Peptides (v.32, #6)

Identification of PSA peptide mimotopes using phage display peptide library by Arulkumaran Shanmugam; Robert Suriano; Devyani Chaudhuri; Shilpi Rajoria; Andrea George; Abraham Mittelman; Raj K. Tiwari (1097-1102).
► Identification of cyclical PSA peptide mimotopes. ► Detection of non-cross reactive peptide mimotopes. ► Recognition of immunological reactive PSA peptide mimotope. ► Neutralization of PSA by peptide specific antibody.Prostate cancer (PCa) is one of the most common types of cancer in men in the United States and is the second leading cause of cancer related death in men. Clinically, secreted prostate specific antigen (PSA) has gained recognition because of its proteolytic activity being directly linked to PCa cell proliferation leading to disease initiation and progression. Using phage display technology, we identified four distinct cyclical peptides. These peptides apart from differences in their amino acid sequence, elicited minimal cross reactive antibody responses against each other. One of the four peptides analyzed produced an antibody response that recognizes the PSA protein. We demonstrate that the synthetic PSA peptide mimics identified in our study are immunologically active and produce neutralizing activity and this has relevance and utility for prostate cancer disease progression.
Keywords: PSA; Peptide mimotope; Phage display; Prostate cancer;

Isolation, antiproliferation on tumor cell and immunomodulatory activity of BSP-I, a novel bursal peptide from chicken humoral immune system by Xiuli Feng; Taoqing Liu; Fangquan Wang; Ruibing Cao; Bin Zhou; Yu Zhang; Xiang Mao; Puyan Chen; Hui Zhang (1103-1109).
► A novel bursal septpeptide I (BSP-I) is isolated from BF unique to birds. ► BSP-I played antiproliferation on tumor cell, not on normal cell. ► BSP-I enhanced antitumor factor p53 and Bax protein expression. ► BSP-I induced humoral and cellular immune response from immunized mice.The bursa of Fabricius (BF) is acknowledged as central humoral immune organ unique to birds. Our purpose was to identify the potential function of a novel bursal-derived bioactive peptide. A bursal septpeptide (BSP-I), EPASGMM, first isolated from BF, reduced MCF and Hela tumor cells proliferation, and enhanced antitumor factor p53 luciferase activity and protein expression. Further, we found the significantly immune inducing function of BSP-I on antigen-specific immune response in BALB/c mice intraperitoneally immunized with inactivated avian influence virus (AIV, H9N2 subtype) vaccine, including of enhancing the antibody (IgG, the isotypes IgG1 and IgG2a) production, and stimulating cytokines IL-4 and IFN-γ level, and inducing T cell immunophenotyping and lymphocyte proliferation. These results suggested that as the bioactive peptide from avian humoral immune system, various biological function of BSP-I may have far-reaching implication on immune system significance, which might provide novel insight on linking between humoral immune system and development of effective immunotherapeutic strategies for treating human cancers diseases.
Keywords: Bursal septpeptide (BSP-I); Antiproliferation on tumor cell; Antitumor factor p53; Humoral immune response; Cellular-mediated immunity;

Pardaxin-induced apoptosis enhances antitumor activity in HeLa cells by Jung-Chieh Hsu; Li-Ching Lin; Jason T.C. Tzen; Jyh-Yih Chen (1110-1116).
► Pardaxin induced apoptosis with treatment. ► Pardaxin inhibited tumor cell growth. ► DNA fragmentation and increases in the subG1 phase and caspase 8 activity suggest that pardaxin caused HeLa cell death by inducing apoptosis, but had a different mechanism in HT1080 cells.Pardaxin, a pore-forming antimicrobial peptide that encodes 33 amino acids was isolated from the Red Sea Moses sole, Pardachirus mamoratus. In this study, we investigated its antitumor activity in human fibrosarcoma (HT-1080) cells and epithelial carcinoma (HeLa) cells. In vitro results showed that the synthetic pardaxin peptide had antitumor activity in these two types of cancer cells and that 15 μg/ml pardaxin did not lyse human red blood cells. Moreover, this synthetic pardaxin inhibited the proliferation of HT1080 cells in a dose-dependent manner and induced programmed cell death in HeLa cells. DNA fragmentation and increases in the subG1 phase and caspase 8 activities suggest that pardaxin caused HeLa cell death by inducing apoptosis, but had a different mechanism in HT1080 cells.
Keywords: Antimicrobial peptides (AMPs); Pardaxin; Cytotoxicity; Pores;

Effects of cathelicidin and its fragments on three key enzymes of HIV-1 by Jack Ho Wong; Anna Legowska; Krzysztof Rolka; Tzi Bun Ng; Mamie Hui; Chi Hin Cho; Wendy Wai Ling Lam; Shannon Wing Ngor Au; Oscar Wangang. Gu; David Chi Cheong Wan (1117-1122).
► Human cathelicidin LL37 and its fragments LL13-37 and LL17-32 inhibited HIV-1 reverse transcriptase dose-dependently with an IC50 value of 15 μM, 7 μM, and 70 μM, respectively. ► Human cathelicidin LL37 and its fragments LL13-37 and LL17-32 inhibited HIV-1 protease with a weak potency. ► The peptides did not exert toxicity on human peripheral blood mononuclear cells.Cathelicidins exhibit anti-HIV activity but it is not known if they reduce the activity of enzymes crucial to the life cycle of the retrovirus. It is shown in this investigation that human cathelicidin LL37 and its fragments LL13-37 and LL17-32 inhibited HIV-1 reverse transcriptase dose-dependently with an IC50 value of 15 μM, 7 μM, and 70 μM, respectively. The three peptides inhibited HIV-1 protease with a weak potency, achieving 20–30% inhibition at 100 μM. The mechanism of inhibition was protein–protein interaction as revealed by surface plasmon resonance. The peptides were devoid of the ability to inhibit translocation of HIV-1 integrase, which has been labeled with green fluorescent protein, into the nucleus. The peptides did not exert toxicity on human peripheral blood mononuclear cells.
Keywords: Cathelicidin; Fragments; HIV reverse transcriptase; Protease; Integrase;

► BMAP-18 and its analogs displayed much higher cell selectivity (about 4–97-fold increased) as compared to BMAP-27. ► BMAP-18 and its analogs exhibit lethality toward microbes due to their ability to form small channels, but not by the membrane-disruption/perturbation mode. ► There was a significant linear correlation between the increase in the hydrophobicity of peptides and LPS-neutralizing activity. ► Other important parameters of antimicrobial peptides may be involved in their LPS-neutralizing activity, as well as positive charge and hydrophobicity.To develop novel antimicrobial peptides (AMPs) with improved cell selectivity and potent LPS-neutralizing activity, we synthesized an 18 N-terminal residues peptide (BAMP-18) of bovine myeloid antimicrobial peptide-27 (BMAP-27) and its analogs (BMAP-18-W, BMAP-18-L, BMAP-18-I and BMAP-18-f). BMAP-18 and its analogs displayed much higher cell selectivity (about 4–97-fold increased) as compared to parental BMAP-27 because of their decreased hemolytic activity and retained antimicrobial activity. BMAP-27 caused near-complete dye leakage from bacterial-membrane-mimicking vesicles even at very low concentration of 0.5 μM, whereas BMAP-18 and its analogs induced very little dye leakage (less than 40%) even at 16 μM. These peptides induced near-complete membrane depolarization of Staphylococcus aureus cells under their MIC (4 μM). These results suggests that BMAP-18 and its analogs exhibit lethality toward microbes due to their ability to form small channels that permit the transit of ions or protons, but not molecules as large as calcein, and not by the membrane-disruption/perturbation mode. BMAP-18 and its analogs significantly inhibited nitric oxide (NO) production or tumor necrosis factor-α (TNF-α) release in LPS-stimulated mouse macrophage RAW264.7 cells at 10 μM. In particular, BMAP-18-W showed LPS-neutralizing activity comparable to that of BMAP-27. There was a significant linear correlation between the increase in the hydrophobicity of peptides and LPS-neutralizing activity. Although BMAP-18-W has lower hydrophobicity than BMAP-18-L, it showed higher LPS-neutralizing activity as compared to BMAP-18-L. This result suggests other important parameters of AMPs may be involved in their LPS-neutralizing activity, as well as positive charge and hydrophobicity.
Keywords: Bovine myeloid antimicrobial peptide-27 (BMAP-27); Bovine myeloid antimicrobial peptide-18 (BMAP-18); Cell selectivity; Killing mechanism; LPS-neutralizing activity;

The antibacterial activity of BF-30 in vitro and in infected burned rats is through interference with cytoplasmic membrane integrity by Huimin Zhou; Jie Dou; Jing Wang; Lili Chen; Hui Wang; Weidong Zhou; Yunman Li; Changlin Zhou (1131-1138).
► We examine in vitro activity of BF-30 to kill drug-resistant bacteria. ► We identify protection of BF-30 against Pseudomonas aeruginosa in infected burns. ► We delineate the antibacterial mechanism of BF-30. ► BF-30 killed bacteria by interfering with cytoplasmic membrane. ► BF-30 inhibited the infection of P. aeruginosa in infected burns.Cathelicidin-BF (BF-30) is found in the venom of the snake Bungarus fasciatus and exhibits broad antimicrobial activity against bacteria and fungi. Nevertheless, its antibacterial activity in vivo and antibacterial mechanism is unknown. In the present study, we examined the antibacterial activity of BF-30 in vitro against drug-resistant Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, first identifying its protection against P. aeruginosa in infected burns and then delineating the antimicrobial mechanism of BF-30. The data showed that BF-30 had stronger antimicrobial activities against a broad spectrum of microorganisms than gentamicin, ampicillin or bacitracin. The killing curves of BF-30 against P. aeruginosa and S. aureus showed that CFU counts rapidly decreased by almost 2 logs within 6 min, and it took just less than 2 h to kill all the bacteria. In addition, we investigated whether BF-30 had antibacterial activity in a burn/acute infection rat model. Dose–response (0.75, 3, 12 mg/kg/day) studies indicated that BF-30 significantly reduced the colonization of P. aeruginosa in the burn eschars, lungs and liver of burn injured rats and that it could prevent subsequent systemic infection and development of inflammation. The peptide induced chaotic membrane morphology and cell debris, as determined by electron microscopy, and caused the cytoplasmic membrane to crack, resulting in β-galactosidase leakage and EtBr accumulation. This suggests that the antimicrobial activity of BF-30 is based on cytoplasmic membrane permeability. Taken together, our data demonstrate that antibacterial activity of BF-30 has potential therapeutic value for the prevention and treatment of burn and wound infections.
Keywords: Cathelicidin-BF; Antimicrobial peptides; Drug-resistant microorganisms; Infected burns;

The activity of antimicrobial peptide S-thanatin is independent on multidrug-resistant spectrum of bacteria by Guoqiu Wu; Xiaofang Li; Xiaobo Fan; Hongbin Wu; Shenglan Wang; Zilong Shen; Tao Xi (1139-1145).
► Activity of S-thanatin to MDR bacteria. ► S-thanatin was more effective toward Gram-negative strains, especially for E. coli and K. pneumoniae. ► The activity of S-thanatin was independent on multi-drug resistance of bacteria. ► S-thanatin may be a promising candidate for alternative antimicrobial drug to various multi-resistant bacteria.In this study, the activity of S-thanatin (an analog of antimicrobial peptide derived from thanatin) against different bacterial pathogens frequently which can cause therapeutic problems was tested. The result showed minimal inhibitory concentrations (MICs) of S-thanatin against all isolates of the Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella ornithinolytica and Klebsiella oxytoca were in the range of 4–16 μg/ml, no matter which antibiotic the bacterial was resistant or susceptible, while almost all MICs to Gram-positive bacterial were >128 μg/ml except Enterococcus faecium. S-thanatin was more effective toward Gram-negative strains, especially for Enterobacter and Klebsiella. The MICs of S-thanatin were no significantly different in the same species regardless of antibiotic sensitive or -resistant isolates to single or multiple antibiotic (P  > 0.05). Likewise, no notable difference could be observed between E. coli, K. pneumoniae, E. cloacae, E. aerogenes, K. ornithinolytica which were sensitive to S-thanatin (P  > 0.05). It was implied that the antimicrobial activity of S-thanatin was independent on multi-drug resistance spectrum of bacteria.
Keywords: Antimicrobial peptide; Thanatin; Antimicrobial activity; Clinical isolate;

A novel antimicrobial peptide from skin secretions of the earthworm, Pheretima guillelmi (Michaelsen) by Wenliang Li; Sisi Li; Jian Zhong; Zhu Zhu; Jingze Liu; Wenhong Wang (1146-1150).
► A novel lumbricin-like antimicrobial peptide named lumbricin-PG was isolated from skin secretions of the earthworm, P. guillelmi. ► Its amino acid sequence was determined as FSRYARMRDSRPWSDRKNNYSGPQFTYPPEKAPPEKLIKWNNEGSPIFEMPAEGGHIEP. ► The predicted protein from the cDNA sequence was composed of 73 amino acid residues including a mature lumbricin-PG and predicted signal peptide. ► Purified lumbricin-PG exerted potential antimicrobial activities against bacteria and fungi; it showed weak hemolysis activity against human and rabbit red cells.A novel lumbricin-like antimicrobial peptide named lumbricin-PG was isolated from skin secretions of the earthworm, Pheretima guillelmi (Michaelsen), using a procedure of one step Sephadex G-50 gel filtration and one step C8 reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined as FSRYARMRDSRPWSDRKNNYSGPQFTYPPEKAPPEKLIKWNN EGSPIFEMPAEGGHIEP by Edman degradation combined with cDNA cloning and mass spectrometry analysis. The cDNA encoding lumbricin-PG was cloned by cDNA library screening. The predicted protein from the cDNA sequence was composed of 73 amino acid residues including a mature lumbricin-PG and predicted signal peptide. It showed similarity with lumbricin antimicrobial peptide from the earthworm, Lumbricus rubellus by BLAST search. Purified lumbricin-PG exerted potential antimicrobial activities against bacteria and fungi; it showed weak hemolysis activity against human and rabbit red cells.
Keywords: Earthworm; Antimicrobial peptide; Skin secretion; Lumbricin; Pheretima guillelmi;

An antifungal peptide from Fagopyrum tataricum seeds by Jing-Jun Ruan; Hui Chen; Ji-Rong Shao; Qi Wu; Xue-Yi Han (1151-1158).
► The complete amino acid sequence of FtTI has been established by automatic Edman degradation and mass spectrometry. ► The molecule of inhibitor consists of 86 amino acid residues containing two disulfide bonds which connect Cys8 to Cys65 and Cys49 to Cys58. ► The active site of the inhibitor contains an Asp66–Arg67 bond. ► Dixon plots revealed a competitive inhibition of trypsin with inhibition constants (Ki) of 1.6 nM. ► FtTI exhibited strong inhibitory activity against phytopathogenic fungi.A major trypsin inhibitor was isolated and characterized from the seeds of the tartary buckwheat (Fagopyrum tataricum) (FtTI) by ammonium sulfate precipitation, ion exchange chromatography and centrifugal ultrafiltration. SDS-PAGE analysis under reducing condition showed that FtTI is a single polypeptide chain with a molecular mass of approximately 14 kDa. The complete amino acid sequence of FtTI was established by automatic Edman degradation and mass spectrometry. It was found that the trypsin inhibitor molecule consists of 86 amino acid residues containing two disulfide bonds which connect Cys8 to Cys65 and Cys49 to Cys58. The active site of the inhibitor was found to contain an Asp66–Arg67 bond. MALDI-TOF analysis showed that FtTI has two isoforms (Mr: 11.487 and 13.838 kDa). Dixon plots revealed a competitive inhibition of trypsin with inhibition constants (Ki) of 1.6 nM. Analysis of the amino acid sequence suggests that FtTI is a member of the protease inhibitor I family. What is more, FtTI exhibited strong inhibitory activity against phytopathogenic fungi.
Keywords: Fagopyrum tartaricum; Trypsin inhibitor; Phytopathogenic fungi; Purification;

A novel conotoxin, qc16a, with a unique cysteine framework and folding by Mingyu Ye; Jing Hong; Mi Zhou; Lijun Huang; Xiaoxia Shao; Youshan Yang; Fred J. Sigworth; Chengwu Chi; Donghai Lin; Chunguang Wang (1159-1165).
► qc16a is a novel conotoxin with a rare C–C–CC framework. ► qc16a adopts a ribbon conformation with I–IV, II–III disulfide connectivity. ► Asp1, His7 and Asn8 are all essential for the activity of qc16a.A novel conotoxin, qc16a, was identified from the venom of vermivorous Conus quercinus. qc16a has only 11 amino acid residues, DCQPCGHNVCC, with a unique cysteine pattern. Its disulfide connectivity was determined to be I–IV, II–III. The NMR structure shows that qc16a adopts a ribbon conformation with a simple beta-turn motif formed by residues Gly6, His7 and Asn8. qc16a causes depression symptom in mice when injected intracranially. Point mutation results showed that Asp1, His7 and Asn8 are all essential for the activity of qc16a. Electrophysiologically, qc16a has no strong effect on the whole-cell currents of neurons and the currents of Drosophila Shaker channels, human BK channels and NaV1.7 channels. Its specific target still remains to be identified.
Keywords: Conotoxin; Conus quercinus; Disulfide connectivity; Mutation; NMR; Electrophysiology;

► First cloning of natriuretic peptide/helokinestatin precursor from Heloderma horridum. ► Identification of two novel helokinestatin peptides. ► Establishment of widespread occurrence of helokinestatin-like peptides in other lizards. ► Predicted mature peptides located in HPLC fractions of venom and their primary structures confirmed. ► Peptides and encoding precursor cDNA derived from same venom sample.Natriuretic peptides are common components of reptile venoms and molecular cloning of their biosynthetic precursors has revealed that in snakes, they co-encode bradykinin-potentiating peptides and in venomous lizards, some co-encode bradykinin inhibitory peptides such as the helokinestatins. The common natriuretic peptide/helokinestatin precursor of the Gila Monster, Heloderma suspectum, encodes five helokinestatins of differing primary structures. Here we report the molecular cloning of a natriuretic peptide/helokinestatin precursor cDNA from a venom-derived cDNA library of the Mexican beaded lizard (Heloderma horridum). Deduction of the primary structure of the encoded precursor protein from this cloned cDNA template revealed that it consisted of 196 amino acid residues encoding a single natriuretic peptide and five helokinestatins. While the natriuretic peptide was of identical primary structure to its Gila Monster (H. suspectum) homolog, the encoded helokinestatins were not, with this region of the common precursor displaying some significant differences to its H. suspectum homolog. The helokinestatin-encoding region contained a single copy of helokinestatin-1, 2 copies of helokinestatin-3 and single copies of 2 novel peptides, (Phe)5-helokinestatin-2 (VPPAFVPLVPR) and helokinestatin-6 (GPPFNPPPFVDYEPR). All predicted peptides were found in reverse phase HPLC fractions of the same venom. Synthetic replicates of both novel helokinestatins were found to antagonize the relaxing effect of bradykinin on rat tail artery smooth muscle. Thus lizard venom continues to provide a source of novel biologically active peptides.
Keywords: Lizard; Venom; Peptide; Precursor; Smooth muscle; Molecular cloning;

Oxidative stress augments the secretion of atrial natriuretic peptide in isolated rat atria by Amin Shah; Shan Gao; Young-Bin Oh; Woo Hyun Park; Suhn Hee Kim (1172-1178).
► Pyrogallol and H2O2 stimulated ANP secretion and decreased atrial contractility. ► Pretreatment with ascorbic acid and cariporide attenuated the stimulatory effect of pyrogallol and H2O2 on ANP secretion. ► Antioxidant did not cause any changes in atrial parameters. ► Intracellular – formed ROS stimulates ANP secretion partly through MAPKerk pathway and Na+/H+ exchanger.Reactive oxygen species (ROS) play a role in cardiovascular diseases such as hypertension and heart failure. The objective of the present study was to investigate the role of endogenous ROS in atrial hemodynamics and ANP secretion in isolated perfused beating rat atria. Pyrogallol (a generator of superoxide anion, 0.1, 1 mM) or hydrogen peroxide (0.1, 1, 10, 30 mM) was perfused into atria paced at 1.2 Hz. Pyrogallol and hydrogen peroxide stimulated ANP secretion and concentration in a dose-dependent manner and dramatically decreased atrial contractility and translocation of extracellular fluid. The stimulatory effect of pyrogallol and hydrogen peroxide on ANP secretion was attenuated by the pretreatment with ascorbic acid (an antioxidant; 1 mM) and cariporide (an inhibitor of the Na+/H+ exchanger; 1 μM) but negative inotropic effect was not changed. U120 (a MAPKerk pathway inhibitor; 10 μM) attenuated the stimulatory effect of hydrogen peroxide on ANP secretion. However, U120 augmented negative inotropic effect and stimulatory effect of ANP concentration induced by pyrogallol. Antioxidant such as N-acetyl cystein, gallate, propyl gallate, or ellagic acid did not cause any significant changes in atrial parameters. These results suggest that intracellular – formed ROS stimulates ANP secretion partly through activation of MAPKerk pathway and Na+/H+ exchanger.
Keywords: ROS; Hydrogen peroxide; Pyrogallol; N-acetyl cystein; Polyphenol; Gallate; Antioxidant; Ascorbic acid; Atrial natriuretic peptide; MAPKerk;

Suppression of ANP secretion by somatostatin through somatostatin receptor type 2 by Shan Gao; Young-bin Oh; Amin Shah; Woo Hyun Park; Suhn Hee Kim (1179-1186).
► Somatostatin decreased ANP secretion via AC/PKC pathway. ► Somatostatin-induced ANP secretion was attenuated by SSTR type 2 antagonist. ► SSTR type 2 agonist but not type 5 reduced ANP secretion. ► The suppressive effect of somatostatin on ANP secretion was attenuated in diabetic rat atria. ► The SSTR 2 mRNA and protein was decreased in diabetic rat atria.Somatostatin is a cyclic-14 amino acid peptide which mainly distributed in digestive system and brain. Somatostatin receptor (SSTR) is a G-protein coupled receptor and all five SSTR subtypes are expressed in cardiomyocytes. The aim of this study was to investigate the effect of somatostatin on atrial natriuretic peptide (ANP) secretion and its signaling pathway. Somatostatin (0.01 and 0.1 nM) decreased ANP secretion in isolated beating rat atrium in a dose-dependent manner. But atrial contractility and translocation of extracellular fluid were not changed. Somatostatin-induced decrease in ANP secretion was significantly attenuated by the pretreatment with CYN 154806 (SSTR type 2 antagonist; 0.1 μM), but not by BIM 23056 (SSTR type 5 antagonist; 0.1 μM) and urantide (urotensin II receptor antagonist; 0.1 μM). When pretreated with an agonist for SSTR type 2 (Seglitide, 0.1 nM) and SSTR type 5 (L 817818, 0.1 nM), only Seglitide reduced ANP secretion similar to that of somatostatin. The suppressive effect of somatostatin on ANP secretion was attenuated by the pretreatment with an inhibitor for adenylyl cyclase (MDL-12330A, 5 μM) or protein kinase A (KT 5720, 0.1 μM). In diabetic rat atria, the suppressive effect of somatostatin on ANP secretion and concentration was attenuated. Real time-PCR and western blot shows the decreased level of SSTR type 2 mRNA and protein in diabetic rat atria. These data suggest that somatostatin decreased ANP secretion through SSTR type 2 and an attenuation of suppressive effect of somatostatin on ANP secretion in diabetic rat atria is due to a down-regulation of SSTR type 2.
Keywords: Somatostatin; Somatostatin receptor; ANP; Diabetes;

Inhibition of kallikrein-related peptidases by the serine protease inhibitor of Kazal-type 6 by Tomasz Kantyka; Jan Fischer; Zhihong Wu; Wim Declercq; Karina Reiss; Jens-Michael Schröder; Ulf Meyer-Hoffert (1187-1192).
► SPINK6 inhibits KLK4. ► SPINK6 inhibits KLK6. ► SPINK6 inhibits KLK12. ► SPINK6 inhibits KLK13. ► SPINK6 does not inhibit KLK3 and KLK11.Kallikrein-related peptidases (KLKs) are a group of serine proteases, expressed in several tissues. Their activity is regulated by inhibitors including members of the serine protease of Kazal-type (SPINK) family. Recently, we discovered that SPINK6 is expressed in human skin and inhibits KLK5, KLK7, KLK14 but not KLK8. In this study we tested whether SPINK6 inhibits other members of the KLK family and caspase-14. Using chromogenic substrates, SPINK6 exhibited inhibitory activity against KLK12 and KLK13 with K i around 1 nM, KLK4 with K i  = 27.3 nM, KLK6 with K i  = 140 nM, caspase-14 with a K i approximating 1 μM and no activity against KLK1, KLK3 and KLK11. Taken together, SPINK6 is a potent inhibitor of distinct KLKs members.
Keywords: Atopic dermatitis; Prostate cancer; Epidermal barrier; Protease inhibitor;

Kinin generation from exogenous kininogens at the surface of retinoic acid-differentiated human neuroblastoma IMR-32 cells after stimulation with interferon-γ by Ibeth Guevara-Lora; Magdalena Majkucinska; Anna Barbasz; Alexander Faussner; Andrzej Kozik (1193-1200).
► Kininogens bind to retinoic acid-differentiated human neuroblastoma IMR-32 cells. ► These neuronal cells produce kinins and des-Arg-kinins from exogenous kininogens. ► The production of these peptides increases after cell stimulation with interferon-γ. ► Stimulated cells express tissue kallikrein, carboxypeptidase M and kinin receptors. ► Particularly, the des-Arg-kinin-specific B1-type receptors are highly induced.Bradykinin-related peptides, kinins, ubiquitously occur in the nervous system and together with other pro-inflammatory mediators contribute to pathological states of that tissue such as edema and chronic pain. In the current work we characterized the kinin-forming system of neuronal cells obtained by differentiation of human neuroblastoma cell line IMR-32 with retinoic acid. These cells were shown to concentrate exogenous kinin precursors, kininogens, on the surface, release kinins from kininogens and subsequently convert kinins to their des-Arg metabolites. Significantly higher amounts of kinins and des-Arg-kinins were produced after cell stimulation with interferon-γ, a potent pro-inflammatory mediator involved in many neurological disorders. The expression of the major tissue kininogenase (the human kallikrein 1) and the major cell membrane-bound kininase (the carboxypeptidase M) also increased after cell stimulation with interferon-γ, suggesting the involvement of these enzymes in the kinin production and degradation, respectively. Interferon-γ was also able to up-regulate the expression of two known subtypes of kinin receptors. On the protein level, the changes were only observed in the expression of the des-Arg-kinin-specific type 1 receptor which functions in the propagation of the inflammatory state. Taken together, these results suggest a novel way for local kinin and des-Arg-kinin generation in the nervous tissue during pathological states accompanied by interferon-γ release.
Keywords: Neuronal cells; Kininogen binding; Human kallikrein 1; Carboxypeptidase M; Prolylcarboxypeptidase; Kinin receptors;

PE-11, a peptide derived from chromogranin B, in the rat eye by Katrin Lorenz; Josef Troger; Oliver Gramlich; Franz Grus; Rosa Hattmannstorfer; Reiner Fischer-Colbrie; Stephanie Joachim; Eduard Schmid; Barbara Teuchner; Gertrud Haas; Nikolaos Bechrakis (1201-1206).
► PE-11 is a peptide which is generated in vivo by proteolytic processing of chromogranin B. ► PE-11 is a constituent of capsaicin-sensitive sensory neurons in the peripheral innervation of the rat eye. ► The presence of PE-11 in the rat retina in glia is atypical for neuropeptides and unique.The aim of the study was to investigate the presence and distribution of PE-11, a peptide derived from chromogranin B, in the rat eye. For this purpose, newborn rats were injected with a single dosage of 50 mg/kg capsaicin subcutaneously under the neck fold and after three months, particular eye tissues were dissected and the concentration of PE-11-like immunoreactivity was determined by radioimmunoassay. Furthermore, PE-11-like immunoreactivities were characterized in an extract of the rat eye by reversed phase HPLC. Then, the distribution pattern of PE-11 was investigated in the rat eye and rat trigeminal ganglion by immunofluorescence. As a result, PE-11 was present in each tissue of the rat eye and capsaicin pretreatment led to a 88.05% (±7.07) and a 64.26% (±14.17) decrease of the levels of PE-11 in the cornea and choroid/sclera, respectively, and to a complete loss in the iris/ciliary body complex. Approximately 70% of immunoreactivities detected by the PE-11 antiserum have been found to represent authentic PE-11. Sparse nerve fibers were visualized in the corneal and uveal stroma, surrounding blood vessels at the limbus, ciliary body and choroid and in association with the dilator and sphincter muscle. Furthermore, immunoreactivity was present in the corneal endothelium. In the retina and optic nerve, glia was labeled. In the rat trigeminal ganglion, PE-11-immunoreactivity was visualized in small and medium sized ganglion cells with a diameter of up to 30 μm. In conclusion, there is unequivocal evidence that PE-11 is a constituent of capsaicin-sensitive sensory neurons innervating the rat eye and the distribution pattern is typically peptidergic in the peripheral innervation but in the retina completely atypical for neuropeptides and unique.
Keywords: PE-11; Chromogranin B; Rat; Eye; Capsaicin;

PACAP and a novel stable analog protect rat brain from ischemia: Insight into the mechanisms of action by Agnieszka Dejda; Tommy Seaborn; Steve Bourgault; Omar Touzani; Alain Fournier; Hubert Vaudry; David Vaudry (1207-1216).
► We identify the lowest dose of PACAP which protects the brain from stroke. ► We examine the mechanisms involved in the protective effect of PACAP after stroke. ► We find that PACAP and its stable analog inhibit apoptosis in MCAO model of stroke. ► We find that PACAP and its stable analog modulate inflammatory response after MCAO.Pituitary adenylate cyclase-activating polypeptide (PACAP) shows potent protective effects in numerous models of neurological insults. However, the use of PACAP as a clinically efficient drug is limited by its poor metabolic stability. By combining identification of enzymatic cleavage sites with targeted chemical modifications, a metabolically stable and potent PACAP38 analog was recently developed. The neuroprotective activity of this novel compound was for the first time evaluated and compared to the native peptide using a rat model of middle cerebral artery occlusion (MCAO). Our results show that as low as picomolar doses of PACAP38 and its analog strongly reduce infarct volume and improve neurological impairment induced by stroke. In particular, these peptides inhibit the expression of Bcl-2-associated death promoter, caspase 3, macrophage inflammatory protein-1α, inducible nitric oxide synthase 2, tumor necrosis factor-α mRNAs, and increase extracellular signal-regulated kinase 2, B-cell CLL/lymphoma 2 and interleukin 6 mRNA levels. These results indicate that the neuroprotective effect of PACAP after MCAO is not only due to its ability to inhibit apoptosis but also to modulate the inflammatory response. The present study highlights the potential therapeutic efficacy of very low concentrations of PACAP or its metabolically stable derivative for the treatment of stroke.
Keywords: Stroke; Pituitary adenylate cyclase-activating polypeptide (PACAP); Metabolically stable analog; Neuroprotection; Apoptosis; Inflammation;

Activity-dependent neuroprotective protein (ADNP)-derived peptide (NAP) ameliorates hypobaric hypoxia induced oxidative stress in rat brain by Narendra K. Sharma; Niroj K. Sethy; Ram Niwas Meena; Govindsamy Ilavazhagan; Mainak Das; Kalpana Bhargava (1217-1224).
► Intranasal administration of activity-dependent neuroprotective protein (ADNP) derived peptide (NAP) reaches cortex during hypobaric hypoxia. ► NAP limits hypoxia-induced oxidative stress in brain. ► NAP activates Nrf2-mediated oxidative stress response pathway. ► Presence of NAP impairs hypoxia induced memory loss.Hypobaric hypoxia is a socio-economic problem affecting cognitive, memory and behavior functions. Severe oxidative stress caused by hypobaric hypoxia adversely affects brain areas like cortex, hippocampus, basal ganglia, and cerebellum. In the present study, we have investigated the antioxidant and memory protection efficacy of the synthetic NAP peptide (NAPVSIPQ) during long-term chronic hypobaric hypoxia (7, 14, 21 and 28 days, 25,000 ft) in rats. Intranasal supplementation of NAP peptide (2 μg/Kg body weight) improved antioxidant status of brain evaluated by biochemical assays for free radical estimation, lipid peroxidation, GSH and GSSG level. Analysis of expression levels of SOD revealed that NAP significantly activated antioxidant genes as compared to hypoxia exposed rats. We have also observed a significant increased expression of Nrf2, the master regulator of antioxidant defense system and its downstream targets such as HO-1, GST and SOD1 by NAP supplementation, suggesting activation of Nrf2-mediated antioxidant defense response. In corroboration, our results also demonstrate that NAP supplementation improved the memory function assessed with radial arm maze. These cumulative results suggest the therapeutic potential of NAP peptide for ameliorating hypobaric hypoxia-induced oxidative stress.
Keywords: NAP peptide; Hypobaric hypoxia; Brain; Antioxidant status; Nrf2;

Protective effects of l-pGlu-(2-propyl)-l-His-l-ProNH2, a newer thyrotropin releasing hormone analog in in vitro and in vivo models of cerebral ischemia by Satyendra Kumar Rajput; Maqsood Ahmad Siddiqui; Vivek Kumar; Chhuttan Lal Meena; Aditya Bhushan Pant; Rahul Jain; Shyam Sunder Sharma (1225-1231).
► In this study, we have investigated neuroprotective potential of TRH analog NP-647 in cerebral ischemia models. ► NP-647 showed protection against oxygen deprivation, hydrogen peroxide and glutamate-induced cellular injury in PC-12 cells. ► NP-647 protected hippocampal CA1 neuron and improved memory retention in mice subjected to transient global ischemia. ► NP-647 treatment significantly decreased inflammatory cytokines TNF-α and IL-6 as well as lipid peroxidation in ischemic animals. ► Protective effect of NP-647 in cerebral ischemia was attributed to reduction of excitotoxicity, inflammation and oxidative stress.In the present study, the newly synthesized TRH analog (l-pGlu-(2-propyl)-l-His-l-ProNH2; NP-647) was evaluated for its effects in in vitro (oxygen glucose deprivation (OGD)-, glutamate- and H2O2-induced injury in PC-12 cells) and in vivo (transient global ischemia) models of cerebral ischemic injury. PC-12 cells were subjected to oxygen and glucose deprivation for 6 h. Exposure of NP-647 was given before and during OGD. In glutamate and H2O2 induced injury, exposure of NP-647 was given 1, 6 and 24 h prior to exposure of glutamate and H2O2 exposure. NP-647, per se found to be non-toxic in 1–100 μM concentrations. NP-647 showed protection against OGD at the 1 and 10 μM. The concentration-dependent protection was observed in H2O2- and glutamate-induced cellular injury. In in vivo studies, NP-647 treatment showed protection of hippocampal (CA1) neuronal damage in transient global ischemia in mice and subsequent improvement in memory retention was observed using passive avoidance retention test. Moreover, administration of NP-647 resulted in decrease in inflammatory cytokines TNF-α and IL-6 as well as lipid peroxidation. These results suggest potential of NP-647 in the treatment of cerebral ischemia and its neuroprotective effect may be attributed to reduction of excitotoxicity, oxidative stress and inflammation.
Keywords: In vitro-cytotoxicity; L-pGlu-(2-propyl)-L-His-L-ProNH2; Oxygen glucose deprivation; Transient global ischemia;

► Design of new amyloid fiber binding peptides from the sequence of amyloid-beta 1–42. ► Amyloid fiber binding peptides was derived from the C-terminus of amyloid-beta. ► Amyloid plaque binding was demonstrated in vitro on brain of APP/PS1 transgenic mouse. ► Peptides inhibit amyloid fiber formation as shown by thioflavin T aggregation assay.Amyloid plaques are the main molecular hallmark of Alzheimer's disease. Specific carriers are needed for molecular imaging and for specific drug delivery. In order to identify new low molecular weight amyloid plaque-specific ligands, the phage display technology was used to design short peptides that bind specifically to amyloid-beta protein, which is the principal component of amyloid plaques. For this purpose, a phage display library was designed from the amino acid sequence of amyloid-beta 1–42. Then, the diversity was increased by soft oligonucleotide-directed mutagenesis. This library was screened against amyloid-beta 1–42 and several phage clones were isolated. Their genomes were sequenced to identify the displayed peptides and their dissociation constants for amyloid-beta 1–42 binding were evaluated by ELISA. The two best peptides, which are derived from the C-terminus hydrophobic domain of amyloid-beta 1–42 that forms a beta-strand in amyloid fibers, were synthesized and biotinylated. After confirming their binding affinity for amyloid-beta 1–42 by ELISA, the specific interaction with amyloid plaques was validated by immunohistochemistry on brain sections harvested from a mouse model of Alzheimer's disease. The thioflavin T aggregation assay has furthermore shown that our peptides are able to inhibit the amyloid fiber formation. They are not toxic for neurons, and some of them are able to cross the blood–brain barrier after grafting to a magnetic resonance imaging contrast agent. To conclude, these peptides have high potential for molecular targeting of amyloid plaques, either as carriers of molecular imaging and therapeutic compounds or as amyloid fiber disrupting agents.
Keywords: Alzheimer's disease; Amyloid-beta; Phage display; Amyloid plaques; High-throughput screening;

Neuregulin-1β regulates outgrowth of neurites and migration of neurofilament 200 neurons from dorsal root ganglial explants in vitro by Zhen Liu; Wei Gao; Yanping Wang; Weiwei Zhang; Huaxiang Liu; Zhenzhong Li (1244-1248).
► Organotypic rat dorsal root ganglion explant culture model was established. ► Effects of neuregulin-1β on neurite outgrowth and neuronal migration were evaluated. ► Neuregulin-1β promotes neurite outgrowth of the explant. ► Neuregulin-1β promotes neuronal migration from the explant.Neuregulin-1β (NRG-1β) signaling has multiple functions in neurons. To assess NRG-1β on neurite outgrowth and neuronal migration in vitro, organotypic dorsal root ganglion (DRG) neuronal culture model was established. Neurite outgrowth and neuronal migration were evaluated using this culture model in the presence (5 nmol/L, 10 nmol/L, 20 nmol/L) or absence of NRG-1β. Neurofilament 200 (NF-200)-immunoreactive (IR) neurons were determined as the migrating neurons. The number of nerve fiber bundles extended from DRG explant increased significantly in the presence of NRG-1β (5 nmol/L, 23.0 ± 2.2, P  < 0.05; 10 nmol/L, 27.0 ± 2.7, P  < 0.001; 20 nmol/L, 30.8 ± 3.7, P  < 0.001) as compared with that in the absence of NRG-1β (19.0 ± 2.2). The number of neurons migrating from DRG explants increased significantly in the presence of NRG-1β (5 nmol/L, 39.6 ± 5.0, P  < 0.05; 10 nmol/L, 54.6 ± 6.7, P  < 0.001; 20 nmol/L, 62.2 ± 5.7, P  < 0.001) as compared with that in the absence of NRG-1β (31.6 ± 4.0). Moreover, the increase of the number of nerve fiber bundles and the number of migrating NF-200-IR neurons was dose-dependent for NRG-1β addition. The data in this study imply that NRG-1β promotes neurite outgrowth and neuronal migration from DRG explants in vitro.
Keywords: Neuregulin-1β; Neurofilament 200; Neuron; Dorsal root ganglion;

Hypo- and hyperthyroidism affect NEI concentration in discrete brain areas of adult male rats by Carolina Ayala; Susana Ruth Valdez; María Luján Navarra Morero; Marta Soaje; Norma Beatriz Carreño; Mónica Silvina Sanchez; Jakson Cioni Bittencourt; Graciela Alma Jahn; María Ester Celis (1249-1254).
► Thyroid hormones modify NEI content in specific areas of the CNS related to neuroendocrine control and reproduction. ► Long-term hypothyroidism (24 days) induces reductions in NEI concentration, in the PeFLH and PVH in the morning and afternoon, in the OVLT + AVPV and ME + Arc only in the morning, and in the POA just in the afternoon. ► NEI content changes during hyperthyroidism, principally in POA at morning and in ME + Arc and Pi at afternoon. ► NEI concentration changes in specific brain areas secondary to hypo- and hyperthyroidism may have consequences itself on reproduction.To date, there has been only one in vitro study of the relationship between neuropeptide EI (NEI) and the hypothalamic–pituitary–thyroid (HPT) axis. To investigate the possible relationship between NEI and the HPT axis, we developed a rat model of hypothyroidism and hyperthyroidism that allows us to determine whether NEI content is altered in selected brain areas after treatment, as well as whether such alterations are related to the time of day. Hypothyroidism and hyperthyroidism, induced in male rats, with 6-propyl-1-thiouracil and l-thyroxine, respectively, were confirmed by determination of triiodothyronine, total thyroxine, and thyrotropin levels. All groups were studied at the morning and the afternoon. In rats with hypothyroidism, NEI concentration, evaluated on postinduction days 7 and 24, was unchanged or slightly elevated on day 7 but was decreased on day 24. In rats with hyperthyroidism, NEI content, which was evaluated after 4 days of l-thyroxine administration, was slightly elevated, principally in the preoptic area in the morning and in the median eminence-arcuate nucleus and pineal gland in the afternoon, the morning and afternoon NEI contents being similar in the controls. These results provide the bases to pursue the study of the interaction between NEI and the HPT axis.
Keywords: Neuropeptide glutamic acid-isoleucine-amide (NEI); Brain areas; Hypothalamic–pituitary–thyroid (HPT) axis; Hypothyroidism; Hyperthyroidism; Adult male rat;

Oxytocin in the periaqueductal gray participates in pain modulation in the rat by influencing endogenous opiate peptides by Jun Yang; Jin-Ying Liang; Peng Li; Yan-Juan Pan; Pei-Yong Qiu; Jing Zhang; Fang Hao; Da-Xin Wang (1255-1261).
► The concentrations of oxytocin (OXT), leucine-enkephalin (L-Ek), methionine-enkephalin (M-Ek) and β-endorphin (β-Ep), not dynorphin A1–13 (DynA1–13) in the PAG perfusion liquid were increased after the pain stimulation. ► The concentrations of L-Ek, M-Ek and β-Ep, not DynA1–13 in the PAG perfusion liquid were decreased by the OXT receptor antagonist. ► The increased pain threshold induced by the OXT was attenuated by naloxone. ► The concentrations of L-Ek, M-Ek and β-Ep, not DynA1–13 in the PAG perfusion liquid were increased by exogenous OXT administration. ► OXT in the PAG participate in pain modulation by influencing the L-Ek, M-Ek and β-Ep rather than DynA1–13.Periaqueductal gray (PAG) plays a very important role in pain modulation through endogenous opiate peptides including leucine-enkephalin (L-Ek), methionine-enkephalin (M-Ek), β-endorphin (β-Ep) and dynorphin A1–13 (DynA1–13). Our pervious study has demonstrated that intra-PAG injection of oxytocin (OXT) increases the pain threshold, and local administration of OXT receptor antagonist decreases the pain threshold, in which the antinociceptive role of OXT can be reversed by pre-PAG administration of OXT receptor antagonist. The experiment was designed to investigate the effect of OXT on endogenous opiate peptides in the rat PAG during the pain process. The results showed that (1) the concentrations of OXT, L-Ek, M-Ek and β-Ep, not DynA1–13 in the PAG perfusion liquid were increased after the pain stimulation; (2) the concentrations of L-Ek, M-Ek and β-Ep, not DynA1–13 in the PAG perfusion liquid were decreased by the OXT receptor antagonist; (3) the increased pain threshold induced by the OXT was attenuated by naloxone, an opiate receptor antagonist; and (4) the concentrations of L-Ek, M-Ek and β-Ep, not DynA1–13 in the PAG perfusion liquid were increased by exogenous OXT administration. The data suggested that OXT in the PAG could influence the L-Ek, M-Ek and β-Ep rather than DynA1–13 to participate in pain modulation, i.e. OXT in the PAG participate in pain modulation by influencing the L-Ek, M-Ek and β-Ep rather than DynA1–13.
Keywords: Oxytocin; Enkephalin; β-Endorphin; Dynorphin; Periaqueductal gray; Pain modulation;

► Exogenously applied 26RFa attenuated the level of allodynia induced by partial sciatic nerve ligation. ► The effects of 26RFa were not antagonized by BIBP3226, a NPFF antagonist. ► The anti-allodynic effect of 26RFa may be mediated by the activation of GPR103. ► Nerve injury increased the number of QRFP-LI positive neurons in the L5 DRG.26RFa and QRFP are endogenous ligands of GPR103. 26RFa binding sites are widely distributed in the brain and the spinal cord where they are involved in processing pain. In the present study, the effects of intrathecal and intracerebroventricular applications of 26RFa on the level of mechanical allodynia induced by partial sciatic nerve ligation were examined in rats. The level of mechanical allodynia was measured using von Frey filaments. Intrathecal and intracerebroventricular injection of 26RFa attenuated the level of mechanical allodynia. 26RFa has been reported to activate not only GPR103 but also neuropeptide FF2 receptor and the effect of intrathecally and intracerebroventricularly administered 26RFa was not antagonized by BIBP3226, an antagonist of neuropeptide FF receptor. Immunohistochemical examination revealed that QRFP-like immunoreactivity (QRFP-LI) was expressed mainly in the small to medium sized neurons in the L5 dorsal root ganglion (DRG) and that partial sciatic nerve injury increased the percentage of QRFP-LI positive neurons. 7 days after the nerve injury, QRFP-LI positive neurons in the L5 DRG ipsilateral to the partial sciatic nerve injury were larger than those in the L5 DRG ipsilateral to the sham operation. These data suggest that (1) exogenously applied 26RFa modulates nociceptive transmission at the spinal and the supraspinal brain in the neuropathic pain model, (2) the mechanism 26RFa uses to produce an anti-allodynic effect may be mediated by the activation of GPR103, and (3) partial sciatic nerve ligation affects the expression of QRFP-LI in the dorsal root ganglion.
Keywords: Dorsal root ganglion; GPR103; Neuropathic pain; Immunohistochemistry;

► QRFP-26 administration increases the intake of a high fat diet in female rats. ► Prepro-QRFP mRNA levels in the VMH/ARC are increased during proestrus. ► Estrous cycle does not influence GPR103a or GPR103b mRNA levels in the VMH/ARC or LH.Pyroglutamylated arginine–phenylalanineamide peptide (QRFP) is a neuropeptide involved in feeding behavior. Central administration of QRFP selectively increases the intake of a high fat diet in male rats. QRFP administration also stimulates the hypothalamic-pituitary–gonadal axis via gonadotrophin-releasing hormone in male and female rats. Prepro-QRFP mRNA is expressed in localized regions of the mediobasal hypothalamus which are abundant in neurotransmitters, neuropeptides and receptor systems important for food intake regulation and reproductive behaviors. The current experiments were conducted to investigate the effects of centrally administered QRFP-26 on the intake of a high fat diet (HFD, 60% kcal from fat) in female rats and to investigate alterations in hypothalamic prepro-QRFP and its receptors, GPR130a and GPR103b, mRNA levels over the estrous cycle. In Experiment 1, female rats were administered QRFP-26 (intracerebroventricular; 0.3 nmol, 0.5 nmol, 1.0 nmol) in rats consuming either a HFD or a low fat diet. All doses of QRFP-26 selectively increased the intake of the HFD in female rats. These data suggest that QRFP-26 regulates the intake of energy dense foods in female rats, which is similar to previous findings in male rats. In Experiment 2, hypothalamic levels of prepro-QRFP mRNA and its receptors were assessed during diestrus, proestrus, or estrus. The level of prepro-QRFP mRNA in the ventromedial/arcuate nucleus (VMH/ARC) of the hypothalamus was increased during proestrus, which suggests that endogenous estrogen levels regulate QRFP expression in the VMH/ARC. These data suggest that QRFP may play a role in coordinating feeding behaviors with reproductive function when energy demand is increased.
Keywords: prepro-QRFP mRNA; Hypothalamus; High fat diet; Estrous cycle; 26RFa;

Time-dependent changes in the serum levels of prolactin, nesfatin-1 and ghrelin as a marker of epileptic attacks young male patients by Suleyman Aydin; Ersel Dag; Yusuf Ozkan; Ozgur Arslan; Guray Koc; Semai Bek; Serkan Kirbas; Tayfun Kasikci; Dilek Abasli; Zeki Gokcil; Zeki Odabasi; Zekiye Catak (1276-1280).
► Prolactin and nesfatin-1 levels were increased in epileptic patients while ghrelin was decreased. ► The prolactin and nesfatin-1 levels decreased while ghrelin increased in epileptic patients over time after a seizure. ► These findings could be helpful for distinguishing epileptic patients from psychogenic event and control subjects.A relationship between hormones and seizures has been reported in animals and humans. Therefore, the purpose of this study was to investigate the association between serum levels of prolactin, nesfatin-1 and ghrelin measured different times after a seizure or non-epileptic event and compared with controls. The study included a total of 70 subjects, and of whom 18 patients had secondary generalized epilepsy (SGE), 16 patients had primary generalized epilepsy (PGE), 16 patients exhibited paroxysmal event (psychogenic) and 20 healthy males were control subjects. The first sample was taken within 5 min of a seizure, with further samples taken after 1, 24, and 48 h so long as the patient did not exhibit further clinically observable seizures; blood samples were taken once from control subjects. Prolactin was measured immediately using TOSOH Bioscience hormone assays. Nesfatin-1 and ghrelin peptides were measured using a commercial immunoassay kit. Patients suffering from focal epilepsy with secondary generalization and primary generalized epilepsy presented with significantly higher levels of serum prolactin and nesfatin-1 and lower ghrelin levels 5 min, 1 and 24 h after a seizure than patients presenting with paroxysmal events (psychogenic) and control subjects; the data were similar but not statistically significant after 48 h. The present study suggests that increased serum prolactin and nesfatin-1 concentrations, decreased ghrelin concentrations could be used as markers to identify patients that have suffered a recent epileptic seizure or other paroxysmal event (psychogenic).
Keywords: Prolactin; Nesfatin-1; Ghrelin; Epilepsy;

The effects of ghrelin/GHSs on AVP mRNA expression and release in cultured hypothalamic cells in rats by Takahiro Nemoto; Hitoshi Sugihara; Asuka Mano; Toshiko Kano; Tamotsu Shibasaki (1281-1288).
► KP-102 and ghrelin significantly increased AVP mRNA expression and release. ► GHS-R knockdown blocked ghrelin- and KP-102-induced AVP mRNA expression and release. ► NPY significantly increased AVP mRNA expression and release. ► Anti-NPY IgG blocked KP-102-induced AVP mRNA expression and release. ► ACTH secretion by ghrelin/GHSs is induced mainly by hypothalamic AVP via NPY.Ghrelin, the endogenous ligand for growth hormone secretagogues (GHSs) receptor (GHS-R), increases adrenocorticotropin (ACTH) and cortisol (corticosterone) as well as GH secretion in humans and animals. However, the site of GHSs action to induce ACTH secretion is not fully understood. To clarify the mechanisms of the action of ghrelin/GHSs on ACTH secretion, we analyzed the effects of KP-102 and ghrelin on the mRNA expression and release of corticotropin releasing factor (CRF) and arginine vasopressin (AVP), ACTH secretagogues, in monolayer-cultured hypothalamic cells of rats. Incubation of cells with KP-102 for 4 h and 8 h and with ghrelin for 4 h significantly increased AVP mRNA expression and release without changing CRF mRNA expression. CRF levels in culture media were undetectable. Suppression of GHS-R expression by siRNA blocked ghrelin- and KP-102-induced AVP mRNA expression and release. NPY significantly increased AVP mRNA expression and release. Furthermore, treatment of cells with anti-NPY IgG blocked KP-102-induced AVP mRNA expression and release. We previously reported that KP-102 significantly increases NPY mRNA expression in cultured hypothalamic cells. Taken together, these results suggest that ACTH secretion by ghrelin/GHSs is induced mainly through hypothalamic AVP, and that NPY mediates the action of ghrelin/GHSs.
Keywords: Ghrelin; AVP; CRF; NPY;

The short term satiety peptide cholecystokinin reduces meal size and prolongs intermeal interval by Dalya M. Lateef; Martha C. Washington; Ayman I. Sayegh (1289-1295).
► We analyzed the meal patterns by camostat and a single dose of CCK-8. ► Camostat reduced meal size (MS) and prolonged IMI but CCK-8 reduced only MS. ► Camostat reduced food intake by limiting MS and prolonging IMI. ► CCK-8 reduce food intake by only limiting meal size.Camostat mesilate (or mesylate) releases endogenous cholecystokinin (CCK) or CCK-58, the only detectable endocrine form of CCK in the rat, and reduces cumulative food intake by activating CCK1 receptor. However, the literature lacks meal pattern analysis and an appropriate dose–response curve for this peptide. Therefore, the current study determines meal size (MS), intermeal interval (IMI) and satiety ratio (SR) by orogastric gavage of camostat (0, 12.5, 25, 50, 100, 200, 300, 400, 800 mg/kg) and compares them to those previously reported by a single dose of CCK-8 (1 nmol/kg, i.p), the most utilized form of CCK. We found that camostat (200, 300, 400 and 800 mg/kg) and CCK-8 reduced cumulative food intake and the size of the first meal, but only camostat prolonged IMI and increased SR. There was no change in the duration of the first two meals or in rated behaviors such as feeding, grooming, standing and resting in response to camostat and CCK-8, but there was more resting during the IMI in response to camostat. This study provides meal pattern analysis and an appropriate dose–response curve for camostat and CCK-8. Camostat reduces food intake by decreasing MS and prolonging IMI, whereas CCK-8 reduces food intake by reducing only meal size.
Keywords: CCK-58; CCK-8; Meal analysis; Meal size; Intermeal interval;

Ileal interposition attenuates the satiety responses evoked by cholecystokinin-8 and -33 by Shannon A. Metcalf; Martha C. Washington; Thelma A.L. Brown; Carol S. Williams; April D. Strader; Ayman I. Sayegh (1296-1302).
► Ileal interposition (II) may alter the satiety responses evoked by CCK. ► We measured food intake in response to CCK-8 and -33 in these rats. ► Both peptides reduced meal size but more so in the sham group. ► Only CCK-33 prolonged intermeal interval but only in sham rats. ► CCK is less likely responsible for reduction of body weight in this surgery.One of the possible mechanisms by which the weight-reducing surgical procedure ileal interposition (II) works is by increasing circulating levels of lower gut peptides that reduce food intake, such as glucagon like peptide-1 and peptide YY. However, since this surgery involves both lower and upper gut segments, we tested the hypothesis that II alters the satiety responses evoked by the classic upper gut peptide cholecystokinin (CCK). To test this hypothesis, we determined meal size (MS), intermeal interval (IMI) and satiety ratio (SR) evoked by CCK-8 and -33 (0, 1, 3, 5 nmol/kg, i.p.) in two groups of rats, II and sham-operated. CCK-8 and -33 reduced MS more in the sham group than in the II group; CCK-33 prolonged IMI in the sham group and increased SR in both groups. Reduction of cumulative food intake by CCK-8 in II rats was blocked by devazepide, a CCK1 receptor antagonist. In addition, as previously reported, we found that II resulted in a slight reduction in body weight compared to sham-operated rats. Based on these observations, we conclude that ileal interposition attenuates the satiety responses of CCK. Therefore, it is unlikely that this peptide plays a significant role in reduction of body weight by this surgery.
Keywords: Meal size; Intermeal interval; CCK, GLP-1, PYY; Weight loss; Bariatric surgery;

GLP-1 analogs containing disulfide bond exhibited prolonged half-life in vivo than GLP-1 by Ying Li; Xuemin Zheng; Lida Tang; Weiren Xu; Min Gong (1303-1312).
► GLP-1 analog (GLP17057) containing an inter-disulfide bond extended its half-life. ► The analog (GLP17057) remained the biological activity of GLP-1. ► GLP17057 showed better glucose tolerance and higher HbA1c reduction than GLP-1 and Exendin-4. ► GLP17057 might be utilized as a long-lasting drug for type 2 diabetes.The multiple physiological characterizations of glucagon-like peptide-1 (GLP-1) make it a promising drug candidate for the therapy of type 2 diabetes. However, the half-life of GLP-1 is short in vivo due to degradation by dipeptidyl peptidase-IV (DPP-IV) and renal clearance. This indicates that the stabilization of GLP-1 is critical for its utility in drug development. In this study, we developed a cluster of GLP-1 mutants containing an inter-disulfide bond that is predicted to increase the half-life of GLP-1 in vivo. Exendin-4 was also mutated with a disulfide bond similar to the GLP-1 analogs. In this study, the binding capacities of the mutants were determined, the stabilities of the mutants were investigated and the physiological functions of the mutants were compared with those of wild-type GLP-1 and exendin-4 in animals. The results indicated that the mutants remarkably raised the half-life in vivo; they also showed better glucose tolerance and higher HbA1c reduction than GLP-1 and exendin-4 in rodents. These results suggest that GLP-1 and exendin-4 mutants containing disulfide bonds might be utilized as possible potent anti-diabetic drugs in the treatment of type 2 diabetes mellitus.
Keywords: GLP-1 analogs; Disulfide bond; Long-acting GLP-1; Insulin stimulation; HbA1c;

► Ex-9 increased insulin-stimulated glucose uptake in C2C12 cell line. ► Ex-9 promotes oxidative differentiation in myotubes. ► Calcineurin A and PGC-1α probably mediated effects of Ex-9 on C2C12 myotubes.Exendin 1-39 amide (Ex-4) and its truncated form exendin 9-39 amide (Ex-9) are peptides of non-mammalian nature, which act as an agonist and antagonist, respectively, of the glucagon-like peptide-1 (GLP-1) receptor in mammals. GLP-1 is an intestinal peptide that plays an important role in the regulation of glucose metabolism and glucose uptake in skeletal muscle; however, the effects of its two analogs (Ex-4 and Ex-9) on myofiber properties are still unclear. Here, we report the effects of Ex-4 and Ex-9 alone or in combination on the myosin heavy chain (MyHC) type composition and the glucose uptake capacity in differentiated C2C12 myotubes. Neither Ex-4 nor Ex-9 altered basal glucose uptake, whereas Ex-9 significantly increased insulin-stimulated glucose uptake, suggesting enhanced insulin sensitivity. The mRNA expression of MyHC I and 2A as well as the percentage of MyHC I protein was remarkably increased in Ex-9-treated myotubes. In contrast, Ex-4, alone or in combination with Ex-9, caused a significant reduction in MyHC 2A mRNA expression and the percentage of MyHC I protein. Consistent with the MyHC type switching peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α expression in myotubes was remarkably increased by Ex-9 yet was significantly inhibited by Ex-4. In addition, intracellular concentrations of free Ca2+ were increased in all treatment groups, but only Ex-9-treated myotubes showed higher calcineurin A protein content. Taken together, our data suggest that Ex-9 promotes oxidative differentiation in myotubes to improve cell insulin sensitivity, probably through calcineurin and PGC-1α mediated pathways.
Keywords: Ex-4; Ex-9; C2C12; MyHC; PGC-1α; Calcineurin A;

Intraperitoneal injection of neuropeptide Y (NPY) alters neurotrophin rat hypothalamic levels: Implications for NPY potential role in stress-related disorders by Francesca Gelfo; Paola De Bartolo; Paola Tirassa; Nicoletta Croce; Carlo Caltagirone; Laura Petrosini; Francesco Angelucci (1320-1323).
► We characterize the effect of NPY intraperitoneal administration in the hypothalamus. ► NPY treatment decreases BDNF production in the hypothalamus. ► NPY treatment increases NGF production in the hypothalamus. ► First study analyzing the effects of NPY peripheral administration on neurotrophins. ► NPY may have a role in pathogenesis and treatment of stress-related disorders.Neuropeptide Y (NPY) is a 36-amino acid peptide which exerts several regulatory actions within peripheral and central nervous systems. Among NPY actions preclinical and clinical data have suggested that the anxiolytic and antidepressant actions of NPY may be related to its antagonist action on the hypothalamic–pituitary–adrenal (HPA) axis. The neurotrophins brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are proteins involved in the growth, survival and function of neurons. In addition to this, a possible role of neurotrophins, particularly BDNF, in HPA axis hyperactivation has been proposed. To characterize the effect of NPY on the production of neurotrophins in the hypothalamus we exposed young adult rats to NPY intraperitoneal administration for three consecutive days and then evaluated BDNF and NGF synthesis in this brain region. We found that NPY treatment decreased BDNF and increased NGF production in the hypothalamus. Given the role of neurotrophins in the hypothalamus, these findings, although preliminary, provide evidence for a role of NPY as inhibitor of HPA axis and support the idea that NPY might be involved in pathologies characterized by HPA axis dysfunctions.
Keywords: Neuropeptide Y; Hypothalamus; Neurotrophins; BDNF; NGF;

► Neuropeptide Y downregulates phagocytic activity of lizard splenic phagocytes. ► NPY modulated the phagocytosis through Y2 and Y5 receptors. ► Y2 and Y5 receptor-coupled AC–cAMP–PKA pathway involved in regulating phagocytosis.Present in vitro study in the wall lizard Hemidactylus flaviviridis, for the first time in ectothermic vertebrates, demonstrated the immunoregulatory role of neuropeptide Y (NPY) and its receptor-coupled downstream signaling cascade. NPY inhibited the percentage phagocytosis and phagocytic index of splenic phagocytes. The inhibitory effect of NPY on phagocytosis was completely antagonized by Y2 and Y5 receptor antagonists. This suggests that NPY mediated its effect on phagocytosis through Y2 and Y5 receptors. Further, NPY receptor-coupled downstream signaling cascade for NPY effect on phagocytosis was explored using the inhibitors of adenylate cyclase (SQ 22536) and protein kinase A (H-89). The SQ 22536/H-89 in a concentration-related manner decreased the inhibitory effect of NPY on phagocytosis. Further, an increase in intracellular cAMP level was observed in response to NPY. Taken together, it can be concluded that NPY via Y2 and Y5 receptor-coupled AC–cAMP–PKA pathway downregulated the phagocytic activity of lizard splenic phagocytes.
Keywords: Neuropeptide Y; Phagocytes; Reptiles; Signal transduction;

► NPY effects on reproductive functions are chiefly mediated by Y1 receptor (Y1R). ► We investigate here if estrous cycle has some effect on Y1R expression by the use of a transgenic mouse carrying the Y1R promoter linked to a reporter gene. ► Fluctuations in circulating levels of gonadal hormones during the estrous cycle are paralleled by changes in the expression of Y1R. ► Y1R expression is higher during the proestrus in some of the investigated nuclei that are related to both energy balance and reproduction.In the present study we used a transgenic mouse model, carrying the neuropeptide Y (NPY) Y1 receptor gene promoter linked to the LacZ reporter gene (Y1R/LacZ mice) to test the hypothesis of its up-regulation by gonadal hormones. Y1 receptor gene expression was detected by means of histochemical procedures and quantitative image analysis in the paraventricular nucleus, arcuate nucleus, medial preoptic nucleus, ventromedial nucleus and bed nucleus of stria terminalis of two-month-old female mice at different stages of estrous cycle. Qualitative and quantitative analyses showed that Y1R/LacZ transgene expression was higher in the paraventricular, arcuate, and ventromedial nuclei of proestrus mice as compared to mice in the other stages of the estrous cycle. In addition, we performed a comparison with a group of sexually active males. In this comparison a significant difference (less in males) was observed between males and proestrus females in the same nuclei. In conclusion, these data indicate that fluctuations in circulating levels of gonadal hormones, depending by estrous cycle, are paralleled by changes in the expression of NPY Y1 receptor in the hypothalamic nuclei involved in the control of both energy balance and reproduction.

► This review summarizes information on neuropeptide F (NPF) and short NPF (sNPF) in invertebrates. ► They are distinct families of peptides with characteristic prepropeptide organization. ► NPFs and NPF receptors are ancestrally related to vertebrate neuropeptide Y and its receptors. ► Bona fide sNPFs appear to exist only in arthropods. ► NPFs regulate feeding, metabolism and reproduction, whereas NPFs have more pleiotropic functions.Neuropeptides referred to as neuropeptide F (NPF) and short neuropeptide F (sNPF) have been identified in numerous invertebrate species. Sequence information has expanded tremendously due to recent genome sequencing and EST projects. Analysis of sequences of the peptides and prepropeptides strongly suggest that NPFs and sNPFs are not closely related. However, the NPFs are likely to be ancestrally related to the vertebrate family of neuropeptide Y (NPY) peptides. Peptide diversification may have been accomplished by different mechanisms in NPFs and sNPFs; in the former by gene duplications followed by diversification and in the sNPFs by internal duplications resulting in paracopies of peptides. We discuss the distribution and functions of NPFs and their receptors in several model invertebrates. Signaling with sNPF, however, has been investigated mainly in insects, especially in Drosophila. Both in invertebrates and in mammals NPF/NPY play roles in feeding, metabolism, reproduction and stress responses. Several other NPF functions have been studied in Drosophila that may be shared with mammals. In Drosophila sNPFs are widely distributed in numerous neurons of the CNS and some gut endocrines and their functions may be truly pleiotropic. Peptide distribution and experiments suggest roles of sNPF in feeding and growth, stress responses, modulation of locomotion and olfactory inputs, hormone release, as well as learning and memory. Available data indicate that NPF and sNPF signaling systems are distinct and not likely to play redundant roles.
Keywords: Neuropeptide prepropeptide; G-protein-coupled receptor; Feeding; Metabolism; Reproduction; Drosophila; Insect peptides;