and quantified any potential effect of glucagon on insulin kinetics in mice. Prehepatic insulin secretion in mice was first estimated following glucose (0.35 g/kg i.v.) and following glucose plus glucagon (10 μg/kg i.v.) using deconvolution of plasma C-peptide concentrations. Plasma concentrations of glucose, insulin, and glucagon were then measured simultaneously in individual mice following glucose alone or glucose plus glucagon (pre dose and at 1, 5, 10, 20 min post). Using the previously determined insulin secretion profiles and the insulin concentration-time measurements, a population modeling analysis was applied to estimate the one-compartment kinetics of insulin disposition with and without glucagon. Glucagon with glucose significantly enhanced prehepatic insulin secretion (Cmax and AUC0-20) compared to that with glucose alone (p  < 0.0001). From the modeling analysis, the population mean and between-animal SD of insulin clearance was 6.4 ± 0.34 mL/min for glucose alone and 5.8 ± 1.5 mL/min for glucagon plus glucose, with no significant effect of glucagon on mean insulin clearance. Therefore, we conclude that the enhancement of circulating insulin after glucagon administration is solely due to stimulated insulin secretion.
Keywords: Glucagon; Insulin secretion; Insulin clearance; Population analysis;

Synthetic peptide antigens derived from long-chain alpha-neurotoxins: Immunogenicity effect against elapid venoms by Guillermo de la Rosa; Nina Pastor; Alejandro Alagón; Gerardo Corzo (80-86).
Three-finger toxins (3FTXs), especially α-neurotoxins, are the most poorly neutralized elapid snake toxins by current antivenoms. In this work, the conserved structural similarity and motif arrangements of long-chain α-neurotoxins led us to design peptides with consensus sequences. Eight long-chain α-neurotoxins (also known as Type II) were used to generate a consensus sequence from which two peptides were chemically synthesized, LCP1 and LCP2. Rabbit sera raised against them were able to generate partially-neutralizing antibodies, which delayed mice mortality in neutralization assays against Naja haje, Dendrospis polylepis and Ophiophagus hannah venoms.
Keywords: Elapid antivenom; Long-chain α-neurotoxins; Snakebites; Three-finger toxins;

An enzymatic assay based on luciferase Ebola virus-like particles for evaluation of virolytic activity of antimicrobial peptides by Marie Peskova; Zbynek Heger; Petr Janda; Vojtech Adam; Vladimir Pekarik (87-96).
The membrane impermeable luciferase substrate furimazine is outside of the virus-like particles while the reporter enzyme is enclosed inside the viral membrane. While the membrane is intact, the substrate remains separated from the luciferase. After the membrane integrity is compromised by the antimicrobial peptide, a pore is formed that allows an ingress of the substrate to the inside of virus like particle. The substrate gets in contact with the enzyme and furimazine chemical conversion is accompanied by the light emission that can be easily detected.Display OmittedAntimicrobial peptides are currently considered as promising antiviral compounds. Current assays to evaluate the effectivity of peptides against enveloped viruses based on liposomes or hemolysis are encumbered by the artificial nature of liposomes or distinctive membrane composition of used erythrocytes. We propose a novel assay system based on enzymatic Ebola virus-like particles containing sensitive luciferase reporter. The assay was validated with several cationic and anionic peptides and compared with lentivirus inactivation and hemolytic assays. The assay is sensitive and easy to perform in standard biosafety level laboratory with potential for high-throughput screens. The use of virus-like particles in the assay provides a system as closely related to the native viruses as possible eliminating some issues associated with other more artificial set ups.We have identified CAM-W (KWKLWKKIEKWGQGIGAVLKWLTTWL) as a peptide with the greatest antiviral activity against infectious lentiviral vectors and filoviral virus-like particles.
Keywords: Virus-like particles; Cytotoxic peptides; Antimicrobial peptides; Nanoluciferase; Hemolytic activity; Virolytic activity; Lentivirus; Ebola; Marburg;

Duplication of neuropeptide Y and peptide YY in Nile tilapia Oreochromis niloticus and their roles in food intake regulation by Peipei Yan; Jirong Jia; Guokun Yang; Dongfang Wang; Caiyun Sun; Wensheng Li (97-105).
In vertebrates, the neuropeptide Y (NPY) family peptides have been recognized as key players in food intake regulation. NPY centrally promotes feeding, while peptide YY (PYY) and pancreatic polypeptide (PP) mediate satiety. The teleost tetraploidization is well-known to generate duplicates of both NPY and PYY; however, the functional diversification between the duplicate genes, especially in the regulation of food intake, remains unknown. In this study, we identified the two duplicates of NPY and PYY in Nile tilapia (Oreochromis niloticus). Both NPYa and NPYb were primarily expressed in the central nervous system (CNS), but the mRNA levels of NPYb were markedly lower than those of NPYa. Hypothalamic mRNA expression of NPYa, but not NPYb, decreased after feeding and increased after 7-days of fasting. However, both NPYa and NPYb caused a significant increase in food intake after an intracranial injection of 50 ng/g body weight dose. PYYb, one of the duplicates of PYY, had an extremely high expression in the foregut and midgut, whereas another form of duplicate PYYa showed only moderate expression in the CNS. Both hypothalamic PYYa and foregut PYYb mRNA expression increased after feeding and decreased after 7-days of fasting. Furthermore, the intracranial injection of PYYb decreased food intake, but PYYa had no significant effect. Our results suggested that although the mature peptides of NPYa and NPYb can both stimulate food intake, NPYa is the main endogenous functional NPY for feeding regulation. A functional division has been identified in the duplicates of PYY, which deems PYYb as a gut-derived anorexigenic peptide and PYYa as a CNS-specific PYY in Nile tilapia.
Keywords: Neuropeptide Y; Peptide YY; Food intake; Nile tilapia;

Urotensin II (UII) is a vasoactive neuropeptide involved in migration and invasion in various cell types. However, the effects of UII on human hepatoma cells still remain unclear. The aim of this study was to investigate the role and mechanism of UII on migration and invasion in human hepatoma cells.Migration was measured by wound healing assays and a Transwell® methodology, and invasion was analyzed using Matrigel® invasion chambers. Reactive oxygen species (ROS) levels were detected using a 2′, 7′-dichlorofluorescein diacetate probe, and flow cytometry, and protein expression levels were evaluated by western blotting. Cell proliferation and actin polymerization were examined using cell proliferation reagent WST-1 and F-actin immunohistochemistry staining.Exposure to UII promoted migration and invasion in hepatoma cells compared with that in cells without UII. UII also increased matrix metalloproteinase-2 (MMP2) expression in a time-independent manner. Furthermore, UII markedly enhanced ROS generation and NADPH oxidase subunit expression, and consequently facilitated the phosphorylation of c-Jun N-terminal kinase (JNK). The UT antagonist urantide or the antioxidant/NADPH oxidase inhibitor apocynin decreased UII-induced ROS production. JNK phosphorylation, migration, invasion, and MMP9/2 expression were also reversed by pretreatment with apocynin. Urantide and JNK inhibitor SP600125 abrogated migration, invasion, or MMP9/2 expression in response to UII. UII induced actin polymerization and fascin protein expression, and could be reversed by apocynin and SP600125.Exogenous UII induced migration and invasion in hepatoma cells that mainly involved NADPH oxidase-derived ROS through JNK activation. UT played an additional role in regulating hepatoma cells migration and invasion. Thus, our data suggested an important effect of UII in hepatocellular carcinoma metastasis.
Keywords: Urotensin II; Migration; Invasion; NADPH oxidase; JNK; Human hepatoma cells;

Design of novel antimicrobial peptide dimer analogues with enhanced antimicrobial activity in vitro and in vivo by intermolecular triazole bridge strategy by Beijun Liu; Haifeng Huang; Zhibin Yang; Beiyin Liu; Sanhu Gou; Chao Zhong; Xiufeng Han; Yun Zhang; Jingman Ni; Rui Wang (115-125).
Currently, antimicrobial peptides have attracted considerable attention because of their broad-sprectum activity and low prognostic to induce antibiotic resistance. In our study, for the first time, a series of side-chain hybrid dimer peptides J-AA (Anoplin-Anoplin), J-RR (RW-RW), and J-AR (Anoplin-RW) based on the wasp peptide Anoplin and the arginine- and tryptophan-rich hexapeptide RW were designed and synthesized by click chemistry, with the intent to improve the antimicrobial efficacy of peptides against bacterial pathogens. The results showed that all dimer analogues exhibited up to a 4–16 fold increase in antimicrobial activity compared to the parental peptides against bacterial strains. Furthermore, the antimicrobial activity was confirmed by time-killing kinetics assay with two strains which showed that these dimer analogues at 1, 2 × MIC were rapidly bactericidal and reduced the initial inoculum significantly during the first 2–6 h. Notably, dimer peptides showed synergy and additivity effects when used in combination with conventional antibiotics rifampin or penicillin respectively against the multidrug-resistant strains. In the Escherichia coli-infected mouse model, all of hybrid dimer analogues had significantly lower degree of bacterial load than the untreated control group when injected once i.p. at 5 mg/kg. In addition, the infected mice by methicillin-resistant (MRSA) strain could be effectively treated with J-RR. All of dimer analogues had membrane-active action mode. And the membrane-dependent mode of action signifies that peptides functions freely and without regard to conventional resistant mechanisms. Circular dichroism analyses of all dimer analogues showed a general predominance of α-helix conformation in 50% trifluoroethanol (TFE). Additionally, the acute toxicities study indicated that J-RR or J-AR did not show the signs of toxicity when adult mice exposed to concentration up to 120 mg/kg. The 50% lethal dose (LD50) of J-AA was 53.6 mg/kg. In conclusion, to design and synthesize side chain-hybrid dimer analogues via click chemistry may offer a new strategy for antibacterial therapeutic option.
Keywords: Click chemistry; Dimer analogues; Antimicrobial activity; α Helical structure; Drug resistance;

Endogenous Opiates and Behavior: 2015 by Richard J. Bodnar (126-188).
This paper is the thirty-eighth consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2015 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, and the roles of these opioid peptides and receptors in pain and analgesia, stress and social status, tolerance and dependence, learning and memory, eating and drinking, drug abuse and alcohol, sexual activity and hormones, pregnancy, development and endocrinology, mental illness and mood, seizures and neurologic disorders, electrical-related activity and neurophysiology, general activity and locomotion, gastrointestinal, renal and hepatic functions, cardiovascular responses, respiration and thermoregulation, and immunological responses.
Keywords: Mu opioid receptor; Delta opioid receptor; Kappa opioid receptor; ORL-1 receptor; Enkephalins; Beta-endorphin; Endomorphins; Nociception; Morphine; Naloxone;

Patients with spinal cord injury (SCI) develop chronic pain that severely compromises their quality of life. We have previously reported that progesterone (PG), a neuroprotective steroid, could offer a promising therapeutic strategy for neuropathic pain. In the present study, we explored temporal changes in the expression of the neuropeptides galanin and tyrosine (NPY) and their receptors (GalR1 and GalR2; Y1R and Y2R, respectively) in the injured spinal cord and evaluated the impact of PG administration on both neuropeptide systems and neuropathic behavior. Male rats were subjected to spinal cord hemisection at T13 level, received daily subcutaneous injections of PG or vehicle, and were evaluated for signs of mechanical and thermal allodynia. Real time PCR was used to determine relative mRNA levels of neuropeptides and receptors, both in the acute (1 day) and chronic (28 days) phases after injury. A significant increase in Y1R and Y2R expression, as well as a significant downregulation in GalR2 mRNA levels, was observed 1 day after SCI. Interestingly, PG early treatment prevented Y1R upregulation and resulted in lower NPY, Y2R and GalR1 mRNA levels. In the chronic phase, injured rats showed well-established mechanical and cold allodynia and significant increases in galanin, NPY, GalR1 and Y1R mRNAs, while maintaining reduced GalR2 expression. Animals receiving PG treatment showed basal expression levels of galanin, NPY, GalR1 and Y1R, and reduced Y2R mRNA levels. Also, and in line with previously published observations, PG-treated animals did not develop mechanical allodynia and showed reduced sensitivity to cold stimulation. Altogether, we show that SCI leads to considerable changes in the spinal expression of galanin, NPY and their associated receptors, and that early and sustained PG administration prevents them. Moreover, our data suggest the participation of galaninergic and NPYergic systems in the plastic changes associated with SCI-induced neuropathic pain, and further supports the therapeutic potential of PG- or neuropeptide-based therapies to prevent and/or treat chronic pain after central injuries.
Keywords: Allodynia; Chronic pain; Galanin; Neuropeptide tyrosine; Progesterone; Spinal cord;

Inhibition of endoplasmic reticulum stress by neuregulin-1 protects against myocardial ischemia/reperfusion injury by Shan-Juan Fang; Peng-Yang Li; Chun-Mei Wang; Yi Xin; Wei-Wei Lu; Xiao-Xia Zhang; Song Zuo; Chang-Sheng Ma; Chao-Shu Tang; Shao-Ping Nie; Yong-Fen Qi (196-207).
Neuregulin-1 (NRG-1), an endogenously produced polypeptide, is the ligand of cardiomyocyte ErbB receptors, with cardiovascular protective effects. In the present study, we explored whether the cardioprotective effect of NRG-1 against I/R injury is mediated by inhibiting myocardial endoplasmic reticulum (ER) stress. In vitro, NRG-1 directly inhibited the upregulation of ER stress markers such as glucose-regulated protein 78, CCAAT/enhancer binding protein homologous protein and cleaved caspase-12 induced by the ER stress inducers tunicamycin or dithiothreitol in both neonatal and adult ventricular myocytes. Attenuating ErbB signals by an ErbB inhibitor AG1478 or ErbB4 knockdown and preincubation with phosphoinositide 3-kinase inhibitors all reversed the effect of NRG-1 inhibiting ER stress in cultured neonatal rat cardiomyocytes. Concurrently, cardiomyocyte ER stress and apoptosis induced by hypoxia-reoxygenation were decreased by NRG-1 treatment in vitro. Furthermore, in an in vivo rat model of myocardium ischemia/reperfusion (I/R), intravenous NRG-1 administration significantly decreased ER stress and myocardial infarct size induced by I/R. NRG-1 could protect the heart against I/R injury by inhibiting myocardial ER stress, which might be mediated by the phosphoinositide 3-kinase/Akt signaling pathway.
Keywords: Neuregulin-1; Ischemia/reperfusion injury; Endoplasmic reticulum stress; Apoptosis; Heart;