Peptides (v.42, #C)

Evidence that kinin B2 receptor expression is upregulated by endothelial overexpression of B1 receptors by Eliete S. Rodrigues; Rafael F. Silva; Renan P. Martin; Suzana M. Oliveira; Clovis R. Nakaie; Regiane A. Sabatini; Vanessa F. Merino; João B. Pesquero; Michael Bader; Suma I. Shimuta (1-7).
► Rat overexpressing the B1R specifically in the vascular endothelium were assessed. ► The BK-induced relaxations were significantly increased in the transgenic rats. ► The expression level of the B2R in the transgenic rats was highly increased. ► The ACE activity and mRNA ACE expression was not altered. ► We report that the B2R expression is upregulated by the overexpression of B1R.Bradykinin (BK) and des-Arg9-bradykinin (DBK) of kallikrein-kinin system exert its effects mediated by the B2 (B2R) and B1 (B1R) receptors, respectively. It was already shown that the deletion of kinin B1R or of B2R induces upregulation of the remaining receptor subtype [10,12,16,28,36]. However studies on overexpression of B1R or B2R in transgenic animals have supported the importance of the overexpressed receptor but the expression of another receptor subtype has not been determined [17,19,33]. Previous study described a marked vasodilatation and increased susceptibility to endotoxic shock which was associated with increased mortality in response to DBK in thoracic aorta from transgenic rat overexpressing the kinin B1R (TGR(Tie2B1)) exclusively in the endothelium. In another study, mice overexpressing B1R in multiple tissues were shown to present high susceptibility to inflammation and to lipopolysaccharide-induced endotoxic shock. Therefore the role of B2R was investigated in the thoracic aorta isolated from TGR(Tie2B1) rats overexpressing the B1R exclusively in the vascular endothelium. Our findings provided evidence for highly increased expression level of the B2R in the transgenic rats. It was reported that under endotoxic shock, these rats exhibited exaggerated hypotension, bradycardia and mortality. It can be suggested that the high mortality during the pathogenesis of endotoxic shock provoked in the transgenic TGR(Tie2B1) rats could be due to the enhanced expression of B2R associated with the overexpression of the B1R.
Keywords: AngiotensinII; Bradykinin; des-Arg9-bradykinin; Kinin receptors; ACE;

Peripheral administration of TAT-obestatin can influence the expression of liporegulatory genes but fails to affect food intake in mice by Guangcai Ren; Zuyong He; Peiqing Cong; Hu Chen; Yunxue Guo; Jingwei Yu; Zhiguo Liu; Qianqian Ji; Zhenwei Song; Yaosheng Chen (8-14).
► TAT-Obestatin can cross the BBB into mouse brain. ► Food intake and body weight generally was not affected by TAT-obestatin. ► TAT-obestatin may play a role in adiposegenesis regulation.Obestatin is a 23-amino-acid peptide originally regarded as an anorexigenic factor. However, most of the subsequent studies failed to confirm the initially reported anorexigenic properties of obestatin. Obestatin is incapable of crossing the blood brain barrier (BBB), which may affect its biological function. Here, we report the physiological effects of obestatin in mice after intraperitoneal administration of obestatin conjugated to the cell-permeable peptide TAT, which is capable of delivering different types of proteins through the BBB. Acute peripheral administration of 1 μmol/kg of TAT-obestatin did not influence the 24 h cumulative food intake and body weight gain of mice that were fasted for 18 h. Fed mice were injected intraperitoneally with 100 nmol/kg of TAT-obestatin daily for 25 d. Compared with control groups, on day 3, the gain in body weight was significantly altered; on day 7, abdominal fat mass was remarkably reduced; however, on day 25, there was a surprisingly notable increase in abdominal and epididymal fat mass. In comparison with control groups, on day 25, the expression levels of adiponectin, ADD1, C/EBPα, PPARG and GLUT4 were significantly up-regulated in liver tissues; in white adipose tissue, the expression level of C/EBPα was significantly up-regulated, but adiponectin and GLUT4 were significantly down-regulated. In addition, GPR39, the suspected receptor of obestatin, was up-regulated in white adipose tissue on day 25. These findings suggest that TAT-obestatin might play a role in white adipose tissue metabolism, but its physiological effects on food intake and body weight gain regulation remain unclear.
Keywords: Obestatin; Cell-permeable peptides; Food intake; Adipose tissue; Gene expression;

Gonadotropin-releasing hormone neuropeptides and receptor in human breast cancer: Correlation to poor prognosis parameters by Kalliopi Pazaitou-Panayiotou; Christina Chemonidou; Aliki Poupi; Maria Koureta; Athina Kaprara; Maria Lambropoulou; Theodoros C. Constantinidis; Grammati Galaktidou; Maria Koffa; Anastasia Kiziridou; Stylianos Kakolyris; George Kolios; Alexandros Kortsaris; Ekaterini Chatzaki (15-24).
► GnRH I and II and their receptor were examined in breast cancer biopsies. ► Quantification was performed by a multiplex real-time RT-PCR assay. ► All genes were twice as frequently detected in tumor than adjunct benign tissue. ► Expression of each gene was correlated to the others and poor prognosis parameters.Expression of the two gonadotropin-releasing hormone homologue peptides GnRHI and GnRHII and their receptor GnRHR has been demonstrated in a number of malignancies. In hormone-dependent breast cancer, GnRH analogs are used for therapy in premenopausal women. Gene expression of GnRHI, II and R was studied in breast biopsies from primary breast adenocarcinoma obtained from the tumor and the adjacent benign tissue. Levels were evaluated by a multiplex real-time RT-PCR. GnRHI transcripts were detected in 14.7% of the benign and 29.4% malignant biopsies and GnRHII in 21.2% benign and 44.1% malignant biopsies. GnRHR was also more frequent in the malignant (54.2%) than in the benign (24.0%) biopsies, at similar expression levels. No transcripts were detected in biopsies from healthy individuals. There was a strong correlation between the presence of GnRHI and GnRHII transcripts and their receptor in the benign and the malignant biopsies. GnRHI, II and R expression correlated significantly with poor prognosis pathological parameters. Immunohistochemistry for GnRHR revealed expression in malignant cells and in epithelial cells of mammary ducts of the adjacent area with pre-cancerous features. In contrast, GnRH I and II peptides were rarely expressed at low levels in breast cancer cells. In conclusion GnRH peptides and receptor are expressed more frequently in breast tumors than in the adjacent mammary tissue, representing a malignant feature. Their expression correlated to tumor characteristics of poor prognosis and was therefore related to more aggressive malignancies. Concomitant expression of peptides and receptor supports an autocrine/paracrine regulating role.
Keywords: Gonadotropin releasing hormone; Receptor; Neuropeptide; Human; Breast; Cancer;

Angiotensin-(1-7) inhibits vascular calcification in rats by Yu-Bin Sui; Jin-Rui Chang; Wen-Jia Chen; Lei Zhao; Bao-Hong Zhang; Yan-Rong Yu; Chao-Shu Tang; Xin-Hua Yin; Yong-Fen Qi (25-34).
► The endogenous levels of ACE2 and the Mas receptor were increased in calcified aortas. ► Ang-(1-7) inhibited the development of vascular calcification in rats. ► Ang-(1-7) prevented the loss of lineage markers and attenuated the osteogenic transition of vascular smooth muscle cells. ► Ang-(1-7) reversed the upregulated levels of the ACE/Ang II/AT1 axis in calcified arota.Angiotensin-(1-7) [Ang-(1-7)] is a new bioactive heptapeptide in the renin–angiotensin–aldosterone system (RAAS) with potent protective effects in cardiovascular diseases, opposing many actions of angiotensin II (Ang II) mediated by Ang II type 1 (AT1) receptor. It is produced mainly by the activity of angiotensin-converting enzyme 2 (ACE2) and acts through the Mas receptor. However, the role of Ang-(1-7) in vascular calcification (VC) is still unclear. In this study, we investigated the protective effects of Ang-(1-7) on VC in an in vivo rat VC model induced by vitamin D3 plus nicotine. The levels of ACE2 and the Mas receptor, as well as ACE, AT1 receptor, Ang II type 2 receptor and angiotensinogen, were significantly increased in calcified aortas, and Ang-(1-7) reversed the increased levels. Ang-(1-7) restored the reduced expression of lineage markers, including smooth muscle (SM) α-actin, SM22α, calponin and smoothelin, in vascular smooth muscle cells (VSMCs) and retarded the osteogenic transition of VSMCs by decreasing the expression of bone-associated proteins. It reduced alkaline phosphatase activity and calcium deposition in VC and alleviated the hemodynamic disorders of rats with VC. We provide the first in vivo evidence that Ang-(1-7) can inhibit the development of VC by inhibiting the osteogenic transition of VSMCs, at least in part by decreasing levels of the ACE/Ang II/AT1 axis. The increased expression of ACE2 and the Mas receptor in calcified aortas suggests the involvement of the ACE2/Ang-(1-7)/Mas axis during VC. Ang-(1-7) might be an efficient endogenous vasoprotective factor for VC.
Keywords: Angiotensin-(1-7); Vascular calcification; Angiotensin converting enzyme 2 (ACE2); Mas receptor;

► Chronic CNS ghrelin infusion increases appetite and promote weight gain. ► Chronic CNS ghrelin infusion reduces BP and HR despite weight gain and hyperphagia. ► Ghrelin's effect on BP and HR is associated with reduced cardiac sympathetic tone.Acute studies showed that ghrelin acts on the central nervous system (CNS) to reduce blood pressure (BP), heart rate (HR) and sympathetic activity. However, the long-term CNS cardiovascular actions of ghrelin are still unclear. We tested whether chronic intracerebroventricular (ICV) infusion of ghrelin causes sustained reductions in BP, HR and whether it alters baroreceptor sensitivity (BRS) and autonomic input to the heart. A cannula was placed in the lateral ventricle of male Sprague–Dawley (SD) rats for ICV infusions via osmotic minipump (0.5 μl/h). BP and HR were measured 24-h/day by telemetry. After 5 days of control measurements, ghrelin (0.21 nmol/h) or saline vehicle were infused ICV for 10 days followed by a 5-day post-treatment period. Chronic ICV ghrelin infusion increased food intake (22 ± 3 to 26 ± 1 g/day) leading to ∼50 g body weight gain. BP fell slightly during ghrelin infusion while HR decreased by ∼26 bpm. In control animals BP and HR increased modestly. ICV Ghrelin infusion caused a 50% reduction in sympathetic tone to the heart but did not alter BRS. We also tested if the depressor responses to ICV ghrelin infusion were enhanced in spontaneously hypertensive rats (SHR) due to their high basal sympathetic tone. However, we observed similar BP and HR responses compared to normotensive rats. These results indicate that ghrelin, acting via direct actions on the CNS, has a sustained effect to lower HR and a modest impact to reduce BP in normotensive and hypertensive animals despite increasing appetite and body weight.
Keywords: Ghrelin; Heart rate; Baroreceptor function; Blood pressure; Food intake; Leptin;

Plasma copeptin concentration and outcome after pediatric traumatic brain injury by Chao Lin; Ning Wang; Zhi-Peng Shen; Zheng-Yan Zhao (43-47).
► Plasma copeptin level increased in children with traumatic brain injury. ► Copeptin has high predictive value for long-term clinical outcomes. ► Copeptin may be a good prognostic factor for long-term clinical outcomes.Higher plasma copeptin level has been associated with poor outcomes of critical illness. The present study was undertaken to investigate the plasma copeptin concentrations in children with traumatic brain injury (TBI) and to analyze the correlation of copeptin with disease outcome. Plasma copeptin concentrations of 126 healthy children and 126 children with acute severe TBI were measured by enzyme-linked immunosorbent assay. Twenty-one patients (16.7%) died and 38 patients (30.2%) had an unfavorable outcome (Glasgow Outcome Scale score of 1–3) at 6 months. Plasma copeptin level was obviously higher in patients than in healthy children (46.2 ± 20.8 pmol/L vs. 9.6 ± 3.0 pmol/L, P  < 0.001). Plasma copeptin level was identified as an independent predictor for 6-month mortality [odds ratio (OR) 1.261, 95% confidence interval (CI) 1.112–1.538, P  = 0.005] and unfavorable outcome (OR 1.313, 95% CI 1.146–1.659, P  = 0.003). The predictive value of copeptin was similar to that of Glasgow Coma Scale (GCS) score for 6-month mortality [area under curve (AUC) 0.832, 95% CI 0.755–0.892 vs. AUC 0.873, 95% CI 0.802–0.926, P  = 0.412] and unfavorable outcome (AUC 0.863, 95% CI 0.790–0.918 vs. AUC 0.885, 95% CI 0.816–0.935, P  = 0.596). Copeptin improved the AUC of GCS score for 6-month unfavorable outcome (AUC 0.929, 95% CI 0.869–0.967, P  = 0.013), but not for 6-month mortality (AUC 0.887, 95% CI 0.818–0.936, P  = 0.600). Thus, plasma copeptin level represents a novel biomarker for predicting 6-month clinical outcome in children with TBI.
Keywords: Copeptin; Traumatic brain injury; Functional outcome; Mortality;

Orexin: A potential role in the process of obstructive sleep apnea by Wei Wang; Yongchu Pan; Qingyi Li; Lin Wang (48-54).
► There is a close relationship between orexin and obesity, sex, age, smoking. ► The numbers of orexin neurons decrease with age, but the prevalence of sleep apnea increases with age. ► Obesity in OSA patient may be induced by decreased orexin neurons activity or orexin levels. ► Nicotine in cigarettes may have a positive effect on OSA because of the promotion on orexin neurons.Obstructive sleep apnea (OSA) is a complicated disease with an unrecognized mechanism. Obesity, sex, age, and smoking have been found to be independent correlates of OSA. Orexin (also named hypocretin) mainly secreted by lateral hypothalamus neurons has a wide array of biological functions like regulating sleep, energy levels and breathing. Several clinical studies found ties between orexin and OSA. Because of the close correlation between orexin and obesity, sex, age and smoking (which are the key risk factors for OSA patients), we hypothesize that orexin may play a key role in the pathogenesis of OSA.
Keywords: Orexin; Obstructive sleep apnea; Obesity; Sex; Age; Smoking;

Antimicrobial activity and mechanism of action of a novel cationic α-helical dodecapeptide, a partial sequence of cyanate lyase from rice by Norihiro Takei; Nobuteru Takahashi; Tomohiro Takayanagi; Atsuo Ikeda; Kenji Hashimoto; Masahiro Takagi; Tsutomu Hamada; Eiichi Saitoh; Akihito Ochiai; Takaaki Tanaka; Masayuki Taniguchi (55-62).
Display Omitted► A dodecapeptide from rice was a novel cationic α-helical antimicrobial peptide. ► The peptide contains three arginine and two lysine residues. ► The peptide exhibited the antimicrobial activity against a periodontal pathogen. ► The peptide disrupted giant unilamellar vesicles in a detergent-like manner. ► Arginine residues at positions 15 and 24 are critical for the antimicrobial activity.CL(14-25), a dodecapeptide, that is a partial region near N-terminus of cyanate lyase (CL, EC 4.3.99.1) from rice (Oryza sativa L. japonica), contains three arginine and two lysine residues. It was a novel cationic α-helical antimicrobial peptide. The antimicrobial activity of CL(14-25) against Porphyromonas gingivalis, a periodontal pathogen, was quantitatively evaluated by a chemiluminescence method that measures ATP derived from viable cells. The 50% growth-inhibitory concentration of CL(14-25) against P. gingivalis cells was 145 μM. CL(14-25), even at a concentration of 800 μM, had no hemolytic activity. When giant unilamellar vesicles (GUVs) that mimic the membrane composition of Gram-negative bacteria were used, microscopy image analysis suggested that CL(14-25) disrupted GUVs in a detergent-like manner. Therefore, CL(14-25) appears to exhibit antimicrobial activity through membrane disruption. To investigate the contribution of cationic amino acid residues in CL(14-25) to its antimicrobial activity, we synthesized four truncated CL analogs, in which one or two cationic amino acid residues were deleted from the N- and C- termini of CL(14-25). The degrees of calcein leakage from large unilamellar vesicles (LUVs) and 3,3′-dipropylthiadicarbocyanine iodide (diSC3-5) release from P. gingivalis cells induced by truncated CL analogs were closely related to their antimicrobial activities. CL analogs, which were truncated by removing an arginine residue from the N-terminus and a lysine residue from the C-terminus maintained their antimicrobial activity. However, CL analogs, which were further truncated by removing two arginine residues from the N-terminus, and an arginine and a lysine residue from the C-terminus, rarely exhibited antimicrobial activity.
Keywords: Antimicrobial peptide; Cationic α-helical peptide; Porphyromonas gingivalis; Giant unilamellar vesicle; Membrane-active peptide;

Peptidomic analysis of human reflex tear fluid by Eisuke Hayakawa; Bart Landuyt; Geert Baggerman; Ruud Cuyvers; Rob Lavigne; Walter Luyten; Liliane Schoofs (63-69).
► The peptidome of human reflex tear fluid was analyzed. ► Thirty endogenous peptides were identified by using off-line LC-MALDI-TOF–TOF. ► Twenty-six peptides are derived from proline-rich protein 4. ► Four peptides are derived from the polymeric immunoglobulin receptor. ► These peptides may contribute to the protective environment of the ocular surface.Tear fluid is a complex mixture of biological compounds, including carbohydrates, lipids, electrolytes, proteins, and peptides. Despite the physiological importance of tear fluid, little is known about the identity of its endogenous peptides. In this study, we analyzed and identified naturally occurring peptide molecules in human reflex tear fluid by means of LC-MALDI-TOF–TOF. Tandem MS analyses revealed 30 peptides, most of which have not been identified before. Twenty-six peptides are derived from the proline-rich protein 4 and 4 peptides are derived from the polymeric immunoglobulin receptor. Based on their structural characteristics, we suggest that the identified tear fluid peptides contribute to the protective environment of the ocular surface.
Keywords: Peptidomics; Endogenous peptide; Tear; MALDI-TOF–TOF; Proline-rich protein 4; Polymeric immunoglobulin receptor;

► Brain activation of sst subtypes blunts various components of the stress response. ► Brain sst2/5 inhibits stress-induced CRF-ACTH release. ► Brain sst2 prevents stress-related anorexia and anxiogenic-like responses ► Brain sst5 suppresses stress-induced sympathetic activation and gastric stasis. ► Brain sst1 blocks stress-induced stimulation of colonic motor function.Somatostatin-14 was discovered in 1973 in the hypothalamus as a peptide inhibiting growth hormone release. Somatostatin interacts with five receptor subtypes (sst1−5) which are widely distributed in the brain with a distinct, but overlapping, expression pattern. During the last few years, the development of highly selective peptide agonists and antagonists provided new insight to characterize the role of somatostatin receptor subtypes in the pleiotropic actions of somatostatin. Recent evidence in rodents indicates that the activation of selective somatostatin receptor subtypes in the brain blunts stress-corticotropin-releasing factor (CRF) related ACTH release (sst2/5), sympathetic-adrenal activaton (sst5), stimulation of colonic motility (sst1), delayed gastric emptying (sst5), suppression of food intake (sst2) and the anxiogenic-like (sst2) response. These findings suggest that brain somatostatin signaling pathways may play an important role in dampening CRF-mediated endocrine, sympathetic, behavioral and visceral responses to stress.
Keywords: ACTH; Anxiety; Autonomic nervous system; Catecholamines; CRF; Food intake; Gastrointestinal motility; Octreotide; ODT8-SST; Stress;

Antimicrobial activity of the synthetic peptide Lys-a1 against oral streptococci by Bruno Rocha da Silva; Victor Aragão Abreu de Freitas; Victor Alves Carneiro; Francisco Vassiliepe Sousa Arruda; Esteban Nicolás Lorenzón; Andréa Silvia Walter de Aguiar; Eduardo Maffud Cilli; Benildo Sousa Cavada; Edson Holanda Teixeira (78-83).
► The MIC values ranged from 3.9 to 125 μg mL−1, while the MBC values ranged from 3.9 to 500 μg mL−1. ► The peptide Lys-a1 demonstrated the potential to inhibit biofilm formation in all bacterial species tested. ► Streptococcus parasanguinis showed the greatest susceptibility to the peptide, reducing the number of viable cells at 1.9 μg mL−1. ► The antimicrobial peptide Lys-a1 has shown remarkable antimicrobial and antibiofilm activity.The peptide LYS-[TRP6]-Hy-A1 (Lys-a1) is a synthetic derivative of the peptide Hy-A1, initially isolated from the frog species Hypsiboas albopunctatus. According to previous research, it is a molecule with broad antimicrobial activity. The objective of this study was to evaluate the antimicrobial activity of the synthetic peptide Lys-a1 (KIFGAIWPLALGALKNLIK-NH2) on the planktonic and biofilm growth of oral bacteria. The methods used to evaluate antimicrobial activity include the following: determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in microtiter plates for growth in suspension and quantification of biomass by crystal violet staining and counting of colony forming units for biofilm growth. The microorganisms Streptococcus oralis, Streptococcus sanguinis, Streptococcus parasanguinis, Streptococcus salivarius, Streptococcus mutans and Streptococcus sobrinus were grown in Brain Heart Infusion broth at 37 °C under atmospheric pressure with 10% CO2. The peptide was solubilized in 0.1% acetic acid (v/v) at various concentrations (500–1.9 μg mL−1). Chlorhexidine gluconate 0.12% was used as the positive control, and BHI culture medium was used as the negative control. The tested peptide demonstrated a remarkable antimicrobial effect, inhibiting the planktonic and biofilm growth of all strains tested, even at low concentrations. Thus, the peptide Lys-a1 is an important source for potential antimicrobial agents, especially for the control and prevention of microbial biofilms, which is one of the most important factors in cariogenic processes.
Keywords: Antimicrobial peptide; Biofilms; Streptococcus mutans; Dental caries;

► Plasma IMD and BNP levels were markedly higher in ACS patients than that in controls. ► The increased plasma IMD and BNP levels were positively correlated with degree of coronary stenosis in ACS patients ► Plasma levels of IMD were positively correlated with BNP levels in all subjects.Intermedin (IMD) is a newly discovered peptide with increased levels in plasma and cardiac tissue in mice with ischemia/reperfusion. Continuous administration of low dose IMD markedly elevated the mRNA abundance of myocardial BNP in rats. Plasma BNP levels may reflect the severity of degree of coronary stenosis in patients with acute coronary syndrome (ACS). However, the role of circulating IMD in coronary heart disease remains unclear. We aimed to examine the plasma content of IMD and brain natriuretic peptide (BNP) and its clinical significance in patients with ACS. We collected plasma samples from 41 patients with ACS and 31 controls and measured IMD and BNP levels by radioimmunoassay. The severity of coronary artery stenosis for patients with ACS was measured by coronary angiography. Plasma IMD and BNP levels were markedly higher in ACS patients than that in controls (P  < 0.05). The increased plasma IMD and BNP were positively correlated with degree of coronary stenosis in ACS patients (r  = 0.263 and r  = 0.238, respectively, both P  < 0.05). In addition, plasma levels of IMD were positively correlated with BNP levels.
Keywords: Acute coronary syndrome; Intermedin; Brain natriuretic peptide;

► Study investigated allosteric peptide, PAOPA, specific for the dopamine D2 receptor. ► Study of PAOPA distribution showed it to effectively cross the blood–brain barrier. ► Pharmacokinetic profile of PAOPA was examined in rats across various time points. ► Toxic effects were examined through necropsy, histopathology, and blood analyses. ► Study showed robust drug safety, and thus platforms its progress to clinical trials.Schizophrenia is a mental illness characterized by a breakdown in cognition and emotion. Over the years, drug treatment for this disorder has mainly been compromised of orthosteric ligands that antagonize the active site of the dopamine D2 receptor. However, these drugs are limited in their use and often lead to the development of adverse movement and metabolic side effects. Allosteric modulators are an emerging class of therapeutics with significant advantages over orthosteric ligands, including an improved therapeutic and safety profile. This study investigates our newly developed allosteric modulator, PAOPA, which is a specific modulator of the dopamine D2 receptor. Previous studies have shown PAOPA to attenuate schizophrenia-like behavioral abnormalities in preclinical models. To advance this newly developed allosteric drug from the preclinical to clinical stage, this study examines the pharmacokinetic behavior and toxicological profile of PAOPA. Results from this study prove the effectiveness of PAOPA in reaching the implicated regions of the brain for therapeutic action, particularly the striatum. Pharmacokinetic parameters of PAOPA were found to be comparable to current market antipsychotic drugs. Necropsy and histopathological analyses showed no abnormalities in all examined organs. Acute and chronic treatment of PAOPA indicated no movement abnormalities commonly found with the use of current typical antipsychotic drugs. Moreover, acute and chronic PAOPA treatment revealed no hematological or metabolic abnormalities classically found with the use of atypical antipsychotic drugs. Findings from this study demonstrate a better safety profile of PAOPA, and necessitates the progression of this newly developed therapeutic for the treatment of schizophrenia.
Keywords: Allosteric; D2 receptor; Pharmacokinetics; Toxicology; Antipsychotic; Schizophrenia;

Thr21Met (T21M) but not Ser89Asn (S89N) polymorphisms of the urotensin-II (UTS-II) gene are associated with Behcet's disease (BD) by Serdar Oztuzcu; Mustafa Ulasli; Yavuz Pehlivan; Muhammer Özgür Çevik; Beyhan Cengiz; Bulent Gogebakan; Yusuf Ziya Igci; Seydi Okumuş; Ahmet Arslan; Ahmet Mesut Onat (97-100).
► Study enrolled Behcet's disease (BD) patients (198) and healthy controls (275). ► Thr21Met and Ser89Asn gene polymorphisms of urotensin-II gene were screened. ► Thr21Met but not Ser89Asn polymorphisms were frequently encountered in BD patients.Behcet's disease (BD) is multisytemic vasculitis or chronic inflammation that may lead to various autoimmune and autoinflammatory syndromes. Exact etiopathogenesis of BD has not been clarified yet. Urotensin II (UTS-II) is predominantly a vasoactive peptide and Thr21Met polymorphism in UTS-II gene was proved to increasing in some autoimmune diseases. Considering these, our objective was to evaluate whether two UTS-II gene polymorphisms (Thr21Met and Ser89Asn) were responsible in genetic susceptibility to BD in a Turkish population. A total of 198 patients with BD and 275 healthy controls were enrolled. We analyzed the genotype and allele frequencies of two UTS-II gene polymorphisms, Thr21Met and Ser89Asn, in BD patients and in controls. We found that Thr21Met but not Ser89Asn polymorphisms of the UTS-II gene were markedly associated with the risk of developing BD (p  < 0.0001), The Met21Met genotype was less common among BD patients (6.1% in patients vs. 17.1% in controls; p  < 0.0001). There was also an increase in the 21Thr allele (54.8% in BD patients vs. 43.8% in controls) and a decrease in 21Met allele frequencies (45.2% in controls vs. 56.2% in patients) in the BD groups (p  < 0.0044). To the best of our knowledge, for the first time in the literature, our study claims that there is an association between Thr21Met, and not between Ser89Asn polymorphisms in the UTS-II gene and BD. These results put a new player to the field of undiscovered pathogenesis of BD and hopefully provide new insights to the treatment options.
Keywords: Urotensin-II gene; Polymorphisms; Behcet's disease;

Prognostic significance of plasma visfatin levels in patients with ischemic stroke by Cong-Guo Yin; Lin Jiang; Bo Tang; Hao Zhang; Qi Qian; Guo-Zhong Niu (101-104).
► Plasma visfatin level increased in ischemic stroke. ► Visfatin emerged as an independent predictor for 6-month clinical outcomes. ► Visfatin had high prognostic performance for 6-month clinical outcomes.Plasma visfatin concentration has been enhanced in ischemic stroke. The aim of the current investigation was to test whether determination of visfatin in plasma is associated with 6-month clinical outcomes including mortality and unfavorable outcome (modified Rankin Scale score > 2) in the patients with ischemic stroke. Between July 2009 and January 2012, plasma visfatin concentrations of 186 patients and 100 healthy individuals were quantified by enzyme-linked immunosorbent assay. Plasma visfatin concentrations were higher in patients than in healthy individuals (108.5 ± 41.1 ng/mL vs. 13.8 ± 3.9 ng/mL, P  < 0.001). A logistic regression analysis selected plasma visfatin concentration as an independent predictor for 6-month clinical outcomes (both P  < 0.01). Using receiver operating characteristic curve analysis, plasma visfatin concentration was found to predict 6-month clinical outcomes with the high predictive performance. The predictive value of visfatin was in the range of National Institutes of Health Stroke Scale score (both P>  0.05). Combined use of visfatin and National Institutes of Health Stroke Scale score did not improve the predictive significance (both P>  0.05). Thus, visfatin may help in the prediction of long-term clinical outcomes in patients with ischemic stroke.
Keywords: Ischemic stroke; Outcome; Visfatin;

► VIP promotes the proliferation of endothelial cell in RBMECs exposed to OGD. ► VIP increases OGD-induced expression of VEGF and cAMP levels. ► The enhanced VEGF mediates the stimulatory effect of VIP on endothelial cell proliferation. ► VIP-regulated VEGF expression may be related to the activation of cAMP/PKA pathway.Vasoactive intestinal peptide (VIP) enhances angiogenesis in rats with focal cerebral ischemia. In the present study, we investigated the molecular mechanism of the proangiogenic action of VIP using an in vitro ischemic model, in which rat brain microvascular endothelial cells (RBMECs) are subjected to oxygen and glucose deprivation (OGD). Western blotting and immunocytochemistry were carried out to examine the expression of VIP receptors and vascular endothelial growth factor (VEGF) in cultured RBMECs. The cell proliferation was assessed by the MTT assay. Cyclic adenosine monophosphate (cAMP) and VEGF levels were measured by using the enzyme-linked immunosorbent assay. The cultured RBMECs expressed VPAC1, VPAC2 and PAC1 receptors. Treatment with VIP significantly promoted the proliferation of RBMECs and increased OGD-induced expression of VEGF, and this effect was antagonized by the VPAC receptor antagonist VIP6-28 and VEGF antibody. VIP significantly increased contents of cAMP in RBMECs and VEGF in the culture medium. The VIP-induced VEGF production was blocked by H89, a protein kinase A (PKA) inhibitor. These data suggest that treatment with VIP promotes VEGF-mediated endothelial cell proliferation after ischemic insult in vitro, and this effect appears to be initiated by the VPAC receptors leading to activation of the cAMP/PKA pathway.
Keywords: Vasoactive intestinal peptide; Vascular endothelial growth factor; CAMP; Brain microvascular endothelial cells; Oxygen and glucose deprivation;

► The Ghr acted to promote NO release and had an inhibitory effect on hypoxia-induced HPAECs apoptosis. ► Ghr-mediated protective effect was accompanied by the phosphorylation of Akt and eNOS. ► The PI3 K/Akt pathway plays a key role in hypoxia-induced dysfunction in HPAECs. ► The Ghr demonstrates a significant potential to prevent and treat PAH.Endothelial injury and diminished NO release induced by hypoxia is thought to be a critical factor in the development of pulmonary artery hypertension (PAH). Ghrelin (Ghr) is a well-characterized hormone and has protective effects on the cardiovascular system, specifically by promoting the vascular endothelial cell function. The aim of this study was to investigate the effect of the Ghr on the hypoxia-induced injury in human pulmonary artery endothelial cells (HPAECs) and on the involved transduction pathway. Effects were investigated by treating cells with varying concentrations of Ghr in the absence or presence of inhibitors that target phosphoinositide 3-kinase (PI3K), in normoxic or hypoxic conditions for 24 h. Our results indicated that the treatment with 10−7  mol/l Ghr significantly enhanced cell viability (P  < 0.05, n  = 5) and upregulated the ratio of Bcl-2/Bax under hypoxic condition (P  < 0.05, n  = 4), as compared with the hypoxic condition alone. However, an addition of the PI3K/Akt inhibitor LY294002 inhibited these Ghr-mediated effects. Moreover, the Ghr (10−7  mol/l) significantly increased NO secretion and eNOS phosphorylation in comparison with the hypoxia or normoxia alone group (P  < 0.05, n  = 4). Nevertheless, the treatment with LY294002 (20 μ mol/l) decreased the Ghr-induced NO release as well as the eNOS activity. In conclusion, the Ghr could inhibit hypoxia-mediated HPAECs dysfunction via the PI3K/Akt pathway, and the bcl-2/bax ratio was also involved in the protective action of the Ghr in HPAECs. As such, the Ghr demonstrates a significant potential to prevent and treat PAH affected by the endothelial dysfunction.
Keywords: Human pulmonary artery endothelial cell; Ghrelin; Hypoxia; PI3K;

Responses of proenkephalin Peptide F to aerobic exercise stress in the plasma and white blood cell biocompartments by William J. Kraemer; Maren S. Fragala; Wendy R.H. Beijersbergen van Henegouwen; Scott E. Gordon; Jill A. Bush; Jeff S. Volek; N. Travis Triplett; Courtenay Dunn-Lewis; Brett A. Comstock; Tunde K. Szivak; Shawn D. Flanagan; David R. Hooper; Hui-Ying Luk; Andrea M. Mastro (118-124).
► Peptide F is bound to white blood cells in the blood. ► Exercise alters Peptide F in the plasma and white blood cell blood fractions. ► Immune cells show typical responses to exercise stress. ► White blood cells are a target tissue for binding of Peptide F.Proenkephalin Peptide F [107–140] is an enkephalin-containing peptide found predominantly within the adrenal medulla, co-packaged with epinephrine within the chromaffin granules. In vivo studies indicate that Peptide F has classic opioid analgesia effects; in vitro studies suggest potential immune cell interactions. In this investigation we examined patterns of Peptide F concentrations in different bio-compartments of the blood at rest and following sub-maximal cycle exercise to determine if Peptide F interacts with the white blood cell (WBC) bio-compartment during aerobic exercise. Eight physically active men (n  = 8) performed sub-maximal (80–85% V ˙ O 2 peak ) cycle ergometer exercise for 30 min. Plasma Peptide F and WBC Peptide F immunoreactivity were examined pre-exercise, mid-exercise and immediately post-, 5-min post-, 15-min post-, 30-min post- and 60-min post-exercise and at similar time-points during a control condition (30 min rest). Peptide F concentrations significantly (p  < 0.05) increased at 5 and 60 min post-exercise, compared to pre-exercise concentrations. No significant increases in Peptide F concentrations in the WBC fraction were observed during or after exercise. However, a significant decrease was observed at 30 min post-exercise. An ultradian pattern of Peptide F distribution was apparent during rest. Furthermore, concentrations of T cells, B cells, NK cells, and total WBCs demonstrated significant changes in response to aerobic exercise. Data indicated that Peptide F was bound in significant molar concentrations in the WBC fraction and that this biocompartment may be one of the tissue targets for binding interactions. These data indicate that Peptide F is involved with immune cell modulation in the white blood circulatory biocompartment of blood.
Keywords: Enkephalin-containing peptides; Opioid responses to exercise; Buffy coat; Exercise; Immune function;

Effect of PACAP treatment on kidney morphology and cytokine expression in rat diabetic nephropathy by E. Banki; P. Degrell; P. Kiss; K. Kovacs; A. Kemeny; K. Csanaky; A. Duh; D. Nagy; G. Toth; A. Tamas; D. Reglodi (125-130).
► PACAP has protective effects in diabetic nephropathy in rats. ► PACAP attenuates the morphological alterations in the diabetic kidney. ► PACAP conteracts alterations in cytokine expression in diabetic nephropathy.Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide, exerting diverse effects. One of its frequently examined functions is cell protection, which is achieved mainly via inhibiting apoptotic, inflammatory and oxidative processes. All its three receptors (PAC1, VPAC1, VPAC2) are expressed in the kidney and PACAP has been shown to have protective effects against different renal pathologies. Diabetic nephropathy is the leading cause of end stage renal disease. The aim of the present study was to investigate the possible ameliorative effect of PACAP in streptozotocin-induced diabetic nephropathy and to evaluate its anti-inflammatory effect in this model. Diabetes was induced by a single intravenous injection of streptozotocin (65 mg/kg) in male Wistar rats. PACAP-treated animals were administered ip. 20 μg PACAP every second day, while untreated animals were given vehicle. Kidneys were removed after 8-weeks survival. Besides the complex histological analysis (glomerular PAS positive area/glomerulus area, tubular damage, arteriolar hyalinosis), expression of several cytokines was evaluated by cytokine array and Luminex assay. Histological analysis revealed severe diabetic changes in kidneys of control diabetic animals (glomerular PAS-positive area expansion, tubular damage, Armanni-Ebstein phenomenon). PACAP treatment significantly diminished the damage. Diabetic kidneys showed significant cytokine activation compared to their healthy controls. PACAP was effective in downregulation of several cytokines including CINC-1, TIMP-1, LIX, MIG, s-ICAM. To conclude, PACAP is effective in ameliorating diabetic nephropathy at least partly through its well-known anti-inflammatory effect. These results raise the opportunity for the use of PACAP as a possible therapeutic or preventive method in treating the complications of diabetes.

Angiotensin III stimulates high stretch-induced ANP secretion via angiotensin type 2 receptor by Byung Mun Park; Young-Bin Oh; Shan Gao; Seung Ah Cha; Kyung Pyo Kang; Suhn Hee Kim (131-137).
► Angiotensin III stimulated high stretch-inducedANP secretion. ► These effects were mediated through AT2 receptor/PI3K/Akt/NO/PKG pathway. ► Intravenous infusion of subpressor dose of angiotensin III increased plasma ANP level whereas angiotensin II decreased it.Angiotensin III (Ang III) is metabolized from Ang II by aminopeptidase (AP) A and in turn, Ang III is metabolized to Ang IV by APN. Ang III is known to have a similar effect to Ang II on aldosterone secretion, but the effect of Ang III on atrial natriuretic peptide (ANP) secretion from cardiac atria is not known. The aim of the present study is to define the effect of Ang III on ANP secretion and its receptor subtype using isolated perfused beating atria. The volume load was achieved by elevating the height of outflow catheter connected with isolated atria from 5 cmH2O to 7.5 cmH2O. Atrial stretch by volume load increased atrial contractility and ANP secretion. Ang III stimulated stretch-induced ANP secretion in a dose-dependent manner without change in atrial contractility. The stimulated effect of Ang III (1 μM) on stretch-induced ANP secretion was blocked by the pretreatment of Ang II type 2 (AT2) receptor antagonist but not by AT1 or Mas receptor antagonist. Pretreatment with inhibitor of phosphoinositide 3-kinase (PI3K), Akt, nitric oxide synthase, soluble guanylyl cyclase, or protein kinase G (PKG) attenuated Ang III-stimulated ANP secretion. When Ang III (40 nM) or Ang II (4 nM) was infused for 10 min into anesthetized rats, mean arterial pressure was increased about 10%. However, Ang III increased plasma ANP level by 35.81 ± 10.19% but Ang II decreased plasma ANP level by 30.41 ± 7.27%. Therefore, we suggest that Ang III, opposite to Ang II, stimulated stretch-induced ANP secretion through AT2 receptor/PI3K/Akt/nitric oxide/PKG pathway.
Keywords: Hypertension; Hypertrophy; Atrial natriuretic peptide; Angiotensin III; Angiotensin II; Receptor; Atrial stretch;

Serum chemerin levels during normal human pregnancy by Maria F. Garces; Elizabeth Sanchez; Ariel I. Ruíz-Parra; Jorge Andrés Rubio-Romero; Edith Angel-Müller; Miguel A. Suarez; Luisa F. Bohórquez; Susana B. Bravo; Rubén Nogueiras; Carlos Diéguez; Jorge E. Caminos (138-143).
► A cross-sectional study was carried out with 60 pregnant women and a control group. ► Serum concentrations of biochemistries, chemerin, and adiponectin were measured. ► Chemerin levels did not vary in the early follicular and midluteal phases. ► Chemerin rose significantly and is negatively associated with adiponectin levels. ► Further study needed to investigate the use of chemerin as a clinical biomarker.During gestation there are important changes in maternal metabolism and an increase in insulin resistance, coinciding with an increase in adiposity. Chemerin is an adipocytokine which is expressed and secreted in various tissues, including placenta, and may play an important role in metabolic regulation during pregnancy. The aim of this study was to determine serum levels of chemerin during gestation and compare them to other indicators of insulin resistance. A cross-sectional study was carried out analyzing serum chemerin levels of 20 pregnant women during three gestational periods, early, middle, and late (between the 10th and 14th, the 23rd and 26th, and the 34th and 37th week) and 20 non-pregnant women were used as a control group. An analysis of chemerin levels during the menstrual cycle was performed in an eumenorrheic group (n  = 16) in the early follicular (cycle day 4 ± 1) and the midluteal phase (cycle day 22 ± 1), demonstrating that serum chemerin levels did not fluctuate significantly. Serum levels of chemerin were significantly elevated during late gestation when compared to early (P  < 0.001) and middle (P  = 0.001) gestation and a negative correlation between serum chemerin and adiponectin levels (r  = −0.1643) became more significant when the non-pregnant group was included in the calculations (r  = −0.2471). There was no significant association of triglycerides, total cholesterol, LDL, HDL, insulin, and HOMA levels with chemerin. Although chemerin rose significantly and is negatively associated with adiponectin levels, it is not correlated with other markers of insulin sensitivity, suggesting that more study is needed to determine whether chemerin is useful in predicting insulin resistance during gestation.
Keywords: Chemerin; Human; Gestation; Adipokines; Adiponectin;

In vivo antimicrobial evaluation of an alanine-rich peptide derived from Pleuronectes americanus by Leandro D. Teixeira; Osmar N. Silva; Ludovico Migliolo; Isabel C.M. Fensterseifer; Octavio L. Franco (144-148).
Display Omitted► An antimicrobial peptide Pa-MAP exhibited in vivo activity against Escherichia coli.Pa-MAP has an effective activity at lower concentrations when compared to other antibiotics. ► Pa-MAP prevents weight loss during infected mice treatment. ► No immunomodulatory activity of Pa-MAP was observed in this study.In several organisms, the first barrier against microbial infections consists of antimicrobial peptides (AMPs) which are molecules that act as components of the innate immune system. Recent studies have demonstrated that AMPs can perform various functions in different tissues or physiological conditions. In this view, this study was carried out in order to evaluate the multifunctional activity in vivo of an alanine-rich peptide, known as Pa-MAP, derived from the polar fish Pleuronectes americanus. Pa-MAP was evaluated in intraperitoneally infected mice with a sub-lethal concentration of Escherichia coli at standard concentrations of 1 and 5 mg kg−1. At both concentrations, Pa-MAPs exhibited an ability to prevent E. coli infection and increase mice survival, similar to the result observed in mice treated with ampicillin at 2 mg kg−1. In addition, mice were monitored for weight loss. The results showed that mice treated with Pa-MAPs at 1 mg kg−1 gained 0.8% of body weight during the 72 h of experiment. The same was observed with Pa-MAP at 5 mg kg−1, which had a gain of 0.5% in body weight during the treatment. Mice treated with ampicillin at 2 mg kg−1 show a significant weight loss of 5.6% of body weight. The untreated group exhibited a 5.5% loss of body weight. The immunomodulatory effects were also evaluated by the quantification of IL-10, IL-12, TNF-α, IFN-γ and nitric oxide cytokines in serum, but no immunomodulatory activity was observed. Data presented here suggest that Pa-MAP should be used as a novel antibiotic against infection control.
Keywords: Antimicrobial peptides; Promiscuity; Immunomodulatory; in vivo infections;