Peptides (v.38, #1)

Inhibition of emetic and superantigenic activities of staphylococcal enterotoxin A by synthetic peptides by Edward K. Maina; Dong-Liang Hu; Krisana Asano; Akio Nakane (1-7).
► Peptide A2, A3, A6 and A10 inhibited SEA-induced IFN-γ production of spleen cells. ► Peptide A2, A3, A6 and A10 attenuated SEA-induced lethal shock in animal model. ► Peptide A2, A3, A6 and A10 inhibited SEA-induced proliferation of shrew spleen cells. ► Peptide A2, A3 and A6 but not A10 inhibited SEA-induced emesis in house musk shrews. Staphylococcus aureus is a major human pathogen producing different types of toxins. Enterotoxin A (SEA) is the most common type among clinical and food-related strains. The aim of the present study was to estimate functional regions of SEA that are responsible for emetic and superantigenic activities using synthetic peptides. A series of 13 synthetic peptides corresponding to specific regions of SEA were synthesized, and the effect of these peptides on superantigenic activity of SEA including interferon γ (IFN-γ) production in mouse spleen cells, SEA-induced lethal shock in mice, spleen cell proliferation in house musk shrew, and emetic activity in shrews were assessed. Pre-treatment of spleen cells with synthetic peptides corresponding to the regions 21–40, 35–50, 81–100, or 161–180 of SEA significantly inhibited SEA-induced IFN-γ production and cell proliferation. These peptides also inhibited SEA-induced lethal shock. Interestingly, peptides corresponding to regions 21–40, 35–50 and 81–100 significantly inhibited SEA-induced emesis in house musk shrews, but region 161–180 did not. These findings indicated that regions 21–50 and 81–100 of SEA are important for both superantigenic and emetic activities of SEA molecule while region 161–180 is involved in superantigenic activity but not emetic activity of SEA. These regions could be important targets for therapeutic intervention against SEA exposure.
Keywords: Staphylococcal enterotoxin; Superantigen; Emesis; Synthetic peptide; House musk shrew;

Change in plasma visfatin level after severe traumatic brain injury by Jun Chen; Jian-Feng Weng; Wei-Cong Hong; Li-Feng Luo; Wei Yu; Shi-Da Luo (8-12).
► Plasma visfatin level increased after severe traumatic brain injury. ► Visfatin could possibly serve as a novel biomarker in traumatic brain injury. ► Visfatin may be a good prognostic factor for 6-month clinical outcomes of severe traumatic brain injury patients.Higher plasma visfatin concentration has been associated with ischemic stroke. Thus, we sought to investigate change in plasma visfatin level after traumatic brain injury and to evaluate its relation with disease outcome. Seventy-six healthy controls and 98 patients with acute severe traumatic brain injury were recruited. Twenty-seven patients (27.6%) died and 48 patients (49.0%) suffered from unfavorable outcome (Glasgow outcome scale score of 1–3) in 6 months. On admission, plasma visfatin level was increased in patients than in healthy controls and was highly correlated with Glasgow Coma Scale score. A multivariate analysis identified plasma visfatin level as an independent predictor for 6-month mortality and unfavorable outcome. According to receiver operating characteristic curve analysis, the predictive value of the plasma visfatin concentration was similar to Glasgow Coma Scale score's. In a combined logistic-regression model, visfatin did not improve the predictive value of Glasgow Coma Scale score. Thus, increased plasma visfatin level is associated with 6-month clinical outcomes after severe traumatic brain injury.
Keywords: Visfatin; Traumatic brain injury; Prognosis;

► Two enzymatic conditions of TSG were optimized by orthogonal experiment. ► A double-enzyme hydrolysis was applied to obtain the antioxidant hydrolysate (TSGH). ► Two peptides with high antioxidant activity were isolated from TSGH. ► The structures of peptides were determined as Glu-Gly-Leu and Tyr-Gly-Asp-Glu-Tyr. ► Their IC50 values on hydroxyl radical scavenging activity were 4.61 and 6.45 μg mL−1.To obtain hydrolysates with high degree of hydrolysis (DH) and scavenging radical activity, tilapia skin gelatin (TSG) was hydrolyzed by properase E and multifect neutral. The optimum hydrolysis condition of each enzyme was determined using the orthogonal experiment, and double-enzyme hydrolysis was further applied. The results showed the tilapia skin gelatin hydrolysate (TSGH) obtained by progressive hydrolysis using multifect neutral and properase E had the highest DH and hydroxyl radical scavenging activity. The IC50 values of TSGH on scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide anion radical (•O2) and hydroxyl radical (•OH) activities were also determined. TSGH was further purified using gel filtration chromatography, ion exchange chromatography, and RP-HPLC. The peptides were identified using nano-LC-ESI mass spectrometry. Finally, two antioxidant peptides were identified and the amino acid sequences were Glu-Gly-Leu (317.33 Da) and Tyr-Gly-Asp-Glu-Tyr (645.21 Da), respectively. The IC50 values of two peptides on hydroxyl radical scavenging activities were 4.61 μg mL−1and 6.45 μg mL−1, respectively. Therefore, the results demonstrated that the hydrolysates of TSG prepared by multifect neutral and properase E could serve as a source of peptides with high antioxidant activity. It provided a scientific basis for the preparation of antioxidant peptides.
Keywords: Tilapia skin gelatin; Antioxidant peptide; Orthogonal experiment; Purification and identification; Amino acid sequence;

Antifungal properties of Canavalia ensiformis urease and derived peptides by Melissa Postal; Anne H.S. Martinelli; Arlete B. Becker-Ritt; Rodrigo Ligabue-Braun; Diogo R. Demartini; Suzanna F.F. Ribeiro; Giancarlo Pasquali; Valdirene M. Gomes; Celia R. Carlini (22-32).
► Ureases have biological effects independent of their enzyme activity. ► Urease shows homology to plant antifungal defensins, chitinases and thaumatin. ► Jackbean urease is fungicidal to yeasts at submicromolar doses. ► Fungitoxic effects include permeabilization, altered metabolism and cell death. ► The urease-derived peptide Jaburetox is fungitoxic to yeast and filamentous fungi.Ureases (EC are metalloenzymes that hydrolyze urea into ammonia and CO2. These proteins have insecticidal and fungicidal effects not related to their enzymatic activity. The insecticidal activity of urease is mostly dependent on the release of internal peptides after hydrolysis by insect digestive cathepsins. Jaburetox is a recombinant version of one of these peptides, expressed in Escherichia coli. The antifungal activity of ureases in filamentous fungi occurs at submicromolar doses, with damage to the cell membranes. Here we evaluated the toxic effect of Canavalia ensiformis urease (JBU) on different yeast species and carried out studies aiming to identify antifungal domain(s) of JBU. Data showed that toxicity of JBU varied according to the genus and species of yeasts, causing inhibition of proliferation, induction of morphological alterations with formation of pseudohyphae, changes in the transport of H+ and carbohydrate metabolism, and permeabilization of membranes, which eventually lead to cell death. Hydrolysis of JBU with papain resulted in fungitoxic peptides (∼10 kDa), which analyzed by mass spectrometry, revealed the presence of a fragment containing the N-terminal sequence of the entomotoxic peptide Jaburetox. Tests with Jaburetox on yeasts and filamentous fungi indicated a fungitoxic activity similar to ureases. Plant ureases, such as JBU, and its derived peptides, may represent a new alternative to control medically important mycoses as well as phytopathogenic fungi, especially considering their potent activity in the range of 10−6–10−7  M.
Keywords: Jaburetox; Pseudohyphae; Yeast; Glucose-induced acidification; Membrane permeabilization; Growth inhibition;

Defense peptides from barnyard grass (Echinochloa crusgalli L.) seeds by E.A. Rogozhin; D.Y. Ryazantsev; E.V. Grishin; T.A. Egorov; S.K. Zavriev (33-40).
► A spectrum of novel defense peptides was isolated from Echinochloa crusgalli seeds. ► N-terminal primary sequences of these peptides were determined and it was shown that these molecules belong to cystein-rich plant peptides. ► There are peptides from three AMP families and two protease inhibitor families in barnyard grass seeds. ► The biological activity of isolated peptides against several phytopatogenic bacterial species and oomycete Phytophthora infestans was studied.A number of defense polypeptides from latent seeds of weed cereal barnyard grass (Echinochloa crusgalli L.) has been isolated and characterized using an acidic extraction and high performance liquid chromatography methods in combination with MALDI-TOF mass spectrometry and Edman sequencing. Members of three antimicrobial peptide families and two protease inhibitor families were found to be localized in barnyard grass seeds. Their biological activity concerning to Gram-Positive and Gram-Negative phytopathogenic bacteria, as well as oomycete Phytophthora infestans, has been investigated. Diversity of barnyard grass defense peptides is a significant factor that provides a resistance of E. crusgalli seeds to germination and latent phases.
Keywords: Plant defense peptides; Echinochloa crusgalli seeds; Plant immunity;

Characterization of diverse antimicrobial peptides in skin secretions of Chungan torrent frog Amolops chunganensis by Xiaohong Yang; Jiangnan Xia; Zhijun Yu; Yuhong Hu; Fengjiao Li; Hao Meng; Shujie Yang; Jingze Liu; Hui Wang (41-53).
► cDNAs encoding 11 antimicrobial peptides were cloned. ► Seven antimicrobial peptides were first purified from skin secretions of Amolops chunganensis. ► Effects of antimicrobial peptides on microorganism and human erythrocytes were observed by SEM. ► Phylogenetic analysis of A. chunganensis based upon the primary structures of brevinin-1, brevinin-2, and esculentin-2 families antimicrobial peptides.We have cloned, synthesized, and characterized 11 novel antimicrobial peptides from a skin derived cDNA library of the Chungan torrent frog, Amolops chunganensis. Seven of the 11 antimicrobial peptides were present in authentic A. chunganensis skin secretions. Sequence analysis indicated that the 11 peptides belonged to the temporin, esculentin-2, palustrin-2, brevinin-1, and brevinin-2 families. The peptides displayed potent antimicrobial activities against several strains of microorganisms. One peptide, brevinin-1CG5, demonstrated antimicrobial activity against all tested Gram-positive and Gram-negative bacteria and fungi, and showed high antimicrobial potency (MIC = 0.6 μM) against Gram-positive bacterium Rhodococcus rhodochrous. Some peptides also demonstrated weak hemolytic activity against human erythrocytes in vitro. Phylogenetic analysis based on the amino acid sequences of brevinin-1, brevinin-2, and esculentin-2 peptides from family Ranidae confirmed that the current taxonomic status of A. chunganensis is correct.
Keywords: Amphibian; Amolops chunganensis; Frog skin; Antimicrobial peptides;

Exercise induces renin–angiotensin system unbalance and high collagen expression in the heart of Mas-deficient mice by Gislaine G. Guimarães; Sérgio H.S. Santos; Marilene L. Oliveira; Elizabeth P. Pimenta-Velloso; Daisy F. Motta; Almir S. Martins; Natalia Alenina; Michael Bader; Robson A.S. Santos; Maria Jose Campagnole-Santos (54-61).
► Exercise in Mas-KO mice induced a pronounced increase in collagen mRNA expression. ► Exercise in Mas-KO mice shifted the balance between RAS arms favoring Ang II. ► Ang-(1–7)/Mas mediates the cardiac adaptations in physical training.The renin–angiotensin system (RAS) is involved in the cardiac and vascular remodeling associated with cardiovascular diseases. Angiotensin (Ang) II/AT1 axis is known to promote cardiac hypertrophy and collagen deposition. In contrast, Ang-(1–7)/Mas axis opposes Ang II effects in the heart producing anti-trophic and anti-fibrotic effects. Exercise training is known to induce cardiac remodeling with physiological hypertrophy without fibrosis. We hypothesize that cardiac remodeling induced by chronic exercise depends on the action of Ang-(1–7)/Mas axis. Thus, we evaluated the effect of exercise training on collagen deposition and RAS components in the heart of FVB/N mice lacking Mas receptor (Mas-KO). Male wild-type and Mas-KO mice were subjected to a moderate-intense swimming exercise training for 6 weeks. The left ventricle (LV) of the animals was sectioned and submitted to qRT-PCR and histological analysis. Circulating and tissue angiotensin peptides were measured by RIA. Sedentary Mas-KO presented a higher circulating Ang II/Ang-(1–7) ratio and an increased ACE2 expression in the LV. Physical training induced in Mas-KO and WT a similar cardiac hypertrophy accompanied by a pronounced increase in collagen I and III mRNA expression. Trained Mas-KO and trained WT presented increased Ang-(1–7) in the blood. However, only in trained-WT there was an increase in Ang-(1–7) in the LV. In summary, we showed that deletion of Mas in FVB/N mice produced an unbalance in RAS equilibrium increasing Ang II/AT1 arm and inducing deleterious cardiac effects as deposition of extracellular matrix proteins. These data indicate that Ang-(1–7)/Mas axis is an important counter-regulatory mechanism in physical training mediate cardiac adaptations.
Keywords: Angiotensin-(1–7); Mas receptor; Exercise training; Extracellular matrix proteins; Collagen; Renin–angiotensin system;

Expression of corticotropin releasing factor receptor type 1 (CRF1) in the human gastrointestinal tract and upregulation in the colonic mucosa in patients with ulcerative colitis by Pu-Qing Yuan; S. Vincent Wu; Julie Elliott; Peter A. Anton; Ekaterini Chatzaki; Mulugeta Million; Yvette Taché (62-69).
► CRF1 is expressed in the whole GI tract in healthy subjects with distinct patterns in the different segments. ► CRF1 is located in the colonic mucosal lamina propria, epithelia and enteric neurons. ► 79% of CRF1 immunoreactive (IR) cells in colonic lamina propria occurred in macrophages. ► CRF1 IR cells and CRF1 IR macrophages are significant increased by 4.2 and 4.0 folds in colonic biopsies of UC patients.Brain corticotropin-releasing factor (CRF) acting on CRF receptor type 1 (CRF1) is a main signaling pathway in the stress response. CRF is also produced in a variety of peripheral sites and acts locally as a proinflammatory mediator. We investigated CRF1 mRNA expression in the human gastrointestinal tract, and localized CRF1 immunoreactive cells in the colonic mucosa of healthy subjects and patients with ulcerative colitis (UC). In 4 male healthy subjects (24–29 years), CRF1 transcript was detected by RT-PCR throughout the gastrointestinal tract with the highest levels in the ileum and rectum and the lowest level in the colon. Immunohistochemistry on whole thickness sigmoid colon sections showed that CRF1 was localized in the lamina propria and epithelial cells and enteric neurons. In sigmoid colonic biopsies, immunohistochemically double-labeled cells with CRF1 and CD163, a marker for macrophages, represent 79% of total CRF1 immunoreactive (IR) cells in healthy subjects. In 10 UC patients, the total number of CRF1 IR cells and CRF1/CD163 double-labeled macrophages was increased by 4.2 and 4.0 folds respectively compared to healthy subjects. These findings indicate that CRF1 is distributed throughout the GI tract of healthy human subjects. The increase of CRF1 IR cells prominently in macrophages of the sigmoid colonic mucosa of UC patients provides anatomical support for a role of CRF1 signaling in modulating the immune-inflammatory process of UC.
Keywords: Colon; CRF; CRF1 receptor; Gut; Sigmoid biopsy; Macrophage; Ulcerative colitis;

Effects of endokinin A/B and endokinin C/D on the antinociception properties of hemopressin in mice by Lanxia Zhou; Qiaoying Jin; Yinliang Yang; Zhengkun Liu; Xiaoli Li; Shouliang Dong; Li Zhao (70-80).
► Hemopressin (HP) could fully antagonize the hyperalgesia induced by endokinin A/B. ► HP could extend the analgesic duration of endokinin C/D (EKC/D). ► Co-injection of EKC/D and HP showed significant dose-dependently analgesic effects. ► SR140333B and SR142801 were used for mechanism studies.The current study evaluated the effects of hemopressin (HP) on pain modulation by endokinin A/B (EKA/B) and endokinin C/D (EKC/D) at the supraspinal level in mice. Intracerebroventricular administration of HP (10 nmol) fully antagonized the hyperalgesia induced by EKA/B (10, 30, and 100 pmol), and induced a dose-dependent potent analgesic effect. HP at different concentrations (10 pmol, 100 pmol, and 1 nmol) showed varying effects on the analgesic effect of EKA/B (3 nmol). HP extended the duration of the analgesic effect of EKC/D (3 nmol). Moreover, HP at different concentrations (10 pmol, 5 pmol, 1 pmol, and 100 fmol) co-administered with EKC/D (30 pmol) induced significant analgesia at two different time points: 5 min and 50 min. To investigate the antinociceptive mechanism, we used SR140333B and SR142801. HP (1 pmol) potentiated the analgesic effect of SR140333B (100 pmol) + EKA/B (30 pmol) in 5–10 min, while HP (100 pmol) had no effect in the analgesia induced by SR140333B (3 nmol) + EKA/B (3 nmol). HP (1 nmol) fully inhibited the analgesic effect of SR140333B (3 nmol) + EKC/D (3 nmol) or SR142801 (3 nmol) + EKC/D (3 nmol). HP (1 pmol) weakened the analgesic effect of SR142801 (100 pmol) + EKA/B (30 pmol), but HP (100 pmol) strengthened the analgesic effect of SR142801 (3 nmol) + EKA/B (3 nmol). These findings may pave the way for a new strategy on investigating the interaction between tachykinins and opioids on pain modulation.
Keywords: Pain modulation; Hemopressin; Endokinin A/B; Endokinin C/D;

Central administration of oxytocin reduces hyperalgesia in mice: Implication for cannabinoid and opioid systems by R. Russo; G. D’Agostino; G. Mattace Raso; C. Avagliano; C. Cristiano; R. Meli; A. Calignano (81-88).
► Icv oxytocin administration reduces carrageenan-induced hyperalgesia. ► CB1 receptor is involved in oxytocin-induced antihyperalgesic effect. ► μ- and κ-opioid receptor subtypes play a key role in OXT-induced antihyperalgesic effect. ► Central oxytocin does not reduce carrageenan-induced paw edema.The neuropeptide oxytocin (OXT) contributes to the regulation of diverse cognitive and physiological functions including nociception. Indeed, OXT has been reported to be analgesic when administered directly into the brain, the spinal cord, or systemically. Although many authors have reported the analgesic effects of OXT, its mechanism has not been well elucidated. Recently, it has been also hypothesize that OXT, increasing intracellular concentration of calcium, could regulate the production of mediators, like endocannabinoids (eCB). It has been well documented that eCB are able to suppress pain pathways. The present study investigates the effect of OXT in paw carrageenan-induced pain. Intracerebroventricular (icv) administration of OXT, but neither intraperitoneal nor intraplantar route, induces an antihyperalgesic effect increasing paw withdrawal latency to mechanical or thermal stimuli. Our results clearly demonstrate that 3 and 6 h following carrageenan challenge, central administration of OXT (30 ng/mouse) shows a significant antihyperalgesic activity. Moreover, for the first time, we demonstrate that CB1 receptor plays a key role in the antihyperalgesic effect of OXT. In fact our results show CB1 antagonist, but not the specific CB2 antagonist reduce OXT-induced antihyperalgesic effect. In addition, our data show that central OXT administration is able to reduce carrageenan-induced hyperalgesia but does not modify carrageenan-induced paw edema. Finally, using opioid antagonists we confirm an important role of opioid receptors. In conclusion, our experiments suggest that central administration of OXT reduces hyperalgesia induced by intraplantar injection of carrageenan, and this effect may work via cannabinoid and opioid systems.
Keywords: Oxytocin; Endocannabinoid system; Opioid receptors; Inflammation; Pain;

Biophysical characterization of the proton-coupled oligopeptide transporter YjdL by Johanne Mørch Jensen; Fie C. Simonsen; Amir Mastali; Helle Hald; Ida Lillebro; Frederik Diness; Lars Olsen; Osman Mirza (89-93).
► Direct binding of dipeptide ligands to the purified peptide transporter YjdL has been established. ► Binding of Ala-Lys can be detected whereas Ala-Gln, which has similar potency, is undetectable. ► Binding is undetectable to a Gln-variant of the highly conserved Glu388.Proton-coupled oligopeptide transporters (POTs) utilize the electrochemical proton gradient to facilitate uptake of di- or tripeptide molecules. YjdL is one of four POTs found in Escherichia coli. It has shown an extraordinary preference for di- rather than tripeptides, and is therefore significantly different from prototypical POTs such as the human hPepT1. Nonetheless YjdL contains several highly conserved POT residues, which include Glu388 that is located in the putative substrate binding cavity. Here we present biophysical characterization of WT-YjdL and Glu388Gln. Isothermal titration calorimetrical studies exhibit a K d of 14 μM for binding of Ala-Lys to WT-YjdL. Expectedly, no binding could be detected for the tripeptide Ala-Ala-Lys. Surprisingly however, binding could not be detected for Ala-Gln, although earlier studies indicated inhibitory potencies of Ala-Gln to be comparable to Ala-Lys (IC50 values of 0.6 compared to 0.3 mM). Finally, Ala-Lys binding to Glu388Gln was also undetectable which may support a previously suggested role in interaction with the ligand peptide N-terminus.
Keywords: Isothermal titration calorimetry; Circular dichroism spectroscopy; Membrane protein; Ligand binding; Secondary active transport;

Intravenous phage display identifies peptide sequences that target the burn-injured intestine by Todd W. Costantini; Brian P. Eliceiri; James G. Putnam; Vishal Bansal; Andrew Baird; Raul Coimbra (94-99).
► Phage display identifies functional targeting ligands and their receptors. ► In vivo biopanning identifies peptides which target the burn-injured intestine. ► Gut-targeting peptides identified in mouse have cross reactivity with human gut.The injured intestine is responsible for significant morbidity and mortality after severe trauma and burn; however, targeting the intestine with therapeutics aimed at decreasing injury has proven difficult. We hypothesized that we could use intravenous phage display technology to identify peptide sequences that target the injured intestinal mucosa in a murine model, and then confirm the cross-reactivity of this peptide sequence with ex vivo human gut. Four hours following 30% TBSA burn we performed an in vivo, intravenous systemic administration of phage library containing 1012 phage in balb/c mice to biopan for gut-targeting peptides. In vivo assessment of the candidate peptide sequences identified after 4 rounds of internalization was performed by injecting 1 × 1012 copies of each selected phage clone into sham or burned animals. Internalization into the gut was assessed using quantitative polymerase chain reaction. We then incubated this gut-targeting peptide sequence with human intestine and visualized fluorescence using confocal microscopy. We identified 3 gut-targeting peptide sequences which caused collapse of the phage library (4–1: SGHQLLLNKMP, 4–5: ILANDLTAPGPR, 4–11: SFKPSGLPAQSL). Sequence 4–5 was internalized into the intestinal mucosa of burned animals 9.3-fold higher than sham animals injected with the same sequence (2.9 × 105 vs. 3.1 × 104 particles per mg tissue). Sequences 4–1 and 4–11 were both internalized into the gut, but did not demonstrate specificity for the injured mucosa. Phage sequence 4–11 demonstrated cross-reactivity with human intestine. In the future, this gut-targeting peptide sequence could serve as a platform for the delivery of biotherapeutics.
Keywords: Burn; Intestinal injury; Gut targeting; Epithelial barrier; Drug delivery;

Effect of intranasal arginine vasopressin on human headache by Jun Yang; Lu Lu; Hong-Chang Wang; He-Qin Zhan; Guang-Fan Hai; Yan-Juan Pan; Qiong-Qing Lv; Da-Xin Wang; Yu-Quan Wu; Ren-Ren Li; Lei Xue; Xin-Hua Wang; Xiao-Ming Deng; Xin-Feng Liu; Yan-Ning Qian; Zhi-Kuan Deng; Zhi-Jian Zhang; Xin-Huan Zhan; Xin-Jian Zhou; Guo-Liang Wang; Jian-Xin Zhai; Jing-Cheng Wang (100-104).
► Arginine vasopressin (AVP) concentration in both plasma and cerebrospinal fluid (CSF) increased in human headache patients, which related with headache level. ► There was a positive relationship between plasma and CSF AVP concentration in human headache patients. ► Intranasal AVP could relieve the human headache in a dose-dependent manner. ► Intranasal AVP could treat human headache and AVP might be potential drug of pain relief.Arginine vasopressin (AVP), a nonapeptide hormone of posterior pituitary, reaches the central nervous system from systemic blood circulation with a difficulty because of the blood–brain barrier (BBB). The interest has been expressed in the use of the nasal route for delivery of AVP to the brain directly, exploiting the olfactory pathway. Our previous study has demonstrated that AVP in the brain rather than the spinal cord and blood circulation plays an important role in rat pain modulation. For understanding the role of AVP on pain modulation in human, the communication tried to investigate the effect of intranasal AVP on human headache. The results showed that (1) AVP concentration in both plasma and cerebrospinal fluid (CSF) increased significantly in headache patients, who related with the headache level; (2) there was a positive relationship between plasma and CSF AVP concentration in headache patients; and (3) intranasal AVP could relieve the human headache in a dose-dependent manner. The data suggested that intranasal AVP, which was delivered to the brain through olfactory region, could treat human headache and AVP might be a potential drug of pain relief by intranasal administration.
Keywords: Arginine vasopressin; Headache; Intranasal administration; Human;

Epitopic hexapeptide sequences from Baltic cod parvalbumin beta (allergen Gad c 1) are common in the universal proteome by Piotr Minkiewicz; Justyna Bucholska; Małgorzata Darewicz; Justyna Borawska (105-109).
► Gad c 1 hexapeptides were used as query sequences for UniProt database screening. ► Hexapeptides occur in proteins which are not homologs of parvalbumin. ► Hexapeptides occur in proteins from organisms involved in food technology ► Hexapeptides occur in proteins from human symbionts, commensals and pathogens. ► Coverage between epitopes from allergens and pathogens should be avoided in vaccines.The aim of this study was to analyze the distribution of hexapeptide fragments considered as epitopes of Baltic cod parvalbumin beta (allergen Gad c 1) in the universal proteome. Cod (Gadus morhua subsp. callarias) parvalbumin hexapeptides cataloged in the Immune Epitope Database were used as query sequences. The UniProt database was screened using the WU-BLAST 2 program. The distribution of hexapeptide fragments was investigated in various protein families, classified according to the presence of the appropriate domains, and in proteins of plant, animal and microbial species. Hexapeptides from cod parvalbumin were found in the proteins of plants and animals which are food sources, microorganisms with various applications in food technology and biotechnology, microorganisms which are human symbionts and commensals as well as human pathogens. In the last case possible coverage between epitopes from pathogens and allergens should be avoided during vaccine design.

Neuropeptide FF activates ERK and NF kappa B signal pathways in differentiated SH-SY5Y cells by Yu-long Sun; Xiao-yuan Zhang; Ning He; Tao Sun; Yan Zhuang; Quan Fang; Kai-rong Wang; Rui Wang (110-117).
► We first report a differentiated SH-SY5Y cell line dSH-SY5Y endogenously expresses functional hNPFF2 receptor. ► NPFF activates the ERK signal pathway in a PKA- and nitric oxide synthase-dependent manner. ► NPFF stimulates the NF kappa B signal pathway in an ERK-independent way.Neuropeptide FF (NPFF) has been reported to play important roles in regulating diverse biological processes. However, little attention has been focused on the downstream signal transduction pathway of NPFF. Here, we used the differentiated neuroblastoma cell line, dSH-SY5Y, which endogenously expresses hNPFF2 receptor, to investigate the signal transduction downstream of NPFF. In particular we investigated the regulation of the extracellular signal-regulated protein kinase (ERK) and the nuclear factor kappa B (NF-κB) pathways by NPFF in these cells. NPFF rapidly and transiently stimulated ERK. H89, a selective inhibitor of cyclic AMP-dependent protein kinase A (PKA), inhibited the NPFF-activated ERK pathway, indicating the involvement of PKA in the NPFF-induced ERK activation. Down-regulation of nitric oxide synthases also attenuated NPFF-induced ERK activation, suggesting that a nitric oxide synthase-dependent pathway is involved. Moreover, the core upstream components of the NF-κB pathway were also significantly activated in response to NPFF, suggesting that the NF-κB pathway is involved in the signal transduction pathway of NPFF. Collectively, these data demonstrate that nitric oxide synthases are involved in the signal transduction pathway of NPFF, and provide the first evidence for the interaction between NPFF and the NF-κB pathway. These advances in our interpretation of the NPFF pathway mechanism will aid the comprehensive understanding of its function and provide novel molecular insight for further study of the NPFF system.
Keywords: Neuropeptide FF; NPFF2 receptor; Nitric oxide synthase; SH-SY5Y; ERK; NF kappa B;

The impact of confounders on the test performance of natriuretic peptides for cardiac dysfunction in subjects aged 80 and older by Bert Vaes; Damien Gruson; Gijs Van Pottelbergh; Agnes Pasquet; Catharina Matheï; Wim Adriaensen; Nawel Rezzoug; Jean-Louis Vanoverschelde; Jan Degryse (118-126).
► Multiple functional and structural echocardiographic parameters are related to natriuretic peptides in people aged 80 and older. ► Adjusting for confounders only improves the diagnostic accuracy of natriuretic peptides for cardiac dysfunction in subjects with CAF or PM. ► Different cut-off values in strata of individual confounders could be used to optimize the diagnostic characteristics of natriuretic peptides.The hypothesis that natriuretic peptides could be used to identify ‘pancardiac’ damage has been proposed. However, multiple factors are known to influence circulating levels of natriuretic peptides, especially in the very old. Therefore, the impact of confounders on the association between natriuretic peptide levels and cardiac dysfunction was further explored in subjects aged 80 and older. A diagnostic cross-sectional study embedded within the BELFRAIL study (n  = 567) was performed. Baseline BNP and NT-proBNP levels were measured and echocardiograms were performed at the subject's home. Cardiac dysfunction was defined as systolic dysfunction, valvular heart disease or isolated severe diastolic dysfunction. Several functional and structural echocardiographic parameters were independently related to circulating levels of natriuretic peptides. Cystatin C, BMI, β blockers, diabetes, heart frequency, usCRP, age and sex were identified as confounders. The prevalence of cardiac dysfunction was 17.1% in the subjects without and 30.8% in the subjects with chronic atrial fibrillation (CAF) or pacemaker (PM). Only in subjects with CAF or PM the C statistic for cardiac dysfunction improved after correcting for confounders. The post-test probability for a negative test (PTP−) ranged from 3.7% to 12.2% and the PTP+ ranged from 21.9% to 62.2% in different strata of confounders. According to these data adjusting for identified confounders does not improve the diagnostic accuracy of the natriuretic peptides for cardiac dysfunction, except in subjects with CAF or PM. Stratifying for individual confounders showed that different cut-off values could be used to optimize the diagnostic characteristics of natriuretic peptides.
Keywords: Sensitivity and specificity; Aged; 80 and over; Cardiac dysfunction; Natriuretic peptides;

In silico identification of novel hevein-like peptide precursors by William F. Porto; Valéria A. Souza; Diego O. Nolasco; Octávio L. Franco (127-136).
Display Omitted► The first fungal and three novel plant hevein-like peptides were identified. ► The novel hevein-like peptides were predicted as antimicrobial peptides. ► Their predicted structures are similar to previous reported lectins structures. ► The fungal hevein-like peptide may have an unusual disulfide bond in its structure. ► The novel hevein-like peptides are predicted to bind to chitin oligomers.Lectins are proteins with ability to bind reversibly and non-enzymatically to a specific carbohydrate. They are involved in numerous biological processes and show enormous biotechnological potential. Among plant lectins, the hevein domain is extremely common, being observed in several kinds of lectins. Moreover, this domain is also observed in an important class of antimicrobial peptides named hevein-like peptides. Due to higher cysteine residues conservation, hevein-like peptides could be mined among the sequence databases. By using the pattern CX(4,5)CC[GS]X(2)GXCGX[GST]X(2,3)[FWY]C[GS]X[AGS] novel hevein-like peptide precursors were found from three different plants: Oryza sativa, Vitis vinifera and Selaginella moellendorffii. In addition, an hevein-like peptide precursor from the phytopathogenic fungus Phaeosphaeria nodorum was also identified. The molecular models indicate that they have the same scaffold as others, composed of an antiparallel β-sheet and short helices. Nonetheless, the fungal hevein-like peptide probably has a different disulfide bond pattern. Despite this difference, the complexes between peptide and N,N,N-triacetylglucosamine are stable, according to molecular dynamics simulations. This is the first report of an hevein-like peptide from an organism outside the plant kingdom. The exact role of an hevein-like peptide in the fungal biology must be clarified, while in plants they are clearly involved in plant defense. In summary, data here reported clear shows that an in silico strategy could lead to the identification of novel hevein-like peptides that could be used as biotechnological tools in the fields of health and agribusiness.
Keywords: Hevein domain; Regular expression; Data mining; Molecular modeling; Molecular dynamics simulation; Fungal hevein-like peptide;

GLP-1 expression in von Ebner's gland of diabetic rats by Xiao-hong Feng; Li-hong Zhou; Dong Wang; Xiao Yuan (137-141).
► We successfully verified GLP-1 expression in von Ebner's gland of rats. ► The expression of GLP-1 in diabetic rat von Ebner's gland was higher than that in normal controls. ► The ultrastructure changes of von Ebner's gland in diabetes rats were observed.GLP-1, a peptidergic endocrine hormone, which associate with appetite control, glucose homeostasis and satiety. It might play an important role in the gustatory system. We tried to investigate the expression of GLP-1 in von Ebner's gland of diabetic and control rats, and the ultrastructure changes on von Ebner's gland of diabetes rats. GLP-1 expression in von Ebner's gland was evaluated by immunohistochemistry. The number of GLP-1 positive cells in diabetic rat von Ebner's gland was significantly higher than that in normal controls. Electron micrographs showed that a series of pathologic changes in von Ebner's gland of diabetes rats. The results suggest that GLP-1 have some effects within the gustatory systems, and elevated von Ebner's gland GLP-1 expression may be one cause of diabetic taste impairment.
Keywords: Glucagon-like peptide-1; von Ebner's gland; Diabetes; Rats;

► A novel peptide PACAP-TAT was produced by tagging TAT at PACAP's C-terminus. ► PACAP-TAT displayed increased traversing biological barriers ability. ► PACAP-TAT attenuated lung injury significantly more effectively than PACAP. ► PACAP-TAT may be potent substitute for PACAP.Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potential therapeutic peptide with anti-inflammatory and anti-oxidative effects. In order to increase the efficiency of traversing biological barriers, a novel fusion peptide PACAP-TAT was produced by tagging PACAP at its C-terminus with 11-amino acid TAT protein transduction domain. The results of characteristic assays showed that PACAP-TAT activated PACAP specific receptor PAC1 with the same potency as PACAP and PACAP-TAT crossed blood–brain barrier (BBB), blood–air barrier (BAB) and blood–testis barrier (BTB) with the efficiency about 2.5-fold higher than that of PACAP. Both PACAP-TAT and PACAP were used treat the mice with lung injury induced by repeated smoke inhalation. It was shown that both PACAP-TAT and PACAP decreased the mortality, increased the body weight and inhibited the edema and vascular permeability in the lungs of the mice received repeated smoke inhalation, while PACAP-TAT displayed more marked effects than PACAP. PACAP-TAT decreased myeloperoxidase (MPO) activity, increased catalase (CAT) activity and down-regulated interleukin 6 (IL-6) and malondialdehyde (MDA) levels in the lungs with a significantly higher efficiency than PACAP. The histopathological analysis also showed that PACAP-TAT attenuated the cell filtration and bronchi epithelial hyperplasia more significantly than PACAP. Moreover the leukocyte count in blood and the serum superoxide dismutase (SOD) activity in the mice treated with PACAP-TAT were significantly different from that in mice treated with PACAP (p  < 0.05). All these data indicated that PACAP-TAT with increased traversing ability was more effective than PACAP in protecting the mice from the lung injury induced by repeated smoke inhalation.
Keywords: PACAP; Protein transduction domain; Biological barriers; Lung injury; Smoke inhalation;

Hemodynamic actions and mechanisms of systemically administered α-MSH analogs in mice by Petteri Rinne; Sanna Tikka; Satu Mäkelä; Tomi Streng; Eriika Savontaus (150-158).
► Hemodynamic actions of systemically administered α-MSH analogs were studied in mice. ► α-MSH analogs markedly increased blood pressure and heart rate in conscious mice. ► The tachycardic effect was more prominent than the effect on blood pressure. ► The effects were blocked by the melanocortin 3/4 receptor antagonist SHU9119. ► The tachycardic effect was attributed to sympathetic activation and withdrawal of vagal tone.α-Melanocyte-stimulating hormone (α-MSH) regulates important physiological functions including energy homeostasis and inflammation. Potent analogs of α-MSH, [Nle4, d-Phe7]-α-MSH (NDP-α-MSH) and melanotan-II (MT-II), are widely used in pharmacological studies, but the hemodynamic effects associated with their systemic administration have not been thoroughly examined. Therefore, we investigated the hemodynamic actions of these compounds in anesthetized and conscious C57Bl/6N mice using peripheral routes of administration. NDP-α-MSH and MT-II induced mild changes in blood pressure and heart rate in anesthetized mice compared to the effects observed in conscious mice, suggesting that anesthesia distorts the hemodynamic actions of α-MSH analogs. In conscious mice, NDP-α-MSH and MT-II increased blood pressure and heart rate in a dose-dependent manner, but the tachycardic effect was more prominent than the pressor effect. Pretreatment with the melanocortin (MC) 3/4 receptor antagonist SHU9119 abolished these hemodynamic effects. Furthermore, the blockade of β1-adrenoceptors with metoprolol prevented the pressor effect and partly the tachycardic action of α-MSH analogs, while the ganglionic blocker hexamethonium abrogated completely the difference in heart rate between vehicle and α-MSH treatments. These findings suggest that the pressor effect is primarily caused by augmentation of cardiac sympathetic activity, but the tachycardic effect seems to involve withdrawal of vagal tone in addition to sympathetic activation. In conclusion, the present results indicate that systemic administration of α-MSH analogs elevates blood pressure and heart rate via activation of MC3/4 receptor pathways. These effects and the consequent increase in cardiac workload should be taken into account when using α-MSH analogs via peripheral routes of administration.
Keywords: Melanocortin; Hemodynamics; Melanocortin receptor; Sympathetic activity;

24-Hour profiles of circulating ghrelin and peptide YY are inversely associated in normal weight premenopausal women by Brenna R. Hill; Mary Jane De Souza; David A. Wagstaff; Rino Sato; Nancy I. Williams (159-162).
► Circulating PYY was a significant predictor of ghrelin over 24 h in humans. ► Increases in circulating PYY were associated with decreases in circulating ghrelin. ► PYY may be modulating ghrelin secretion in normal weight women over a 24 h period. ► Findings substantiate experimental inferences that PYY modulates ghrelin secretion.Peptide YY (PYY) and ghrelin (GHR) may modulate one another's actions within the hypothalamus. Peripheral infusion of PYY in humans acutely suppresses circulating concentrations of GHR. Whether an association between PYY and GHR exists in the peripheral circulation of humans over 24 h is unknown. The purpose of this study was to determine if circulating concentrations of PYY and GHR were significantly associated over 24 h in humans. Participants (n  = 13) were normal weight, moderately active, women ages 18–24 yr. Blood samples were obtained q10 min for 24 h and assayed using RIA for total PYY and total GHR hourly from 0800 to 1000 h and 2000 to 0800 h and q20 min from 1000 to 2000 h. Dietary intake during the 24 h procedure was comprised of 55% carbohydrates, 30% fat, and 15% protein (three meals and a snack). Statistical analyses included linear mixed-effects modeling to test whether PYY predicted GHR concentrations over 24 h. Participants weighed 57.0 ± 1.5 kg and had 26.1 ± 1.5% body fat (15.0 ± 1.1 kg), 42.1 ± 1.1 kg fat free mass, a BMI of 21.3 ± 0.5 kg/m2 and RMR of 1072 ± 28 kcal/24 h. Visually, PYY and GHR exhibited an inverse association over nearly the entire 24 h period. Statistically, circulating concentrations of 24 h PYY predicted 24 h GHR (ghrelin = 1860.51–2.14*PYY; p  = 0.04). Circulating concentrations of PYY are inversely associated with GHR over 24 h. These data provide evidence that PYY may contribute to the modulation of the secretion of GHR in normal weight, premenopausal women over a 24 h period and supports similar inferences from experimental studies in animals and humans.
Keywords: Ghrelin; Peptide YY;

A surfactin cyclopeptide of WH1fungin used as a novel adjuvant for intramuscular and subcutaneous immunization in mice by Zhenqiu Gao; Shengying Wang; Gaofu Qi; Hui Pan; Li Zhang; Xiaohui Zhou; Jingjing Liu; Xiuyun Zhao; Jie Wu (163-171).
► This is the first report that Surfactin can be used as a novel immunoadjuvants. ► Surfactin can markedly enhance the immune response as strong as Freund's adjuvant. ► Surfactin can help to induce both of durable humoral and cellular immune response. ► Surfactin is very easy for use by simply mixture with antigen solution.WH1fungin, a surfactin cyclopeptide from Bacillus amyloliquefaciens WH1, is firstly reported as a novel immunoadjuvant, which can markedly enhance the immune response when given in mixture with antigens. After intramuscular or subcutaneous immunization, WH1fungin can help to induce both of durable humoral and cellular immune response, even as strong as Freund's adjuvant. Both IgG1 and IgG2a antigen-specific antibodies were elicited from the immunizations indicating a mixed Th1/Th2 response. Splenocytes from mice intramuscularly immunized with OVA plus WH1fungin responded to OVA CTL peptide stimulation resulting in an increase in CD8+TNF-α+ and CD8+IFN-γ+ T cell populations, and also an increase in CD4+TNF-α+ T cells and CD4+IFN-γ+ T cell populations was found from mice subcutaneously immunized with OVA plus WH1fungin when responded to OVA Th peptide stimulation. These results further suggest that WH1fungin helps to elicit humoral and cellular responses to OVA. The potential mechanism of WH1fungin as an immunoadjuvant was investigated. In vitro assays showed that WH1fungin could enter into RAW 264.7 cells, induce ROS accumulation, and increase the expression of cell surface markers and cytokines in cells. Further investigation suggested that WH1fungin might exert its adjuvant activity by ligating with TLR-2 in antigen present cells such as RAW 264.7. Taken together, WH1fungin is very potent as a novel adjuvant for development of vaccines in the future.
Keywords: WH1fungin; Surfactin; Adjuvant; Vaccine;

► Cyclic-7mer peptides displaying brain tropism were identified by in vivo phage display. ► 349 unique peptides were associated with gray matter (microvasculature and parenchyma). ► Amino acid distribution suggested a theoretical ideal peptide sequence PP(S/P)SSST. ► Phage displaying SYTSSTM were recovered at highest frequency from brain gray matter. ► SYTSSTM phage showed a 5-fold greater brain accumulation compared to control phage.Peptide motifs that demonstrate tropism for the blood brain barrier (BBB) are of real translational value in developing innovative delivery strategies for biological brain targeted therapies. In vivo peptide-phage display affords peptide selection against the full complement of biological markers within the correct cellular macro- and micro-environments. Here a stringent in vivo biopanning protocol was employed in the rat aimed at identifying cyclic 7-mer peptide motifs that mediate tropism to brain microvasculature. Five rounds of biopanning identified 349 unique peptide motifs in the brain tissue gray matter compartment (microvasculature and parenchyma). While in general no consensus was evident linking peptide physico-chemical properties and brain tropism, peptides bearing c-SxTSSTx-c or c-xxxSSTx-c motifs were found to be present in high abundance. Based on amino acid frequency distribution of the 349 unique peptides sequences a theoretical ‘idealized’ peptide pattern, c-PP(S/P)SSST-c, could be derived. For the most abundant experimental peptide sequence found in brain tissue, c-SYTSSTM-c, an in vivo pharmacokinetic and whole body tissue biodistribution study was performed. Based upon tissue exposure data (i.e. tissue AUC(0–infinity)) the sequence c-SYTSSTM-c efficiently retargeted phage virions to the brain providing an approximate 5-fold greater (P  < 0.05) accumulation in brain over control phage; in all other organs no significant (P  > 0.05) difference in tissue tropism between c-SYTSSTM-c and control phages were evident. This peptide and more generally the peptide motifs, -SxTSSTx- or -xxxSSTx-, warrant further investigation as agents mediating sequence-dependent tropism to brain microvasculature potentially able to deliver biologic cargo to the CNS.
Keywords: Brain tropism; Blood–brain barrier; Phage display; Peptide; Biodistribution; Pharmacokinetics;

Cardiovascular effects of a PEGylated apelin by Z.Q. Jia; L. Hou; A. Leger; I. Wu; A.B. Kudej; J. Stefano; C. Jiang; C.Q. Pan; G.Y. Akita (181-188).
► N-terminal conjugated 40 kDa PEG apelin-36 was successfully produced. ► PEGylation had minimal effect on APJ receptor binding affinity. ► Comparable in vitro bioactivity was observed between PEG–apelin-36 and apelin-36. ► Inotropic effect and circulation time were prolonged with PEG–apelin-36. ► Apelin-36 and PEG–apelin-36 had no effect on blood pressure.Several studies have documented cardiovascular effects of apelin, including enhanced inotropy and vasodilation. However, these cardiovascular effects are short lived due to the predicted short circulating half-life of the apelin peptide. To address this limitation of apelin, we pursued N-terminal PEGylation of apelin and examined the cardiovascular effects of the PEGylated apelin. A 40 kDa PEG conjugated apelin-36 (PEG–apelin-36) was successfully produced with N-terminal conjugation, high purity (>98%) and minimum reduction of APJ receptor binding affinity. Using an adenylate cyclase inhibition assay, comparable in vitro bioactivity was observed between the PEG–apelin-36 and unmodified apelin-36. In vivo evaluation of the PEG–apelin-36 was performed in normal rats and rats with myocardial infarction (MI). Cardiac function was assessed via echocardiography before, during a 20 min IV infusion and up to 100 min post peptide infusion. Similar increases in cardiac ejection fraction (EF) were observed during the infusion of PEG–apelin-36 and apelin-36 in normal rats. However, animals that received PEG–apelin-36 maintained significantly increased EF over the 100 min post infusion monitoring period compared to the animals that received unmodified apelin-36. Interestingly, EF increases observed with PEG–apelin-36 and apelin-36 were greater in the MI rats. PEG–apelin-36 had a prolonged circulating life compared to apelin-36 in rats. There were no changes in aortic blood pressure when PEG–apelin-36 or apelin-36 was administered. To our knowledge this is the first report of apelin PEGylation and documentation of its cardiovascular effects.
Keywords: Apelin; PEGylation; Cardiac function; Rat; Inotropic effect;

Cafeteria diet-induced obesity plus chronic stress alter serum leptin levels by I.C. Macedo; L.F. Medeiros; C. Oliveira; C.M. Oliveira; J.R. Rozisky; V.L. Scarabelot; A. Souza; F.R. Silva; V.S. Santos; S.G. Cioato; W. Caumo; I.L.S. Torres (189-196).
► Animals were subjected to chronic stress and a high-calorie diet for 40 days. ► The parameters evaluated were serum leptin levels and specific adipose tissue weights. ► Stress inhibited weight independently of diet type. ► A hypercaloric diet affected weight parameters and serum leptin levels.Obesity is a disease that has become a serious public health issue worldwide, and chronic stressors, which are a problem for modern society, cause neuroendocrine changes with alterations in food intake. Obesity and chronic stress are associated with the development of cardiovascular diseases and metabolic disorders. In this study, a rat model was used to evaluate the effects of a hypercaloric diet plus chronic restraint stress on the serum leptin and lipids levels and on the weight of specific adipose tissue (mesenteric, MAT; subcutaneous, SAT and visceral, VAT). Wistar rats were divided into the following 4 groups: standard chow (C), hypercaloric diet (HD), stress plus standard chow (S), and stress plus hypercaloric diet (SHD). The animals in the stress groups were subjected to chronic stress (placed inside a 25 cm × 7 cm plastic tube for 1 h per day, 5 days per week for 6 weeks). The following parameters were evaluated: the weight of the liver, adrenal glands and specific adipose tissue; the delta weight; the Lee index; and the serum levels of leptin, corticosterone, glucose, total cholesterol, and triglycerides. The hypercaloric diet induced obesity in rats, increasing the Lee index, weight, leptin, triglycerides, and cholesterol levels. The stress decreased weight gain even in animals fed a hypercaloric diet but did not prevent a significant increase in the Lee index. However, an interaction between the independent factors (hypercaloric diet and stress) was observed, which is demonstrated by the increased serum leptin levels in the animals exposed to both protocols.
Keywords: Adipose tissue; Cafeteria diet; Chronic restraint stress; Hypercaloric diet; Leptin; Obesity;