Peptides (v.33, #2)

► Soybeans are major food allergens. ► Soybeans can inadvertently be present in food products due to cross contamination. ► Mass spectrometry of marker proteolytical fragments of protein allergens is recognized as a new detection method in food control. ► Quantification of soybean at the peptide level is hindered due to limited information regarding specific stable markers derived after proteolytic digestion. ► The peptides 401Val-Arg410 from the G1 glycinin and the 518Gln-Arg528 from the α′ chain of the β-conglycinin proved to be the most stable peptides as identified by MALDI-TOF MS and MS/MS.Soybean (Glycine max) is extensively used all over the world due to its nutritional qualities. However, soybean is included in the “big eight” list of food allergens. According to the EU directive 2007/68/EC, food products containing soybeans have to be labeled in order to protect the allergic consumers. Nevertheless, soybeans can still inadvertently be present in food products. The development of analytical methods for the detection of traces of allergens is important for the protection of allergic consumers. Mass spectrometry of marker proteolytical fragments of protein allergens is growingly recognized as a detection method in food control. However, quantification of soybean at the peptide level is hindered due to limited information regarding specific stable markers derived after proteolytic digestion. The aim of this study was to use MALDI-TOF/MS and MS/MS as a fast screening tool for the identification of stable soybean derived tryptic markers which were still identifiable even if the proteins were subjected to various changes at the molecular level through a number of reactions typically occurring during food processing (denaturation, the Maillard reaction and oxidation). The peptides 401Val-Arg410 from the G1 glycinin (Gly m 6) and the 518Gln-Arg528 from the α′ chain of the β-conglycinin (Gly m 5) proved to be the most stable. These peptides hold potential to be used as targets for the development of new analytical methods for the detection of soybean protein traces in processed foods.
Keywords: Soybean; Stable peptides; Screening; MALDI-TOF/MS and MS/MS; Modified proteins;

Design and characterization of novel hybrid antimicrobial peptides based on cecropin A, LL-37 and magainin II by Marc A. Fox; Joanne E. Thwaite; David O. Ulaeto; Timothy P. Atkins; Helen S. Atkins (197-205).
► Rational design of hybrid peptides from natural parents. ► Direct antimicrobial activity improved over parents. ► Activity against Bacillus anthracis, Francisella tularensis and Burkholderia spp. ► Further modification reduces hemolysis and improves therapeutic index.Antimicrobial peptides (AMPs) are a naturally occurring component of the innate immune response of many organisms and can have activity against both Gram-negative and Gram-positive bacterial species. In order to optimize and improve the direct antimicrobial effect of AMPs against a broad spectrum of bacterial species, novel synthetic hybrids were rationally designed from cecropin A, LL-37 and magainin II. AMPs were selected based on their α-helical secondary structure and fragments of these were analyzed and combined in silico to determine which hybrid peptides would form the best amphipathic cationic α-helices. Four hybrid peptides were synthesized (CaLL, CaMA, LLaMA and MALL) and evaluated for direct antimicrobial activity against a range of bacterial species (Bacillus anthracis, Burkholderia cepacia, Francisella tularensis LVS and Yersinia pseudotuberculosis) alongside the original ‘parent’ AMPs. The hybrid peptides showed greater antimicrobial effects than the parent AMPs (in one case a parent is completely ineffective while a hybrid based on it removes all traces of bacteria by 3 h), although they also demonstrated higher hemolytic properties. Modifications were then carried out to the most toxic hybrid AMP (CaLL) to further improve the therapeutic index. Modifications made to the hybrid lowered hemolytic activity and also lowered antimicrobial activity by various degrees. Overall, this work highlights the potential for rational design and synthesis of improved AMPs that have the capability to be used therapeutically for treatment of bacterial infections.
Keywords: Antimicrobial peptides; Computer-aided rational design; Bacillus; Burkholderia; Francisella;

Purification and characterization of a novel antimicrobial peptide from Brevibacillus laterosporus strain A60 by Jing Zhao; Lihua Guo; Hongmei Zeng; Xiufen Yang; Jingjing Yuan; Huaixing Shi; Yehui Xiong; Mingjia Chen; Lei Han; Dewen Qiu (206-211).
► We purified a peptide, named BL-A60, from extracellular extract of Brevibacillus laterosporus. ► BL-A60 has a molecular weight of 1602.0469 Da. ► BL-A60 contains eleven amino acid residues and a choline connected to the N-terminal and a tenuazonic acid to the C-terminal. ► BL-A60 showed high pH and thermal stability. ► BL-A60 strongly inhibited different stages of the life cycle of Phytophthora capsici.A novel antimicrobial peptide, with molecular mass of 1602.0469 Da, produced by Brevibacillus laterosporus strain A60 was isolated and purified from the soil of mango plants. The purification procedure consisted of ammonium sulfate precipitation, cation exchange chromatography on an HiTrap SP HP column, thin layer chromatography and High Performance Liquid Chromatography (HPLC) on C18 reversed-phase column. After the four isolation procedures, one peptide with antimicrobial activity was obtained and named BL-A60. The determination of the complete amino acid sequences of this peptide showed that it contains eleven amino acid residues, L-Y-K-L-V-K-V-V-L-N-M, and a choline connected to the N-terminal and a tenuazonic acid modified of the C-terminal. This peptide shows relatively low identification to other antimicrobial peptides from bacteria. Purified BL-A60 showed high pH and thermal stability and a strong inhibition of different stages of the life cycle of Phytophthora capsici, including mycelial growth, sporangia formation and cystospore germination, with EC50 values of 7.89, 0.60 and 21.96 μg ml−1, respectively.
Keywords: Antimicrobial peptide; BL-A60; Brevibacillus laterosporus; Purification; Antimicrobial activity;

Effect of a novel antimicrobial peptide chrysophsin-1 on oral pathogens and Streptococcus mutans biofilms by Wei Wang; Rui Tao; Zhongchun Tong; Yonglin Ding; Rong Kuang; Shafei Zhai; Jun Liu; Longxing Ni (212-219).
► The inhibitory effect of chrysophsin-1 against oral pathogens and S. mutans biofilms. ► A preliminary study of the antimicrobial mechanism of chrysophsin-1. ► Chrysophsin-1 had considerable antimicrobial and antibiofilm potential. ► Chrysophsin-1 acted by a lytic mechanism involving pore formation and cell lysis.Dental caries and pulpal diseases are common oral bacterial infectious diseases. Controlling and reducing the causative pathogens, such as Streptococcus mutans and Enterococcus faecalis, is a key step toward prevention and treatment of the two diseases. Chrysophsin-1 is a cationic antimicrobial peptide having broad-spectrum bactericidal activity against both Gram-positive and Gram-negative bacteria. In this study, we investigated the antibacterial activity of chrysophsin-1 against several oral pathogens and S. mutans biofilms and performed a preliminary study of the antimicrobial mechanism. Cytotoxic activity of chrysophsin-1 against human gingival fibroblasts (HGFs) was investigated. Minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and time–kill assay were used to evaluate the killing effect of chrysophsin-1. Scanning electron microscopy (SEM) was used to analyze morphological and membrane change in oral pathogens. Live/Dead staining, in conjunction with confocal scanning laser microscopy (CSLM), was used to observe and analyze S. mutans biofilms. MIC and MBC results demonstrated that chrysophsin-1 had different antimicrobial activities against the tested oral microbes. Lysis and pore formation of the cytomembrane were observed following treatment of the bacteria with chrysophsin-1 for 4 h or 24 h by SEM. Furthermore, CLSM images showed that chrysophsin-1 remarkably reduced the viability of cells within biofilms and had a significantly lethal effect against S. mutans biofilms. Toxicity studies showed that chrysophsin-1 at concentration between 8 μg/ml and 32 μg/ml had little effect on viability of HGFs in 5 min. Our findings suggest that chrysophsin-1 may have potential clinical applications in the prevention and treatment of dental caries and pulpal diseases.
Keywords: Antimicrobial peptides; Chrysophsin-1; Streptococcus mutans; Enterococcus faecalis; Biofilms;

► Peptide variants of Tephrosia villosa defensin (TvD1) such as Alpha TvD1, S32R and D37R were generated through in vitro mutagenesis in the β2−β3 loop region of the peptide. ► Alpha-TvD1 showed enhanced antifungal activity against the fungal pathogens, Fusarium oxysporum and F. culmorum. ► It displayed enhanced insect α-amylase inhibitory activity against the insect Tenebrio molitor compared to wild type TvD1. ► TvD1 did not inhibit human and barley α-amylase activities due to differences in the pocket size and interacting loop.TvD1 is a small, cationic, and highly stable defensin from the weedy legume, Tephrosia villosa with demonstrated in vitro antifungal activity. We show here peptide modifications in TvD1 that lead to enhanced antifungal activities. Three peptide variants, S32R, D37R, and Alpha-TvD1 (-G-M-T-R-T-) with variations in and around the β2–β3 loop region that imposes the two β-strands, β2 and β3 were generated through in vitro mutagenesis. Alpha-TvD1 exhibited enhanced antifungal activity against the fungal pathogens, Fusarium culmorum and Fusarium oxysporum with respective IC50 values of 2.5 μM and 3.0 μM, when compared to S32R (<5.0 μM and >5.0 μM), D37R (5.5 μM and 4.5 μM), and the wild type TvD1 (6.5 μM). Because of the enhanced antifungal activity, this variant peptide was characterized further. Growth of F. culmorum in the presence of Alpha-TvD1 showed deformities in hyphal walls and nuclear damage. With respect to the plant pathogenic bacterium, Pseudomonas syringae pv. tomato strain DC3000, both Alpha-TvD1 and the wild type TvD1 showed comparable antibacterial activity. Both wild type TvD1 and Alpha-TvD1 displayed inhibitory activity against the α-amylase of the mealworm beetle, Tenebrio molitor (TMA) with the latter showing enhanced activity. The human salivary as well as barley α-amylase activities were not inhibited even at concentrations of up to 50 μM, which has been predicted to be due to differences in the pocket size and the size of the interacting loops. Present study shows that the variant Alpha-TvD1 exhibits enhanced antifungal as well as insect α-amylase inhibitory activity.
Keywords: Tephrosia villosa; Wild type TvD1; Alpha-TvD1; Tenebrio molitor; α-amylase inhibition; Protein–protein docking;

Neuropeptide F peptides act through unique signaling pathways to affect cardiac activity by M. Setzu; M. Biolchini; A. Lilliu; M. Manca; P. Muroni; S. Poddighe; C. Bass; A.M. Angioy; R. Nichols (230-239).
► NPF arrested slow phase signal frequency; fast phase frequency was affected lesser. ► NPF decreased the slow phase duration, but increased the fast phase duration. ► sNPF-1 decreased signal frequency of the slow phase; the fast phase was unaffected. ► sNPF-1 decreased the slow phase duration, but increased the fast phase duration. ► NPF and sNPF-1 increased isoelectric period duration.Elucidating how neuropeptides affect physiology may result in delineating peptidergic mechanisms and identifying antagonists for application in basic and translational science. Human neuropeptide Y (NPY) regulates cardiac activity; frequently invertebrates contain orthologs of vertebrate peptides. We report invertebrate NPY-like neuropeptide F (NPF) arrested the signal frequency of the slow phase of the cardiac cycle (EC50 = 1 pM); however, signal frequency of the fast phase was affected only minimally. Neuropeptide F decreased the duration of the slow phase by ∼70% (EC50 = 0.6 pM), but increased the duration of the fast phase by ∼57% (EC50 = 10 nM). Short NPF-1 (sNPF-1) decreased the signal frequency of the slow phase by ∼70% (EC50 = 9 nM); yet, signal frequency of the fast phase was unaffected. Short NPF-1 decreased the duration of the slow phase ∼55% (EC50 ∼50 nM), but increased the duration of the fast phase ∼20% without dose dependency. Neuropeptide F and sNPF-1 increased isoelectric period duration. This novel report demonstrated NPY-like peptides are cardioactive but functionally unique. These data contribute to understanding how invertebrate orthologs affect cardiovascular activity. Dipteran fast and slow phases may be generated from separate pacemakers in the abdominal heart and in the anterior thoracocephalic aorta, respectively. Thus, our research suggests NPF and sNPF-1 act through different mechanisms to regulate cardiac activity. Invertebrate NPY-like peptides act in olfaction and feeding yet mechanisms which are associated with their cardioactive effects remain unknown; our work may provide evidence linking their roles in sensory response and cardiac activity.
Keywords: FMRFamide; Myosuppressin; Neuropeptide Y (NPY); Short neuropeptide F (sNPF); RFamide-related peptide (RFRP);

► Considerable progress in the study of polypeptide toxins resulted in determination of a large number of structurally related sequences. ► So-called rational nomenclature has obvious advantages; however it misses structural peculiarities of toxins. ► We suggest modifications to this nomenclature for cysteine-rich polypeptide toxins identified from sea anemones. ► Cysteine-rich polypeptides may be classified into several structural classes on the basis of specific arrangement of cysteine residues. ► Using this specific arrangement or structural motifs for screening sea anemone EST databases we identified 11 novel toxins.Polypeptide toxins are the main constituents of natural venoms. Considerable progress in the study of these molecules has resulted in the determination of a large number of structurally related sequences. To classify newly discovered molecules, a rational nomenclature for naming peptide toxins was developed, which takes into account toxin biological activity, the species name, and structural peculiarities of the polypeptide. Herein, we suggest modifications to this nomenclature for cysteine-rich polypeptide toxins from sea anemones and describe 11 novel polypeptide structures deduced after common database revision.
Keywords: Sea anemone; Polypeptide toxin; Rational nomenclature; Structural motifs; SRDA; Protein labeling;

Identification and molecular cloning of a novel amphibian Bowman Birk-type trypsin inhibitor from the skin of the Hejiang Odorous Frog; Odorrana hejiangensis by Min Wang; Lei Wang; Tianbao Chen; Brian Walker; Mei Zhou; Dayuan Sui; J. Michael Conlon; Chris Shaw (245-250).
► The manuscript describes a novel Bowman Birk type trypsin inhibitor from the skin of a rare Chinese frog. ► This peptide was devoid of antimicrobial activity and an enhanced cationic analog was only weakly active against E. coli. ► Antimicrobial experiments were carried out in two different labs and the data were in accordance. ► Amphibian skin Bowman Birk protease inhibitors are the first of their class to be found outside the Plant Kingdom.In this study, an amphibian (Odorrana hejiangensis) skin extract was fractionated by reverse phase HPLC and fractions were screened for trypsin inhibitory activity. Using this initial approach, a novel trypsin inhibitory peptide was detected with an apparent protonated molecular mass of 1804.83 Da, as determined by MALDI-TOF mass spectrometry. It was named Hejiang trypsin inhibitor (HJTI) in accordance. The primary structure of the biosynthetic precursor of HJTI was deduced from a cDNA sequence cloned from a skin-derived cDNA library. The primary structure of the encoded predicted mature active peptide was established as: GAPKGCWTKSYPPQPCS (non-protonated monoisotopic molecular mass – 1802.81 Da). On the basis of this unequivocal amino acid sequence, a synthetic replicate was synthesized by solid phase Fmoc chemistry. This replicate displayed a moderately potent trypsin inhibition with a K i of 388 nM. Bioinformatic analysis of the primary structure of this peptide indicated that it was a member of the Bowman-Birk family of protease inhibitors. The substitutions of Gln-14 and Ser-17 by Lys, resulted in an increase in cationicity and a small increase in potency to a K i value of 218 nM. Neither HJTI nor its synthetic analog, possessed any significant antimicrobial activity.
Keywords: Amphibian; Skin; Peptide; Protease; Inhibitor; Cloning;

Hainanenins: A novel family of antimicrobial peptides with strong activity from Hainan cascade-frog, Amolops hainanensis by Songyan Zhang; Huanhuan Guo; Fei Shi; Hui Wang; Lili Li; Xudong Jiao; Yipeng Wang; Haining Yu (251-257).
► 10 novel antimicrobial peptides belonging to 4 families were identified from frog Amolops hainanensis. ► A novel AMP family composed of 5 members was identified and designated as hainanenins. ► Two members of hainanenins were selected for the functional analysis. ► Hainanenins possessed strong and broad-spectrum antimicrobial activities, strong hemolytic activity and slight antioxidant activity.Antimicrobial peptides (AMPs) secreted by amphibian skin represent an important innate immune defense strategy. There are more than 340 species in the family of Ranidae worldwidely, and from which nearly 100 families of AMPs comprising between 8 and 48 amino acid (aa) residues have been characterized. In current work, two novel AMPs were purified from the skin secretion of Hainan cascade-frog, Amolops hainanensis, and 31 cDNA sequences encoding 10 novel AMPs belonging to 4 families were cloned from the constructed skin cDNA library of A. hainanensis. Among these 10 AMPs, 5 peptides represent the prototypes of a novel amphibian AMP family. According to the generic name of the species of origin, they were designated as hainanenin-1–5. Each of them consists of 21 aa residues with a C-terminal disulphide loop of 7 residues between Cys15 and Cys21. Two of them (hainanenin-1 and 5) were then synthesized and their in vitro activities were screened, including antimicrobial, hemolytic and antioxidant activities. The results showed that hainanenin-1 and 5 possessed strong and broad-spectrum antimicrobial activities against Gram-positive, Gram-negative bacteria and fungi, including a large number of clinically isolated drug-resistant pathogenic microorganisms, and slight antioxidant activity. Undesirably, hainanenin-1 and 5 exhibited strong hemolytic activity on human erythrocytes. The discovery of hainanenins and their great antimicrobial potency provides new templates for anti-infective agent design.
Keywords: Amphibian; Antimicrobial peptide (AMP); Hainanenins; Amolops hainanensis;

Characterization and immunomodulatory function comparison of various bursal-derived peptides isolated from the humoral central immune organ by Xiu-Li Feng; Qing-Tao Liu; Rui-Bing Cao; Bin Zhou; Yuan-Peng Zhang; Ke Liu; Xiao-Dong Liu; Jian-Chao Wei; Xin-Feng Li; Pu-Yan Chen (258-264).
► Five novel bursal peptides were isolated from the humoral immune organ BF. ► These peptides exerted different immune inducing roles on antibody production. ► These peptides exerted different immunomodulatory roles on cytokine responses. ► BPP-II induced immune responses in dose-dependent manner. ► BPP-I induced immune responses in dose-dependent manner.The bursa of Fabricius (BF) is the acknowledged central immune organ, which is important to the B cell differentiation and antibody production. However, due to difficult purification, the immunomodulatory peptides from BF were little reported. In this study, the extract samples of BF were taken to a chromatographic analysis by RP-HPLC. Five novel low molecular weight peptides were isolated from BF, with amino acid sequences of YEYAY, RMYEE, GPPAT, AGCCNG, and RRL, and named as Bursal pentapeptide (BPP)-III, -IV, -V, and Bursal hexapeptide (BHP), and Bursal tripeptide (BTP), respectively. BSP-I, BSP-II, BPP-I and BPP-II are recently reported to be the bursal-derived bioactive peptides. In this paper, we analyzed the chemical formula and characteristics of these nine bursal-derived peptides. The immunization comparative experiment verified the different immunomodulatory activity of these nine bursal peptides on antibody and cytokine productions. Furthermore, the results showed that at reachable concentrations, BPP-II and BPP-I induced antibody productions, lymphocyte viabilities and cytokine responses in different dose-dependent manner in the immunized mice model, respectively. These results provided important orientations for the comprehensively understanding and study of the humoral central immune system of human, and provided a novel insight on the treatment of serious disease and immune improvement of human.
Keywords: Bursal-derived peptides; Reversed-phase HPLC; Immune model; Antibody production; Cellular mediated immune response;

Organic anion transporters involved in the excretion of bestatin in the kidney by Yanna Zhu; Qiang Meng; Changyuan Wang; Qi Liu; Huijun Sun; Taiichi Kaku; Kexin Liu (265-271).
► We demonstrate for the first time that bestatin is a substrate of organic anion transporter (OAT) 1 and OAT3. ► The elimination mechanism of bestatin at molecular level was elucidated in vivo and in vitro. ► Drug–drug interactions will occur when bestatin is coadministered with other inhibitors, inducers or substrates of OAT1 and OAT3.Bestatin, a dipeptide, a low molecular weight aminopeptidase inhibitor, has been demonstrated to be an immunomodulator with an antitumor activity. However, the transporter-mediated renal excretion of bestatin is not fully understood. The purpose of this study was to elucidate the transporter-mediated renal excretion mechanism for bestatin. The plasma concentration of bestatin was increased markedly and both the accumulative renal excretion and renal clearance of bestatin were decreased significantly after intravenous administration of bestatin in combination with probenecid. p-Aminohippuric acid (PAH), a substrate of organic anion transporter (OAT) 1, benzylpenicillin (PCG), a substrate of OAT3 and JBP485, a substrate of OAT1 and OAT3, reduced the uptake of bestatin in rat kidney slices and in hOAT1- or hOAT3-HEK 293 cells. The accumulation of bestatin in hOAT1-HEK and hOAT3-HEK 293 cells was significantly greater than that in vector-HEK, and the K m and V max were 0.679 ± 0.007 mM and 0.807 ± 0.006 nmol/mg protein/30 s for OAT1, 0.632 ± 0.014 mM and 1.303 ± 0.015 nmol/mg protein/30 s for OAT3 respectively. PAH and JBP485 inhibited significantly the uptake of bestatin in hOAT1-HEK with the K i values of 92 ± 9 μM and 197 ± 21 μM; and PCG, JBP485 inhibited significantly the uptake of bestatin in hOAT3-HEK 293 cells with the K i values of 88 ± 12 μM and 160 ± 16 μM. Our results are novel in demonstrating for the first time that OAT1 and OAT3 are involved in the renal excretion of bestatin.
Keywords: Bestatin; Dipeptide; Drug interaction; Organic anion transporter; Pharmacokinetics; Renal excretion;

In vitro metabolic stability of iodinated obestatin peptides by Bart De Spiegeleer; Sylvia Van Dorpe; Valentijn Vergote; Evelien Wynendaele; Ewald Pauwels; Christophe Van De Wiele; Pablo Garcia-Solis; Juan Carlos Solis-Sainz (272-278).
Display Omitted► Iodinated peptides are not only hydrolyzed by proteases, but also reductively deiodinated in several mammalian tissues. ► The degree and position of iodination influences its metabolization kinetics and products formed, which is moreover dependent on the tissue. ► Analytical characterization and functional evaluation are required in biomedical investigations using iodinated peptides.Different iodinated mouse obestatin peptides have been characterized toward their in vitro stability in the main metabolic compartments plasma, liver and kidney. Using HPLC–UV for quantification, significant differences in the degradation kinetics of the iodinated peptides, arising from both enzymatic proteolysis and dehalogenation, were found when compared to the native, unmodified peptide. HPLC–MS/MS analysis demonstrated that the cleavage sites were dependent upon the biological matrix and the location of the amino acid residue incorporating the iodine atom(s). The degrading proteases were found to target peptide bonds further away from the iodine incorporation, while proteolytic cleavages of nearby peptide bonds were more limited. Diiodinated amino acid residue containing peptides were found to be more susceptible to deiodination than the mono-iodinated derivative. In plasma, the percentage of peptide degradation solely attributed to deiodinase activity after 20 min incubation reached up to 25% for 2,5-diiodo-H19-obestatin compared to 20% and only 3% for (3,5-diiodo-Y16)- and (3-iodo-Y16) obestatin, respectively. Hence, our results demonstrate that the different iodinated peptides pose significantly different metabolization properties and thus, also different biological activities are expected for peptides upon iodination.
Keywords: Peptide iodination; In vitro metabolic stability; Proteases; Deiodinases; Peptidomimetics;

A new strategy for metabolic stabilization of motilin using the C-terminal part of ghrelin by Naomi Morozumi; Seiji Sato; Sayaka Yoshida; Akira Yamaki; Mayumi Furuya; Norio Inomata; Norio Ohnuma; Yoshiharu Minamitake; Kazuhiro Ohsuye; Kenji Kangawa (279-284).
► The C-terminal(8–28) part of ghrelin is important in the biological activity in vivo. ► Motilin is degraded very rapidly in plasma. ► Motilin(1–12) and ghrelin(12–28) chimeric peptide has an equipotent motilin agonist activity. ► Motilin(1–12) and ghrelin(12–28) chimeric peptide is more stable in vivo than motilin. ► The C-terminal part of ghrelin has a potential to improve the biokinetics of motilin.Ghrelin consists of 28 amino acid residues with an octanoyl modification at the third serine residue. Recently we have found that the C-terminal part of ghrelin protects the ester bond of 3-octanoyled serine from plasma esterases and plays the essential role to prolong the plasma half-life and to show its biological activity in vivo. In the present study, we researched whether the C-terminal part of ghrelin has a potential to prolong the plasma half-life of motilin, by comparing the pharmacokinetics of various chimeric peptides of ghrelin and motilin. Motilin is another gastro-intestinal peptide hormone related with ghrelin structurally, binding to the same family of G protein-coupled receptors. Chimeric peptides were designed to be composed of motilin(1–12) fragment, the active core binding to the motilin receptor, GPR38, and C-terminal part of ghrelin. The modification of motilin(1–12) fragment by C-terminal part of ghrelin hardly influenced its agonist activity to GPR38 and almost all these chimeric peptides showed more than two times longer plasma half-lives than motilin in rats. From the relationship between structures of chimeric peptides and their corresponding plasma half-lives, the mid-region of ghrelin rich in basic amino acids (15RKESKK20) was considered to be the most important in prolonging the plasma half-life of motilin. The deletion of these fragments or replacement of 17th glutamic acid with a neutral amino acid resulted in short plasma half-lives. In conclusion, our data suggested that the C-terminal part of ghrelin has a potential to improve the biokinetics of motilin probably by a metabolic stabilizing effect.
Keywords: Ghrelin; Motilin; Chimeric peptide; Terminal half-life in plasma; Pharmacokinetics; Metabolic stability;

Exenatide and feeding: Possible peripheral neuronal pathways by Jizette V. Hunt; Martha C. Washington; Ayman I. Sayegh (285-290).
► Exenatide reduces food intake. ► We evaluated three possible neuronal pathways for this reduction. ► Exenatide (0.5 μg/kg, i.p.) was given to VGX, CMGX, VGX/CMGX animals. ► VGX attenuated the first meal and all surgeries attenuated the second meal. ► Vagus nerve and splanchnic nerves are necessary for exenatide satiation effect.Intraperitoneal (i.p.) administration of the synthetic agonist of the glucagon like peptide-1 (GLP-1) receptor exenatide reduces food intake. Here, we evaluated possible peripheral pathways for this reduction. Exenatide (0.5 μg/kg, i.p.) was given to three, overnight food-deprived, groups of rats: total subdiaphragmatic vagotomy (VGX, severs the vagus nerve), celiaco-mesenteric ganglionectomy (CMGX, severs the splanchnic nerve) and combined VGX/CMGX. Following the injection, meal sizes (MSs) and intermeal intervals (IMIs) were determined for a total of 120 min. We found that exenatide reduced the sizes of the first two meals but failed to prolong the IMI between them, that VGX attenuated the reduction of the first MS, and that VGX, CMGX and combined VGX/CMGX attenuated the reduction of the second MS by exenatide. Therefore, the vagus nerve appears necessary for the reduction of the first MS by exenatide, whereas both nerves appear necessary for the reduction of the second MS by this peptide.
Keywords: GLP-1; Meal size; Intermeal interval; Sympathetic; Vagus;

Distribution of somatostatin receptor 5 in mouse and bullfrog retinas by Xiao-Hua Wu; Qin-Qin Deng; Shi-Xiang Jiang; Xiong-Li Yang; Yong-Mei Zhong (291-297).
► We examine the distribution of sst5 in mouse and bullfrog retinas. ► Sst5 is expressed in dopaminergic, cholinergic amacrine cells and ganglion cells. ► Sst5 shows a more widespread distribution in bullfrog retina. ► Immunostaining for sst5 is seen in bullfrog photoreceptors, but not in mouse ones.Somatostatin (SRIF), as a neuroactive peptide in the CNS, may act as a neuromodulator through activation of five specific receptor subtypes (sst1–sst5). In this work we conducted a comparative study of the expression of sst5 in mouse and bullfrog retinas by immunofluorescence double labeling. Basically, the expression profiles of sst5 in the retinas of the two species were similar. That is, in the inner retina sst5 was localized to dopaminergic and cholinergic amacrine cells, stained by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) respectively, and cells in the ganglion cell layer, whereas in the outer retina immunostaining for sst5 was observed in horizontal cells. However, a more widespread, abundant distribution of labeling for sst5, as compared to mouse retina, was seen in bullfrog retina: strong labeling for sst5 was diffusely distributed in both outer and inner plexiform layers (OPL and IPL) in the bullfrog retina, but the labeling was only observed in the IPL of the mouse retina. In addition, bullfrog photoreceptors, both rods and cones, but not mouse ones, were labeled by sst5. In combination with the experiments showing that SRIF-immunoreactivity was mainly found in the inner retina, our results suggest that SRIF, released from SRIF-containing cells in the inner retina, may play a neuromodulatory role in both outer and inner retina mediated by volume transmission via sst5 in bullfrog retina, while the SRIF action may be largely restricted to the mouse inner retina.
Keywords: Somatostatin receptor; Immunofluorescence double labeling; Confocal laser scanning microscopy; Retina;

Apelin supports primary rat retinal Müller cells under chemical hypoxia and glucose deprivation by Xin-Lei Wang; Yong Tao; Qiang Lu; Yan-Rong Jiang (298-306).
► Exogenous apelin stimulated Müller cells viability and migration, and prevented cellular apoptosis under chemical hypoxia and glucose deprivation. ► Apelin/APJ showed stronger immunoreactivities in hypoxic Müller cultures. ► Apelin/APJ mRNA expression tended to increase within 6 h in hypoxia, while stayed down-regulated during 4–12 h under glucose-deprivation.Müller cells support the integrity of the blood–retinal barrier, whereas their dysfunction under pathological conditions may contribute to retinal edema formation. The apelin peptide, as the endogenous ligand of G protein-coupled receptor APJ, participates in numbers of physiological and pathological processes. Recent studies highlight its emerging role against ischemic injury. Our study aimed to investigate the potential neuroprotection of apelin for primary rat retinal Müller cells under hypoxia or glucose-deprivation (GD) by cell viability, migration and apoptosis, as well as apelin/APJ immunofluorescence labeling and mRNA expression. The results showed that exogenous apelin significantly stimulated Müller cells viability and migration under normal, hypoxic and glucose-free condition, also prevented apoptosis. Apelin immunoreactivities represented weak and diffuse staining in the cytoplasm, along with restricted nuclear APJ expression. They both appeared stronger immunoreactivities after 12 h hypoxia. Under hypoxic stress, apelin mRNA expression began to increase at 6 h (9.97 folds, p  < 0.01), and APJ mRNA also up-regulated (2 h 6.50 folds, p  < 0.05; 4 h 2.25 folds, p  < 0.05; 6 h 14 folds, p  < 0.01), whereas they both down-regulated during 4–12 h GD. Our results suggested that apelin induced the tolerance of Müller cells to hypoxia and GD. Its administration might be a promising protection for blood–retinal barrier to ischemia.
Keywords: Apelin; APJ; Müller cells; Glucose deprivaion; Hypoxia; Ischemia;

Peripheral and central alterations of pituitary adenylate cyclase activating polypeptide-like immunoreactivity in the rat in response to activation of the trigeminovascular system by Bernadett Tuka; Zsuzsanna Helyes; Adrienn Markovics; Teréz Bagoly; József Németh; László Márk; Réka Brubel; Dóra Reglődi; Árpád Párdutz; János Szolcsányi; László Vécsei; János Tajti (307-316).
► We examined the PACAP-27/38-LI in the activated trigeminovascular system. ► Blood plasma, nerve tissues and liquor were analyzed in two rat models. ► PACAP-27/38-LI increased in the region of brainstem in response to both stimulations. ► PACAP-38-LI elevated in the systemic circulation after electrical TRG activation. ► PACAP may have a role in the pain syndromes connected with trigeminal activation.Pituitary adenylate cyclase activating polypeptide (PACAP) is present in the cranial arteries and trigeminal sensory neurons. We therefore examined the alterations in PACAP-like immunoreactivity (PACAP-LI) in a time-dependent manner in two rat models of trigeminovascular system (TS) activation. In one group chemical stimulation (CS) was performed with i.p. nitroglycerol (NTG), and in the other one the trigeminal ganglia (TRG) were subjected to electrical stimulation (ES). The two biologically active forms, PACAP-38 and PACAP-27, were determined by means of radioimmunoassay (RIA) and mass spectrometry (MS) in the plasma, the cerebrospinal fluid (CSF), the trigeminal nucleus caudalis (TNC), the spinal cord (SC) and the TRG. The tissue concentrations of PACAP-27 were 10 times lower than those of PACAP-38 in the TNC and SC, but about half in the TRG. PACAP-38, but not PACAP-27, was present in the plasma. Neither form could be identified in the CSF. PACAP-38-LI in the plasma, SC and TRG remained unchanged after CS, but it was increased significantly in the TNC 90 and 180 min after NTG injection. In response to ES of the TRG, the level of PACAP-38 in the plasma and the TNC was significantly elevated 90 and 180 min later, but not in the SC or the TRG. The alterations in the levels of PACAP-27 in the tissue homogenates in response to both forms of stimulation were identical to those of PACAP-38. The selective increases in both forms of PACAP in the TNC suggest its important role in the central sensitization involved in migraine-like headache.
Keywords: Trigeminal ganglion; Electrical and chemical stimulation; Nitroglycerol; Blood plasma; Trigeminal nucleus caudalis; Mass spectrometry;

Nicotine evoked improvement in learning and memory is mediated through NPY Y1 receptors in rat model of Alzheimer's disease by Ritesh J. Rangani; Manoj A. Upadhya; Kartik T. Nakhate; Dadasaheb M. Kokare; Nishikant K. Subhedar (317-328).
► Nicotine and NPYergic agents improved AD-like condition in rats. ► While NPY or [Leu31, Pro34]-NPY potentiated, BIBP3226 attenuated the effect of nicotine. ► NPY-immunoreactivity was decreased in AcbSh, CeA, ARC and DG in AD-like condition. ► Nicotine treatment showed restoration of NPY-immunoreactivity in these nuclei. ► NPY, acting via NPY Y1 receptors, interacts with the endogenous cholinergic system.We investigated the role of endogenous neuropeptide Y (NPY) system in nicotine-mediated improvement of learning and memory in rat model of Alzheimer's disease (AD). Intracerebroventricular (icv) colchicine treatment induced AD-like condition in rats and showed increased escape latency (decreased learning), and amnesic condition in probe test in Morris water maze. In these rats, nicotine (0.5 mg/kg, intraperitoneal), NPY (100 ng/rat, icv) or NPY Y1 receptor agonist [Leu31, Pro34]-NPY (0.04 ng/rat, icv) decreased escape latency by 54.76%, 55.81% and 44.18%, respectively, on day 4 of the acquisition. On the other hand, selective NPY Y1 receptor antagonist, BIBP3226 (icv) produced opposite effect (44.18%). In the probe test conducted at 24 h time point, nicotine, NPY or [Leu31, Pro34]-NPY increased the time spent by 72.72%, 44.11% and 26.47%, respectively; while BIBP3226 caused reduction (8.82%). It seems that while NPY or [Leu31, Pro34]-NPY potentiated, BIBP3226 attenuated the learning and memory enhancing effects of nicotine. Brains of colchicine treated rats showed significant reduction in NPY-immunoreactivity in the nucleus accumbens shell (cells 62.23% and fibers 50%), bed nucleus of stria terminalis (fibers 71.58%), central nucleus of amygdala (cells 74.33%), arcuate nucleus (cells 70.97% and fibers 69.65%) and dentate gyrus (cells 58.54%). However, in these rats nicotine treatment for 4 days restored NPY-immunoreactivity to the control level. We suggest that NPY, perhaps acting via NPY Y1 receptors, might interact with the endogenous cholinergic system and play a role in improving the learning and memory processes in the rats with AD-like condition.
Keywords: Alzheimer's disease; Nicotine; Neuropeptide Y; Learning and memory; Immunocytochemistry;

Extracellular signal-regulated kinase 1/2 activation is involved in intermedin1–53 attenuating myocardial oxidative stress injury induced by ischemia/reperfusion by Lei Zhao; Ding-Qiong Peng; Jing Zhang; Jun-Qiu Song; Xu Teng; Yan-Rong Yu; Chao-Shu Tang; Yong-Fen Qi (329-335).
► IMD1–53 attenuated the myocardial injury by inhibiting MDA content and LDH activity, elevating the Mn-SOD and CAT activity and decreasing the expression of gp91phox and p47phox. ► IMD1–53 attenuated H2O2-induced cardiomyocyte apoptosis in vitro. ► IMD1–53 activated the ERK1/2 signaling pathway. ► IMD1–53 may improve the oxidative stress injury by enhancing ERK phosphorylation.Intermedin (IMD)1–53 is a novel member of the calcitonin gene-related peptide superfamily and has potent cardioprotective effects against myocardial injury induced by ischemia–reperfusion (I/R). To explore the mechanism of the IMD1–53 cardioprotective effect, we studied the anti-oxidant effects of IMD1–53 on myocardial injury induced by I/R in vivo in rat and H2O2 treatment in vitro in rat cardiomyocytes. Compared with sham treatment, I/R treatment induced severe lipid peroxidation injury in rat myocardium: plasma malondialdehyde (MDA) content and myocardial LDH activity was increased by 34% and 85% (all P  < 0.01); Mn-superoxide dismutase (Mn-SOD) and catalase (CAT) activity was reduced 80% and 86% (all P  < 0.01), respectively, and the protein levels of the NADPH oxidase complex subunits gp91phox and p47phox were markedly increased, by 86% (P  < 0.05) and 95% (P  < 0.01), respectively; IMD1–53 treatment ameliorated lipid peroxidation injury: plasma MDA content and myocardial LDH activity was decreased by 30% (P  < 0.05) and 36% (P  < 0.01); Mn-SOD and CAT activity was elevated 1.0- and 4.3-fold (all P  < 0.01), respectively; and the protein levels of gp91phox and p47phox were reduced, by 28% and 36% (both P  < 0.05), respectively. Concurrently, IMD1–53 treatment markedly promoted cell viability and inhibited apoptosis in cardiomyocytes as compared with H2O2 treatment alone. Furthermore, IMD1–53 increased the ratio of p-ERK to ERK by 66% (P  < 0.05) as compared with I/R alone, and the protective effect of IMD1–53 on H2O2-induced apoptosis was abolished by preincubation with PD98059, a MEK inhibitor. IMD1–53 may improve the oxidative stress injury induced by I/R via inhibiting the production of reactive oxygen species and enhancing ERK phosphorylation.
Keywords: Intermedin1–53; Ischemia/reperfusion; Oxidative stress; Myocardial injury;

► High plasma copeptin level is found in patients with intracerebral hemorrhage. ► Copeptin could emerge as a new biomarker in intracerebral hemorrhage. ► Copeptin independently predicts early neurological deterioration and long-term outcome in patients with intracerebral hemorrhage.High plasma copeptin levels have been found to be associated with short-term poor outcome after intracerebral hemorrhage (ICH). We furthermore evaluate the relation of plasma copeptin levels to long-term outcome and early neurological deterioration after ICH. Fifty healthy controls and 89 patients with acute spontaneous basal ganglia hemorrhage were recruited in this study. Plasma copeptin concentrations on admission measured by enzyme-linked immunosorbent assay were considerably high in patients than healthy controls. A multivariate analysis identified plasma copeptin level as an independent predictor for 1-year mortality, 1-year unfavorable outcome (modified Rankin Scale score > 2) and early neurological deterioration. A receiver operating characteristic curve showed that the predictive value of plasma copeptin concentration was similar to that of National Institutes of Health Stroke Scale scores for long-term poor outcome and early neurological deterioration. However, copeptin did not obviously improve the predictive values of National Institutes of Health Stroke Scale scores. Thus, increased plasma copeptin level is an independent prognostic marker of 1-year mortality, 1-year unfavorable outcome and early neurological deterioration after ICH.
Keywords: Copeptin; Intracerebral hemorrhage; Early neurological deterioration; 1-Year outcome;

► Mean arterial pressure in conscious rats rises to i.a. vasopressin (AVP) injection. ► These injections result in small, but significant increases in plasma AVP levels. ► A two-three fold increase in plasma AVP levels can alter MAP in conscious rats. ► In vivo manipulations that double plasma AVP levels are capable of altering MAP.We recently reported that neuronostatin, a novel neuropeptide, biphasically increased mean arterial pressure, first through the activation of the sympathetic nervous system followed by the release of vasopressin. In those experiments, we found that centrally administered neuronostatin increased plasma vasopressin levels only 2–3 times greater than levels observed in saline-treated controls, and that the increase in mean arterial pressure (approximately 15 mm Hg) could be blocked by pretreatment with a V1-vasopressin antagonist. Here we report the relationship between two to three fold elevations in plasma vasopressin levels and concomitant changes in mean arterial pressure in conscious, unrestrained male rats. We injected increasing doses of vasopressin (5, 20, and 100 ng/kg, intra-arterially) and measured both changes in plasma vasopressin levels and the elevation in mean arterial pressure achieved. At 5-min post injection, plasma levels of vasopressin and mean arterial pressures were similar to those observed following central neuronostatin administration in our earlier study. Thus we conclude that small increases in circulating vasopressin levels can result in significant elevations in mean arterial pressure at least in the conscious rat.
Keywords: Vasopressin; Blood pressure;

BiP mRNA expression is upregulated by dehydration in vasopressin neurons in the hypothalamus in mice by Daisuke Hagiwara; Hiroshi Arima; Yoshiaki Morishita; Motomitsu Goto; Ryoichi Banno; Yoshihisa Sugimura; Yutaka Oiso (346-350).
► BiP mRNA is expressed in AVP neurons of the SON and PVN in wild-type mice under basal conditions. ► BiP mRNA expression increases in proportion to that of AVP mRNA under dehydration. ► BiP is likely to be involved in the osmoregulation of AVP neurons as an ER chaperone.The immunoglobulin heavy chain binding protein (BiP) is an endoplasmic reticulum (ER) chaperone that facilitates the proper folding of newly synthesized secretory and transmembrane proteins. Here we report that BiP mRNA was expressed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus in wild-type mice under basal conditions. Dual in situ hybridization in the SON and PVN demonstrated that BiP mRNA was expressed in almost all the neurons of arginine vasopressin (AVP), an antidiuretic hormone. BiP mRNA expression levels were increased in proportion to AVP mRNA expression in the SON and PVN under dehydration. These data suggest that BiP is involved in the homeostasis of ER function in the AVP neurons in the SON and PVN.
Keywords: Immunoglobulin heavy chain binding protein; Endoplasmic reticulum stress; Unfolded protein response; Arginine vasopressin; Supraoptic nucleus; Paraventricular nucleus;

Urotensin II and urocortin trigger the expression of myostatin, a negative regulator of cardiac growth, in cardiomyocytes by Damien Gruson; Audrey Ginion; Pascale Lause; Jean-Marie Ketelslegers; Jean-Paul Thissen; Luc Bertrand (351-353).
► We have evaluated the gene expression of myostatin in adult cardiomyocytes stimulated by urotensin II (UII) and urocortin (UCN). ► Myostatin is regarded as a down regulator factor possibly counteracting the effects of hypertrophic stimuli and may also play an active role in cardiac remodeling after injury. ► Our results uncover for the first time that the two hypertrophic peptides UII and UCN stimulate the expression of myostatin.Urotensin II (UII) and urocortin (UCN) are potent contributors to the physiopathology of heart failure. Our study investigated the effects of UII and UCN on the expression of myostatin (Mstn) in primary culture of adult cardiomyocytes. Adult rat cardiomyocytes were stimulated for 48 h with UII and UCN. Cell size and protein content were determined. Mstn gene expression was determined by real time quantitative polymerase chain reaction. Treatment with UII and UCN stimulates hypertrophy of adult cardiomyocytes. This effect was associated with a twofold increase of Mstn gene expression. We have established for the first time that the two hypertrophic peptides UII and UCN stimulate the expression of Mstn.

Impact of glomerular filtration rate on urinary BNP and NT-proBNP levels in heart failure by Raquel Cortés; Manuel Portolés; Esther Roselló-Lletí; Luis Martínez-Dolz; Luis Almenar; Lilian Grigorian; Vicente Bertomeu; Miguel Rivera (354-358).
► Urinary excretion of NT-proBNP and BNP was similar, irrespective of eGFR. ► An inverse an exponential relationship was obtained between filtered load of natriuretic peptides with their urinary levels after normalizing urinary concentrations. ► Glomerular filtration alone does not seem to be the major determinant of the urinary concentration of natriuretic peptides ► Altered tubular handling could be involved in urinary natriuretic peptides clearance in our outpatients diagnosed with HF.B-type natriuretic peptide (BNP) and its inactive amino-terminal fragment (NT-proBNP) are diagnostic tools for heart failure (HF), but less is understood regarding the effects of renal function on their urinary concentrations. The objective was to analyze the influence of renal function, as estimated glomerular filtration rate (eGFR), on BNP and NT-proBNP concentrations in 90 HF outpatients (65 ± 12 years; 73% men), grouped according to eGFR below or above 60 mL/min. Patients with worse eGFR had higher serum NT-proBNP (p  < 0.01) and BNP (p  < 0.01) than patients with higher eGFR: NT-proBNP, but urinary levels did not reach statistical differences. In addition, a direct significant correlation between filtered load of serum NT-proBNP or BNP with their concentrations in urine was found in patients with eGFR above 60 mL/min (r  = 0.66, p  < 0.001 and r  = 0.338, p  < 0.05) and below 60 mL/min (r  = 0.63, p  < 0.001 and r  = 0.406, p  < 0.01). However, after normalizing urinary natriuretic peptide concentrations by their filtered load, we obtained a significant inverse and exponential relation in patients with worse renal function for NT-proBNP and BNP (r  = −0.87, p  = 0.001; and r  = −0.71, p  < 0.001, respectively) and in patients with eGFR > 60 mL/min (r  = −0.84, p  < 0.001; and r  = −0.72, p  < 0.001, respectively). In conclusion, similar urinary NT-proBNP and BNP excretion was obtained in patients with high or low eGFR. Furthermore, despite the direct correlation between filtered load of serum natriuretic peptides with their urinary levels, an inverse an exponential relationship was obtained after normalizing urinary concentrations. Therefore, glomerular filtration does not seem to be the major determinant of both urinary peptide concentrations.