Peptides (v.37, #2)

Intermedin ameliorates atherosclerosis in apoE null mice by modifying lipid profiles by Xin Zhang; Lunda Gu; Xiaoping Chen; Si Wang; Xiaoming Deng; Kai Liu; Zhengbing Lv; Rui Yang; Sen He; Yong Peng; Dejia Huang; Wei Jiang; Kai Wu (189-193).
► High amount of exogenous IMD could relieve atherosclerosis. ► IMD could relieve atherosclerosis via decrease TC and LDL-C or increase HDL-C levels in plasma. ► The mechanism related to improve dyslipidimia.Intermedin (IMD) is a recently discovered vasodilator peptide. We studied the role of IMD in the pathogenesis of atherosclerosis by investigating the ability of exogenous IMD to alter lipid profiles and ameliorate the development of atherogenic-diet induced atherosclerosis in ApoE−/− mice. Ten of eight-week-old male C57BL/6J mice were as control. Thirty of eight-week-old male ApoE−/− mice were fed with an atherogenic diet for 18 weeks. After feeding atherogenic diet for 12 weeks, the mice were equally and randomly divided into three groups. Normal saline was given in group A and C57BL/6J mice. Intermedin was given by mini osmotic pumps at the dosage of 100 ng/kg/h and 500 ng/kg/h in group B and group C respectively. After the treatment of IMD for 6 weeks, aortic ultrasonography of group C showed that IMD prevented the progression of atherosclerotic lesions and the increase of wall thickness in the aorta. Oil-red-O staining of the entire aorta and the atherosclerotic aortic root section showed 2 folds decrease atherogenic plaque (p  < 0.05). Serum lipid profiles were measured, compared with the group A, in group C TC and LDL-C levels were decreased by 86.32% and 89.68%, respectively (both p  < 0.05), meanwhile, HDL-C level was significantly increased by 74.82% (p  < 0.05). These data indicate that exogenous administration of IMD could prevent the progression of atherosclerotic plaque. The possible underlying mechanisms may relate to the improvement of lipid profiles.
Keywords: Intermedin; Atherosclerosis; Dyslipidimia; Cholesterol; LDL-C; HDL-C;

Bombesin-like peptides stimulate growth hormone secretion mediated by the gastrin-releasing peptide receptor in cattle by Hongqiong Zhao; Seinosuke Matsuda; Sint ThanThan; Swe Yannaing; Hideto Kuwayama (194-199).
► Effect of bombesin-like peptides on growth hormone secretion was tested in cattle. ► Growth hormone level was increased by gastrin-releasing peptide (GRP). ► Growth hormone level was unaffected by neuromedin B. ► The GRP-induced growth hormone secretion was mediated by GRP receptors. ► Ghrelin/GHS-R system was not involved in GRP-induced growth hormone secretion.This study was designed to determine the effects of bombesin-like peptides (BLPs) on the secretion of growth hormone (GH) and to characterize the receptor subtypes mediating these effects in cattle. Four experiments were conducted: (1) six steers were randomly assigned to receive intravenous (IV) bolus injections of 0, 0.2, 1.0, 12.5 and 50.0 μg/kg neuromedin C (NMC); (2) seven pre-weaned calves were IV injected with 1.0 μg/kg NMC; (3) six steers were IV injected with 2.5 μg/kg bovine gastrin-releasing peptide (GRP), 1.0 μg/kg NMC combined with 20.0 μg/kg [d-Lys3]-GHRP-6 (an antagonist for the GH secretagogue receptor type 1a [GHS-R1a]), 1.0 μg/kg NMC combined with 20.0 μg/kg N-acetyl-GRP20–26-OCH2CH3 (N-GRP-EE, an antagonist for the GRP receptor), 20.0 μg/kg N-GRP-EE alone, 1.0 μg/kg neuromedin B (NMB); and (4) four rats were IV injected 1.0 μg/kg NMC. A serial blood sample was collected before and after injection. Plasma GH levels dose-dependently increased at 5 min after NMC injection and the minimal effective dose was 1.0 μg/kg. Plasma GH level was elevated by GRP, but not by NMB. The NMC-induced elevation of GH was completely blocked by N-GRP-EE. The administration of NMC elevated GH level in pre-weaned calves but not in rats. Ghrelin level was unaffected by any treatments; and [d-Lys3]-GHRP-6 did not block the NMC-induced elevation of GH. The results indicate BLP-induced elevation of GH levels is mediated by the GRP receptor but not through a ghrelin/GHS-R1a pathway in cattle.
Keywords: Bombesin-like peptide; Gastrin-releasing peptide; Neuromedin C; Growth hormone; GRP receptor; Bovine;

Similarities and differences between effects of angiotensin III and angiotensin II on human prostate cancer cell migration and proliferation by Kamila Domińska; Agnieszka Wanda Piastowska-Ciesielska; Agnieszka Lachowicz-Ochędalska; Tomasz Ochędalski (200-206).
► Ang III and Ang II can regulate cell migration and proliferation in prostate cancer lines. ► The biological properties of angiotensin are associated with the hormonal status and invasion potential of prostate cancer cells. ► Ang II enhances the migratory character of androgen-independent prostate cancer cells. ► Ang III is responsible for stimulating the migration of both androgen-dependent and -independent prostate cancer cell lines. ► The cell response to angiotensin peptides is played by changes in the expression of AT1 and AT2 receptor.Proliferation plays a critical role in tumor growth when cell migration is essential to invasion. The effect of Ang III and Ang II was evaluated on these important processes. Changes in the migration potential of prostate cancer cells were investigated using Wound Healing Test and a Transwell Migration Chamber with a 3 μm pore size. Cell proliferation was measured with a BrdU Assay and Countess Automated Cell Counter, thus determining the influence of angiotensins on hormone-dependent (LNCaP) and hormone-independent (DU-145) human prostate cancer lines. The influence of Ang III and Ang II on classic receptors may be inhibited by Losartan or PD123319. Test peptide modulation of the AT1 and AT2 receptors was examined by Western Blot and fluorescent immunocytochemistry. The results indicate that Ang III promotes the migration of both LNCaP and DU-145 lines, whereas Ang II stimulates this process only in androgen-independent cells. Both angiotensin peptides can induce prostate cancer cell proliferation in a time- and dose-dependent manner. The obtained results show that Ang III and Ang II can modify the expression of classic receptors, particularly AT2. These results suggest that the investigated peptide can modulate cell migration and proliferation in prostate cancer cells. Angiotensins probably have a greater influence on proliferation in the early-stage prostate cancer model than hormone-independent cell lines. Assume also that Ang II can enhance the migration tendency aggressive prostate cancer cells, while Ang III does so more effective in non-metastatic cells.
Keywords: Angiotensin III; Angiotensin II; AT1; AT2; Cell migration; Cell proliferation; Prostate cancer;

Overview on the recent study of antimicrobial peptides: Origins, functions, relative mechanisms and application by Yanmei Li; Qi Xiang; Qihao Zhang; Yadong Huang; Zhijian Su (207-215).
► Antimicrobial peptides (AMPs) produced by several species including insects, other animals, micro organisms and synthesis, are a critical component of the natural defense system. ► With the growing problem of pathogenic organisms resistant to conventional antibiotics, especially with the emergence of NDM-1, there is increased interest in the pharmacological application of AMPs. ► They can protect against a broad array of infectious agents, such as bacteria, fungi, parasite, virus and cancer cells. ► AMPs have a good future in the application in pharmaceuticals industry and food additive.Antimicrobial peptides (AMPs), which are produced by several species including insects, other animals, micro-organisms and synthesis, are a critical component of the natural defense system. With the growing problem of pathogenic organisms resistant to conventional antibiotics, especially with the emergence of NDM-1, there is increased interest in the pharmacological application of AMPs. They can protect against a broad array of infectious agents, such as bacteria, fungi, parasite, virus and cancer cells. AMPs have a very good future in the application in pharmaceuticals industry and food additive. This review focuses on the AMPs from different origins in these recent years, and discusses their various functions and relative mechanisms of action. It will provide some detailed files for clinical research of pharmaceuticals industry and food additive in application.
Keywords: Antimicrobial peptides; Origins; Functions; Mechanisms; Application;

Endothelium dependent expression and underlying mechanisms of des-Arg9-bradykinin-induced B1R-mediated vasoconstriction in rat portal vein by Fernanda L. Basei; Daniela A. Cabrini; Cláudia P. Figueiredo; Stefânia Forner; Daniela B. Hara; Andrey F.Z. Nascimento; Graziela S. Ceravolo; Maria Helena C. Carvalho; Michael Bader; Rodrigo Medeiros; João B. Calixto (216-224).
► Induction of endothelial B1R expression mediates portal vein contraction. ► Endothelial B1R expression is dependent of phosphorylation level of PI3K. ► PKC and PI3K regulate activation of transcription factors, CREB and AP-1, but not NF-κB in portal vein contraction. ► The COX-2 expression and activity are increased in the portal endothelium after in vitro incubation. ► Stimulation of endothelial B1R raises PLA2/COX-2-derived TXA2 levels in the portal vein.Endothelial dysfunction has been implicated in portal vein obstruction, a condition responsible for major complications in chronic portal hypertension. Increased vascular tone due to disruption of endothelial function has been associated with an imbalance in the equilibrium between endothelium-derived relaxing and contracting factors. Herein, we assessed underlying mechanisms by which expression of bradykinin B1 receptor (B1R) is induced in the endothelium and how its stimulation triggers vasoconstriction in the rat portal vein. Prolonged in vitro incubation of portal vein resulted in time- and endothelium-dependent expression of B1R and cyclooxygenase-2 (COX-2). Inhibition of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI3K) significantly reduced expression of B1R through the regulation of transcription factors, activator protein-1 (AP-1) and cAMP response element-binding protein (CREB). Moreover, pharmacological studies showed that B1R-mediated portal vein contraction was reduced by COX-2, but not COX-1, inhibitors. Notably, activation of endothelial B1R increased phospholipase A2/COX-2-derived thromboxane A2 (TXA2) levels, which in turn mediated portal vein contraction through binding to TXA2 receptors expressed in vascular smooth muscle cells. These results provide novel molecular mechanisms involved in the regulation of B1R expression and identify a critical role for the endothelial B1R in the modulation of portal vein vascular tone. Our study suggests a potential role for B1R antagonists as therapeutic tools for diseases where portal hypertension may be involved.

Orexin A in the ventral tegmental area induces conditioned place preference in a dose-dependent manner: Involvement of D1/D2 receptors in the nucleus accumbens by Zahra Taslimi; Reza Arezoomandan; Alireza Omranifard; Mohadeseh Ghalandari-Shamami; Esmail Riahi; Abbas Ali Vafaei; Ali Rashidy-Pour; Abbas Haghparast (225-232).
► Administration of orexin A in the VTA induced CPP in a dose-dependent manner in rat. ► Intra-accumbal D1 receptor antagonist reduced orexin-induced CPP in the VTA. ► Blocking D2 receptors in NAc dose-dependently reduced intra-VTA orexin-induced CPP. ► Intra-NAc administration of D1/D2 receptor antagonists alone could not induce CPP.It has been shown that orexin A in the ventral tegmental area (VTA) is necessary for development of morphine place preference. Additionally, D1 and D2 dopamine receptors in the nucleus accumbens (NAc) have critical roles in motivation and reward. However, little is known about the function of orexin in conditioned place preference (CPP) in rats and involvement of D1/D2 receptors in the NAc. In the present study, we investigated the effect of direct administration of orexin A into the VTA, and examined the role of intra-accumbal dopamine receptors in development (acquisition) of reward-related behaviors in the rats. Adult male Wistar rats were unilaterally implanted by two separate cannulae into the VTA and NAc. The CPP paradigm was used, and, conditioning score and locomotor activity were recorded by Ethovision software. The results showed that unilateral intra-VTA administration of orexin A (27, 53 and 107 ng/0.3 μl saline) during conditioning phase induced CPP in a dose-dependent manner. The most effective dose of intra-VTA orexin-A in eliciting CPP was 107 ng. However, intra-NAc administration of SCH 23390 (0.25, 1 and 4 μg/0.5 μl saline), a D1 receptor antagonist, and sulpiride (0.25, 1 and 4 μg/0.5 μl DMSO), a D2 receptor antagonist, inhibited the development of orexin-induced CPP. The inhibitory effect of D2 but not D1 receptor antagonist was exerted in a dose-dependent manner. It is supposed that the activation of VTA dopaminergic neuron by orexin impresses the D2 receptors more than D1 receptors in the NAc.
Keywords: Reward; Orexin; Ventral tegmental area; Nucleus accumbens; D1 dopamine receptor; D2 dopamine receptor; Conditioned place preference; Rat;

The bioactive peptides salusins and apelin-36 are produced in human arterial and venous tissues and the changes of their levels during cardiopulmonary bypass by Suna Aydin; Mehmet Nesimi Eren; Suleyman Aydin; İbrahim Hanefi Ozercan; Adile Ferda Dagli (233-239).
► Immunoreactive salusin α and β and apelin-36 were detected in human the arteries and veins. ► The locally produced salusin-α and β might regulate the local function of those tissues. ► The amount of salusin-β in the circulation was 5–6 times larger than that of salusin-α. ► Salusins and apelin-36 might be important markers in the cardiopulmonary bypass follow up.This study aimed to examine the effects of CPB on salusin-α, salusin-β and apelin-36 bioactive peptides in people who are planned to undergo coronary artery bypass graft (CABG) operation due to coronary artery disease and to explore whether these peptides are produced in human aortic, saphenous and arterial tissues. The study included age and BMI matched 15 patients who underwent CABG operation by CPB. In order to determine salusin-α, salusin-β and apelin-36 levels, venous blood samples were collected before induction of anesthesia (T1), before CPB (T2), 5 min before the removal of cross-clamp (T3), 5 min after the removal of cross-clamp (T4), upon arrival in the intensive care (T5), at postoperative 24th hour (T6) and 72nd hour (T7). Salusin and apelin expressions of the tissues were shown by immunohistochemical method. Peptide amounts of sera and tissues were measured using ELISA. Salusins production by vessels occurs in fibroblast cells of the media in the aorta and smooth muscle cells of the media in the LIMA and saphena. Apelin is produced by endothelial cells of the intima and fibroblast cells of the media in the aorta and by smooth muscle cells of the media in the LIMA and saphena. Changes in the levels of salusin-β and apelin-36 were significant during CPB. Salusin-α, salusin-β and apelin-36 are locally synthesized in the arteries and veins. Salusins and apelin-36 might be important markers in the CPB, and also that salusin-β was more specific in comparison to salusin-α.
Keywords: Salusin-α; Salusin-β; Apelin-36; Bioactive peptides; Coronary artery bypass; LIMA; Saphena;

Gene expression of C-type natriuretic peptide and of its specific receptor NPR-B in human leukocytes of healthy and heart failure subjects by M. Cabiati; L. Sabatino; R. Caruso; C. Caselli; T. Prescimone; D. Giannessi; S. Del Ry (240-246).
► C-type natriuretic peptide (CNP) is synthesized and secreted from monocytes and macrophages. ► CNP could have a possible role in the immune system. ► CNP and NPR-B were detected by Real-Time PCR in leukocytes obtained from small human whole blood samples.C-type natriuretic peptide (CNP), a member of the family of natriuretic peptides, is synthesized and secreted from monocytes and macrophages that resulted to be a source of CNP at inflammatory sites. This suggests that special attention should be focused on the possible role of CNP in the immune system, in addition to its effects on the cardiovascular system. The aim of this study was to evaluate the possibility of measuring the mRNA expression of CNP and NPR-B, its specific receptor, in human whole blood samples of healthy (N; n  = 7) and heart failure (HF; n  = 7) subjects by Real-Time PCR (RT-PCR). Total RNA was extracted from leukocytes with QIAamp RNA Blood Kit and/or with PAXgene Blood RNA Kit. RT-PCR was performed and optimized for each primer. The experimental results were normalized with the three most stably expressed genes. CNP and NPR-B expression trend was similar in both fresh and frozen human whole blood. Significant higher levels of CNP and NPR-B mRNA expression were found in HF patients with respect to controls (CNP: N  = 1.23 ± 0.33 vs. HF = 6.54 ± 2.09 p  = 0.027; NPR-B: N  = 0.85 ± 0.23 vs. HF = 5.31 ± 1.98 p  = 0.04). A significant correlation between CNP and NPR-B (r  = 0.86, p  < 0.0001) was observed. Further studies are needed to clarify the pathophysiological properties of this peptide but the possibility to measure CNP and NPR-B mRNA expression in human leukocytes with a fast and easy procedure is a useful starting point for future investigation devoted to better understand the biomolecular processes associated to different diseases.
Keywords: C-type natriuretic peptides; Natriuretic peptide receptor; Real-Time PCR; mRNA expression; Blood; Human leukocytes; Heart failure;

Decreased hepatic gluconeogenesis in transgenic rats with increased circulating angiotensin-(1-7) by Victor Bilman; Lucas Mares-Guia; Ana Paula Nadu; Michael Bader; Maria José Campagnole-Santos; Robson Augusto S. Santos; Sérgio Henrique S. Santos (247-251).
► Angiotensin-(1-7) improves glucose metabolism. ► Angiotensin-(1-7) decreases hepatic gluconeogenesis. ► Angiotensin-(1-7) alters liver metabolic function.The renin–angiotensin (Ang) system (RAS) plays an important role in the control of glucose metabolism and glycemia. Several studies demonstrated that the effects of angiotensin-(1-7) are mainly opposite to the actions of biological angiotensin II. Recent studies have demonstrated that rats with increased circulating angiotensin-(1-7), acting through the G protein coupled receptor Mas, have enhanced glucose tolerance and insulin sensitivity, presenting improved metabolic parameters. However, there is no data regarding the role of angiotensin-(1-7)–Mas axis in hepatic glycemic metabolism. In the present study, the gluconeogenesis and glycogenolysis was investigated in Sprague–Dawley (SD) and in TGR(A1-7)3292 (TGR) rats which present approximately twofold increase in plasma Ang-(1-7) levels compared to SD. The pyruvate administration in fasted rats showed a decreased synthesis of glucose in TGR compared to the SD rats, pointing to a downregulation of gluconeogenesis. Supporting this data, the mRNA evaluation of gluconeogenic enzymes showed a significant reduction in phosphoenolpyruvate carboxykinase reinforced by a significantly diminished expression of hepatocyte nuclear factor 4α (HNF-4α), responsible for the regulation of gluconeogenic enzymes. In conclusion our data show that the improved glucose metabolism induced by Ang-(1-7) could be due, at least in part, to a downregulation of hepatic gluconeogenesis.

Secretoneurin, substance P and neuropeptide Y in the oxygen-induced retinopathy in C57Bl/6N mice by Eduard Schmid; Marina Nogalo; Nikolaos E. Bechrakis; Reiner Fischer-Colbrie; Ramon Tasan; Günther Sperk; Markus Theurl; Arno G.E. Beer; Rudolf Kirchmair; Herbert Herzog; Josef Troger (252-257).
► Substance P, secretoneurin and neuropeptide Y feature proangiogenic activities. ► They are not involved in the development of abnormal neovascularizations in OIR. ► Neuropeptide Y might participate in the development of the retina.In this study, we investigated whether the proangiogenic neuropeptides secretoneurin (SN), substance P (SP), and neuropeptide Y (NPY) contribute to the development of abnormal neovascularization in the oxygen-induced retinopathy (OIR) model in mice. By exposing litters of C57Bl/6N mice to 75% oxygen from postnatal day 7 (P7) until postnatal day 11 (P11) and then returning them to normoxic conditions, retinal ischemia and subsequent neovascularization on the retinal surface were induced. Retinae were dissected on P9, P11, P12–P14, P16 and P20, and the concentrations of SN, SP, NPY and VEGF determined by radioimmunoassay or ELISA. The levels of SN and SP increased in controls from P9 until P16 and from P9 until P14, respectively, whereas the levels of NPY were high at P9 and decreased thereafter until P20, suggesting that NPY may participate in the development of the retina. However, dipeptidyl peptidase IV (DPPIV) and the NPY-Y2 receptor were not detectable in the immature retina indicating that NPY is not involved in the physiological vascularization in the retina. Compared to controls, OIR had no effect on the levels of SN, whereas levels of both SP and NPY slightly decreased during hyperoxia. Normalization of the levels of SP, and to a more pronounced extent of NPY, was significantly delayed during relative hypoxia. This clearly indicates that these three neuropeptides are not involved in the pathogenesis of neovascularization in OIR. Moreover, since there were no differences in the expression of two vessel markers in the retina of NPY knockout mice versus controls at P14, NPY is also not involved in the delayed development of the intermediate and deep vascular plexus in the retina in this animal model.
Keywords: Substance P; Secretoneurin; Neuropeptide Y; Oxygen-induced retinopathy; Mice;

C-terminal FGF23 is a strong predictor of survival in systolic heart failure by Damien Gruson; Thibault Lepoutre; Jean-Marie Ketelslegers; Jean Cumps; Sylvie A. Ahn; Michel F. Rousseau (258-262).
► Fibroblast growth factor 23 is an important of phosphate and calcium metabolism. ► C-terminal FGF23 is a 71 amino acids peptide. ► We have evaluated Ct-FGF23 in patients presenting systolic heart failure. ► Ct-FGF23 levels were markedly increased in HF patients. ► Ct-FGF23 is the strongest predictor of long term CV death.Fibroblast growth factor 23 (FGF23) is a bone-derived hormone involved in the regulation of phosphate and calcium metabolism. We have evaluated the levels of C-terminal FGF23 (Ct-FGF23) in 73 patients presenting heart failure with reduced ejection fraction (HF-REF) and assess their potential predictive value for long-term survival through a 6 years follow-up. Ct-FGF23 levels were markedly increased in HF-REF. In univariate proportional hazard model, survival was related to glomerular filtration rate (eGFR), intact parathyroid hormone (PTH), B-type natriuretic peptides (BNP) and Ct-FGF23. In a multivariate analysis including age, EF, PTH, BNP, Ct-FGF23, calcium, phosphorus and eGFR levels, Ct-FGF23 is the strongest predictor of long term CV death.
Keywords: Ct-FGF23; Heart Failure; BNP; Prognosis;

Tryptophan-containing milk protein-derived dipeptides inhibit xanthine oxidase by Alice B. Nongonierma; Richard J. FitzGerald (263-272).
► W, WV and VW were shown to inhibit xanthine oxidase (XO) in a non-competitive fashion. ► W, WV and VW are 200 times less potent than Allopurinol a competitive drug of XO. ► Bovine lactoferrin (LF) hydrolyzed with gastrointestinal enzyme preparations generated W containing peptides could inhibit XO. ► Fractionation of a LF hydrolyzate with an activated carbon SPE cartridge allowed enrichment of W containing peptides. ► One SPE fraction enriched in W containing peptides was more potent than the hydrolyzate.Of twelve dipeptides tested, only the Trp containing peptides Val-Trp and its reverse peptide Trp-Val showed a xanthine oxidase (XO) inhibitory activity. Studies with Val and Trp revealed that XO inhibition was mainly attributed to the Trp residue. No significant difference (P  ≥ 0.05) was found for the XO inhibitory potency (IC50) values for Trp, Val-Trp and Trp-Val, which were about 200 times higher than that for Allopurinol. Lineweaver and Burke analysis demonstrated that Trp, Val-Trp and Trp-Val were non-competitive inhibitors while Allopurinol was a competitive inhibitor. Of the different milk-protein substrates hydrolyzed with gastro-intestinal enzyme activities, only lactoferrin (LF) hydrolyzates displayed XO inhibition. Peptides present in a LF hydrolyzate (GLF-240 min) were adsorbed onto activated carbon followed by subsequent desorption with stepwise elution using acetonitrile (ACN). Separation and detection of Trp containing peptides within the different fractions were achieved using RP-HPLC coupled with fluorescence detection. The desorbed fractions displayed different XO inhibitory properties, with no inhibition in the unbound fraction and highest inhibition in fractions eluted with 30, 40 and 70% ACN. The fraction eluting at 40% ACN was significantly more potent (19.1 ± 2.3% inhibition at 1.25 mg mL−1) than the GLF-240 min hydrolyzate (13.4 ± 0.4% inhibition at 1.25 mg mL−1), showing the potential for enrichment of the bioactive peptides on fractionation with activated carbon.
Keywords: Xanthine oxidase inhibition; Tryptophan; Bioactive peptides; Lactoferrin; Activated carbon; Antioxidant;

Protein transduction in human cells is enhanced by cell-penetrating peptides fused with an endosomolytic HA2 sequence by Ji-Sing Liou; Betty Revon Liu; Adam L. Martin; Yue-Wern Huang; Huey-Jenn Chiang; Han-Jung Lee (273-284).
Display Omitted► DNA plasmids containing coding sequences of CPP, HA2, and RFP were constructed. ► Endocytosis is the major route for cellular uptake of CPP-HA2-tagged RFPs. ► HA2 tag of the CPP-HA2 fusion protein improved cytosolic uptake in human A549 cells. ► CPP-HA2-tagged RFP fusion proteins did not display cytotoxicity. ► The CPP-HA2 tag could be an efficient and safe carrier of bioactive molecules.Endocytosis has been proposed as one of the primary mechanisms for cellular entry of cell-penetrating peptides (CPPs) and their cargoes. However, a major limitation of endocytic pathway is entrapment of the CPP-cargo in intracellular vesicles from which the cargo must escape into the cytoplasm to exert its biological activity. Here we demonstrate that a CPP tagged with an endosomolytic fusion peptide derived from the influenza virus hemagglutinin-2 (HA2) remarkably enhances the cytosolic delivery of proteins in human A549 cells. To determine the endosome-disruptive effects, recombinant DNA plasmids containing coding sequences of HA2, CPPs and red fluorescent proteins (RFPs) were constructed. The fusion proteins were purified from plasmid-transformed Escherichia coli, and their effects on protein transduction were examined using live cell imaging and flow cytometry. Our data indicate that endocytosis is the major route for cellular internalization of CPP-HA2-tagged RFP. Mechanistic studies revealed that the fusogenic HA2 peptide dramatically facilitates CPP-mediated protein entry through the release of endocytosed RFPs from endosomes into the cytoplasm. Furthermore, incorporating the HA2 fusion peptide of the CPP-HA2 fusion protein improved cytosolic uptake without causing cytotoxicity. These findings strongly suggest that the CPP-HA2 tag could be an efficient and safe carrier that overcomes endosomal entrapment of delivered therapeutic drugs.
Keywords: Cell-penetrating peptide; Cytotoxicity; Endosomal escape; Hemagglutinin-2; Membrane fusion;

Expression of (pro)renin receptor in human erythroid cell lines and its increased protein accumulation by interferon-γ by Kiriko Kaneko; Hiroshi Nishiyama; Koji Ohba; Akiko Shibasaki; Takuo Hirose; Kazuhito Totsune; Kazumichi Furuyama; Kazuhiro Takahashi (285-289).
► (Pro)renin receptor is expressed in erythroid cells. ► (Pro)renin receptor protein accumulation in erythroid cells was increased by interferon-γ. ► (Pro)renin receptor may have a role in erythropoiesis and the pathophysiology of certain types of anemia.The renin–angiotensin system is known to enhance erythropoiesis. (Pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, has recently been identified. However, expression of (P)RR in erythroid cells has not been studied. The aim of the present study is to clarify expression of (P)RR in erythroid cells, and the effects of erythropoietin, angiotensin II, transforming growth factor-β1 (TGF-β1), interferon-γ (IFN-γ) and interleukin-1β (IL-1β) on its expression. Western blot analysis showed that (P)RR protein was expressed in human cultured erythroid cell lines, YN-1 and YN-1-0-A (a clonal variant cell line of YN-1). Erythropoietin (1 IU/ml) increased (P)RR mRNA expression levels in YN-1-0-A cells (1.7-fold increase compared with control), but angiotensin II did not. Treatment of YN-1-0-A cells with IFN-γ (10 ng/ml) for 48 h increased the expression levels of (P)RR protein significantly (1.4-fold increase compared with control), whereas it had no significant effects on expression levels of (P)RR mRNA. Treatment of YN-1-0-A cells with TGF-β1 or IL-1β for 24 or 48 h had no significant effects on expression levels of (P)RR. The present study has shown for the first time expression of (P)RR in erythroid cells, raising the possibility that (P)RR may have a role in erythropoiesis and the pathophysiology of certain types of anemia.
Keywords: (Pro)renin receptor; Prorenin; Erythropoiesis; Erythroid; Angiotensin; Cytokine; Interferon; Erythropoietin;

Characterization of a distinctive pattern of periovulatory leptin secretion and its relationship with ovulation rate and luteal function in swine with obesity/leptin resistance by Antonio Gonzalez-Bulnes; Susana Astiz; Teresa Encinas; Pedro Gonzalez-Añover; Mariluz Perez-Solana; Raul Sanchez-Sanchez; Laura Torres-Rovira; Jesus A.F. Tresguerres (290-293).
► Sows with obese genotype have a periovulatory increase in leptinemia, not found in lean dams. ► Such pattern of leptin secretion is independent of changes in body condition and fatness. ► Concentration of leptin around estrus onset is positively related to ovulation rate in obese swine. ► Obese sows with higher leptinemia at estrus have lower progesteronemia in the following cycle.Patterns of leptin secretion during the estrous cycle and the possible relationship of changes in circulating leptin during the periovulatory period with ovarian function in sows of obese (Iberian breed) and lean genotype (Large White × Landrace) were evaluated in two consecutive experiments. Plasma leptin concentrations throughout the estrous cycle in lean sows remain unchanged, but Iberian females showed a periovulatory increase in circulating leptin levels without associated changes in body condition and fatness. In these sows, plasma leptin concentrations at Days −1 and 0 of the cycle were found to be positively correlated with the ovulation rate (r  = 0.943 and r  = 0.987, respectively; P  < 0.05 for both), but the levels of leptin at Day 0 were negatively correlated with the progesterone release from Day 3 (r  = −0.557; P  < 0.05) and, became more evident at Day 5 of the estrous cycle (r  = −0.924; P  < 0.005). Such relationships were not observed in the females of the lean genotype. In conclusion, the present study indicates the existence of a distinctive pattern in the periovulatory leptin secretion in swine with obesity and leptin resistance, which is associated with the number and functionality of the corpora lutea present in the subsequent cycle.
Keywords: Leptin; Luteal-function; Ovulation-rate; Obesity; Swine;

Antimicrobial activity of recombinant Pg-AMP1, a glycine-rich peptide from guava seeds by Letícia Stephan Tavares; João Vitor Rettore; Renata Mendes Freitas; William Farias Porto; Ana Paula do Nascimento Duque; Júnya de Lacorte Singulani; Osmar Nascimento Silva; Michelle de Lima Detoni; Eveline Gomes Vasconcelos; Simoni Campos Dias; Octávio Luiz Franco; Marcelo de Oliveira Santos (294-300).
.Display Omitted► Pg-AMP1 was expressed and purified in heterologous peptide. ► The expressed peptide Pg-AMP1 showed activity against human pathogenic bacteria. ► Theoretical structure was proposed in order to provide mechanistic insights.Antimicrobial peptides (AMPs) are compounds that act in a wide range of physiological defensive mechanisms developed to counteract bacteria, fungi, parasites and viruses. These molecules have become increasingly important as a consequence of remarkable microorganism resistance to common antibiotics. This report shows Escherichia coli expressing the recombinant antimicrobial peptide Pg-AMP1 previously isolated from Psidium guajava seeds. The deduced Pg-AMP1 open reading frame consists in a 168 bp long plus methionine also containing a His6 tag, encoding a predicted 62 amino acid residue peptide with related molecular mass calculated to be 6.98 kDa as a monomer and 13.96 kDa at the dimer form. The recombinant Pg-AMP1 peptide showed inhibitory activity against multiple Gram-negative (E. coli, Klebsiella pneumonia and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Staphylococcus epidermides) bacteria. Moreover, theoretical structure analyses were performed in order to understand the functional differences between natural and recombinant Pg-AMP1 forms. Data here reported suggest that Pg-AMP1 is a promising peptide to be used as a biotechnological tool for control of human infectious diseases.
Keywords: Pg-AMP1; Antimicrobial peptides; Heterologous expression; Psidium guajava;

Predicting antimicrobial peptides from eukaryotic genomes: In silico strategies to develop antibiotics by André C. Amaral; Osmar N. Silva; Nathália C.C.R. Mundim; Maria J.A. de Carvalho; Ludovico Migliolo; Jose R.S.A. Leite; Maura V. Prates; Anamélia L. Bocca; Octávio L. Franco; Maria S.S. Felipe (301-308).
Display Omitted► Four novel antifungal peptides were screened from eukaryotic genomes. ► Molecular modeling studies were realized showing helical structures. ► Val6 and Val8 residues observed seem to be important for peptide fungi-interaction.A remarkable and intriguing challenge for the modern medicine consists in the development of alternative therapies to avoid the problem of microbial resistance. The cationic antimicrobial peptides present a promise to be used to develop more efficient drugs applied to human health. The in silico analysis of genomic databases is a strategy utilized to predict peptides of therapeutic interest. Once the main antimicrobial peptides’ physical–chemical properties are already known, the correlation of those features to search on these databases is a tool to shorten identifying new antibiotics. This study reports the identification of antimicrobial peptides by theoretical analyses by scanning the Paracoccidioides brasiliensis transcriptome and the human genome databases. The identified sequences were synthesized and investigated for hemocompatibility and also antimicrobial activity. Two peptides presented antifungal activity against Candida albicans. Furthermore, three peptides exhibited antibacterial effects against Staphylococcus aureus and Escherichia coli; finally one of them presented high potential to kill both pathogens with superior activity in comparison to chloramphenicol. None of them showed toxicity to mammalian cells. In silico structural analyses were performed in order to better understand function–structure relation, clearly demonstrating the necessity of cationic peptide surfaces and the exposition of hydrophobic amino acid residues. In summary, our results suggest that the use of computational programs in order to identify and evaluate antimicrobial peptides from genomic databases is a remarkable tool that could be used to abbreviate the search of peptides with biotechnological potential from natural resources.
Keywords: In silico analyses; Antimicrobial peptides; Paracoccidioides brasiliensis;

Antimicrobial–immunomodulatory activities of zebrafish phosvitin-derived peptide Pt5 by Yunchao Ding; Xuemei Liu; Lingzhen Bu; Hongyan Li; Shicui Zhang (309-313).
► Pt5 enhances the survival rate of zebrafish challenged by A. hydrophila. ► Pt5 was able to block multiplication/dissemination of A. hydrophila in zebrafish. ► Pt5 suppressed proinflammatory cytokine gene IL-1β, IL-6, TNF-α and IFN-γ expression. ► Pt5 enhanced antiinflammatory cytokine gene IL-10 and IL-4 expression.A phosvitin (Pv)-derived peptide, Pt5, which consists of the C-terminal 55 residues of Pv in zebrafish, has been shown to function as an antimicrobial agent capable of killing microbes in vitro. However, its in vivo role in zebrafish remains unknown. In this study, we clearly demonstrated that Pt5 protected adult zebrafish from pathogenic Aeromonas hydrophila attack, capable of significantly enhancing the survival rate of zebrafish after the pathogenic challenge. Pt5 also caused a marked decrease in the numbers of A. hydrophila in the blood, spleen, kidney, liver and muscle, suggesting that Pt5 was able to block multiplication/dissemination of A. hydrophila in zebrafish. Additionally, Pt5 markedly suppressed the expression of the proinflammatory cytokine genes IL-1β, IL-6, TNF-α and IFN-γ in the spleen and head kidney of A. hydrophila-infected zebrafish, but it considerably enhanced the expressions of the antiinflammatory cytokine genes IL-10 and IL-4 in the same tissues. Taken together, these data indicate that Pt5 plays a dual role in zebrafish as an antimicrobial and immunomodulatory agent, capable of protecting zebrafish against pathogenic A. hydrophila through its antimicrobial activity as well as preventing zebrafish from the detrimental effects of an excessive inflammatory response via modulating immune functions.
Keywords: Zebrafish; Danio rerio; Phosvitin; Antimicrobial peptide; Immunomodulator;

The role of nitric oxide, reactive oxygen species, and protein kinase C in oxytocin-induced cardioprotection in ischemic rat heart by Mahdieh Faghihi; Ali Mohammad Alizadeh; Vahid Khori; Mostafa Latifpour; Saeed Khodayari (314-319).
► There has an increased interest in mechanisms involved in oxytocin cardioprotection in recent years. ► We planned oxytocin effects against I/R via NO, ROS, and PKC in the preconditioned myocardium. ► Oxytocin benefit effects were abolished by L-NAME, chelerythrine, and N-acetylcysteine. ► The beneficial effects of oxytocin were achieved via NO release, PKC activation, and ROS balance.Ischemia–reperfusion injury is a common complication of heart disease that is the leading cause of death worldwide. Here, we plan to elucidate oxytocin cardioprotection effects against ischemia–reperfusion via nitric oxide (NO), reactive oxygen species (ROS), and protein kinase C (PKC) in anesthetized rat preconditioned myocardium. Forty-eight Sprague-Dawley rats were equally divided into eight groups. All animals were subjected to 25 min ischemia and 120 min reperfusion. Oxytocin (OT), L-NAME (LNA, a nitric oxide synthase inhibitor), chelerythrine (CHE, a PKC enzyme inhibitor), and N-acetylcysteine (NAC, a ROS scavenger) were used prior to ischemia. Results showed that mean arterial pressure significantly reduced during the first 10 min of ischemia and reperfusion in IR, LNA, CHE, and NAC groups (p  < 0.05). OT prevented mean arterial pressure decline during early phase of ischemia and reperfusion. Cardioprotective effects of OT in infarct size, plasma levels of creatine kinase-MB and lactate dehydrogenase, severity and incidence of ventricular arrhythmias were abolished by L-NAME, chelerythrine, and N-acetylcysteine (p  < 0.05). The present study showed that OT pretreatment reduces myocardial infarct size and ventricular arrhythmias, and improves mean arterial pressure via NO production, PKC activation, and ROS balance. These findings provide new insight into therapeutic strategies for ischemic heart disease.
Keywords: Oxytocin; Cardioprotection; NO; ROS; PKC;

In vivo, ex vivo, and in vitro studies on apelin's effect on myocardial glucose uptake by Shiming Xu; Pei Han; Mei Huang; Joseph C. Wu; Chingpin Chang; Philip S. Tsao; Patrick Yue (320-326).
► Apelin enhances myocardial glucose uptake in vivo. ► Apelin triggers GLUT 4 translocation form cytosol to cell membrane. ► Apelin increases glucose uptake in cultured H9C2 cells. ► AMPK and IRS-1 participate in apelin's effect on glucose uptake in vivo and in vitro. ► Knockdown AMPK can inhibit apelin's effect on IRS-1 Ser-789 phosphorylation and glucose uptake.Apelin is an endogenous peptide hormone recently implicated in glucose homeostasis. However, whether apelin affects glucose uptake in myocardial tissue remains undetermined. In this study, we utilized in vivo, ex vivo and in vitro methods to study apelin's effect on myocardial glucose uptake. Pyroglutamated apelin-13 (2 mg/kg/day) was administered to C57BL6/J mice for 7 days. In vivo myocardial glucose uptake was measured by FDG-PET scanning, and GLUT4 translocation was assessed by immunofluorescence imaging. For in vitro studies, differentiated H9C2 cardiomyoblasts were exposed to pyroglutamated apelin-13 (100 nM) for 2 h. To test their involvement in apelin-stimulated myocardial glucose uptake, the energy sensing protein kinase AMPK were inhibited by pharmacologic inhibition (compound C) and RNA interference. IRS-1 phosphorylation was assessed by western blotting using an antibody directed against IRS-1 Ser-789-phosphorylated form. We found that apelin increased myocardial glucose uptake and GLUT4 membrane translocation in C57BL6/J mice. Apelin was also sufficient to increase glucose uptake in H9C2 cells. Apelin-mediated glucose uptake was significantly decreased by AMPK inhibition. Finally, apelin increased IRS-1 Ser-789 phosphorylation in an AMPK-dependent manner. The results of our study demonstrated that apelin increases myocardial glucose uptake through a pathway involving AMPK. Apelin also facilitates IRS-1 Ser-789 phosphorylation, suggesting a novel mechanism for its effects on glucose uptake.
Keywords: Glucose uptake; Apelin; AMPK; IRS-1; Heart;

Neurohypophyseal hormones protect against pentylenetetrazole-induced seizures in zebrafish: Role of oxytocin-like and V1a-like receptor by Daniela Braida; Andrea Donzelli; Roberta Martucci; Luisa Ponzoni; Alberto Pauletti; Mariaelvina Sala (327-333).
► We studied the neurohypophyseal hormones on seizures in zebrafish. ► We evaluated the epileptic behavior induced by pentylenetetrazole. ► Oxytocin (OT), vasopressin, isotocin and vasotocin reduced epileptic behavior. ► OT and vasopressin V1a receptor antagonists selectively antagonized the protection. ► These hormones are anticonvulsant agents through a receptor-mediated mechanism.Oxytocin (OT) and arginine-vasopressin (AVP) are involved in the physiological response to different stressors like the occurrence of seizures which is regarded as a severe stress factor. Zebrafish (Danio rerio) is recently featured as a model of epilepsy but the role of neurohypophyseal hormones on this teleost is still unknown. We attempted to determine whether non-mammalian homologues like isotocin (IT) and vasotocin (AVT) affected pentylenetetrazole (PTZ)-induced seizures in adult zebrafish in comparison with OT/AVP. The mechanism was studied using the most selective OT and AVP receptor antagonists. Zebrafish were injected i.m. with increasing doses (0.1–40 ng/kg) of the neuropeptides 10 min before PTZ exposure. DesGly-NH2-d(CH2)5-[D-Tyr2,Thr4]OVT (desglyDTyrOVT) for OT receptor and SR49059 for V1a subtype receptor, were injected together with each agonist 20 min before PTZ exposure. All the peptides significantly decreased the number of seizures, increased the mean latency time to the first seizure and decreased lethality. This protective effect led to a dose–response curve following a U-shaped form. IT was approximately 40 times more active than OT while AVT was 20 times more potent than AVP in reducing the number of seizures. DesglyDTyrOVT was more effective in antagonizing OT/IT, while SR49059 mainly blocked AVP/AVT-induced protection against PTZ-induced seizures. The present findings provide direct evidence of an important involvement of IT/OT and AVP/AVT as anticonvulsant agents against PTZ-induced seizures with a receptor-mediated mechanism in zebrafish. These data reinforce zebrafish as an emerging experimental model to study and identify new antiepileptic drugs.
Keywords: Epilepsy; Anticonvulsant; Neurohypophyseal; Hormone; Teleost;

► α-MSH possesses antimicrobial activity against both MSSA and MRSA. ► α-MSH non-susceptible Staphylococcus aureus strains have shown higher LPG content. ► α-MSH non-susceptible S. aureus strains have comparatively rigid membrane. Staphylococcus aureus (S. aureus), a major human pathogen of hospital and community acquired infections, is becoming resistant to almost all commercially available antibiotics. This has prompted development of antimicrobial peptides as therapeutic options. Alpha melanocyte stimulating hormone (α-MSH) is one such peptide known to possess antimicrobial properties. In the present study, we analyzed the antimicrobial activity of α-MSH against 75 clinical strains of S. aureus including both methicillin susceptible S. aureus (MSSA) and methicillin resistant S. aureus (MRSA) strains. Results of our previous study showed that membrane damage is the major mechanism of staphylocidal activity of α-MSH. In this context, we compared the various bacterial membrane parameters, viz., membrane fluidity, lipid composition, and surface charge of a few selected MSSA and MRSA strains that showed variable susceptibility to the melanocortin peptide. Our results showed that α-MSH killed both type of strains efficiently (≥70% killing in 84% clinical strains after exposure with 6 μM of α-MSH for 1 h). It was observed that compared to the α-MSH-susceptible strains, the α-MSH-non-susceptible strains had a different membrane order and phospholipid pattern. There was no consistent pattern of cell surface charge to distinguish α-MSH-susceptible strain from a non-susceptible strain. In conclusion, α-MSH possessed potential staphylocidal activity for both against MSSA and MRSA strains. S. aureus strains not susceptible to the peptide exhibited a rigid membrane and a higher amount of the cationic phospholipid as compared to the α-MSH-susceptible strains.
Keywords: Alpha-MSH; Staphylococcus aureus; Antimicrobial peptides; Bacterial membrane;