Peptides (v.31, #4)

Anti-peptide antibodies differentiate between plasmodial lactate dehydrogenases by Ramona Hurdayal; Ikechukwu Achilonu; David Choveaux; Theresa H.T. Coetzer; J.P. Dean Goldring (525-532).
Malaria lactate dehydrogenase, a glycolytic enzyme, is a malaria diagnostic target in lateral flow immunochromatographic rapid diagnostic tests. Recombinant Plasmodium yoelii LDH was cloned into the pET-28a vector, expressed and the expressed protein purified from a Ni-NTA affinity matrix. A pan-malarial LDH antibody directed against a common malaria LDH peptide (APGKSDKEWNRDDLL) and two anti-peptide antibodies, each targeting a unique Plasmodium falciparum (LISDAELEAIFDC) and Plasmodium vivax (KITDEEVEGIFDC) LDH peptide were raised in chickens. The antibodies were affinity purified with the appropriate peptide affinity matrix. The affinity purified anti-peptide antibodies detected recombinant P. falciparum, P. vivax and P. yoelii LDH and native P. falciparum and P. yoelii LDH in western blots and immunofluorescence studies. The pan-malarial antibody detected LDH from the three malaria species in western blots. The species-specific anti-peptide antibodies differentiated between P. falciparum and P. vivax LDH. Affinity purified chicken antibodies against recombinant PfLDH, PvLDH and PyLDH proteins each detected the parent and orthologous proteins with similar titers in an ELISA. The study supports an anti-peptide antibody approach to the development of diagnostic reagents.
Keywords: Malaria; Plasmodium; Chicken; Peptides Antibodies; Lactate dehydrogenase;

Synthesis, biophysical, and biological studies of wild-type and mutant psalmopeotoxins—Anti-malarial cysteine knot peptides from Psalmopoeus cambridgei by Pacharin Kamolkijkarn; Thitawan Prasertdee; Chawita Netirojjanakul; Pakornwit Sarnpitak; Somsak Ruchirawat; Songpon Deechongkit (533-540).
Psalmopeotoxin I and II (PcFK1 and PcFK2), an anti-malarial peptide first extracted from Psalmopoeus cambridgei was synthesized and characterized. Both peptides belong to the Inhibitor Cystine Knot (ICK) superfamily, containing three disulfide bridges. The six cysteine residues are conserved similar to other members of the ICK superfamily, suggesting their critical role for either folding or function. In this study, the peptides were synthesized using Fmoc solid-phase peptide synthesis (SPPS). The three disulfide bonds of were constructed by regioselective and random oxidative approaches. The resulting disulfide bond patterns were verified by the HPLC-MS analysis of intact peptides and by the disulfide bond mapping using tryptic digestion. Implications of the disulfide bonds on the biophysical and biological properties of PcFKs were studied using three disulfide mutants in which a particular pair of cysteines was replaced with two isosteric serine residues. Structures and biophysical characteristics of all variants were studied using far-UV CD and fluorescence spectroscopy. Biological activities of all variants were evaluated using antiplasmodial assay against the K1 multi-drug-resistant strain of P. falciparum. The experimental results showed that the three disulfide bridges could not be correctly synthesized by the random oxidative strategy. Structural and biophysical analyses revealed that all variants had similar structures to the twisted β-sheet. However, the studies of disulfide bond removal indicated that each disulfide bond had different effects on both biophysical and biological activities of PcFKs. Correlation of biophysical parameters and biological activities showed that both PcFKs may have different mechanisms of actions for antiplasmodial activity.
Keywords: Anti-malarial peptides; Biological information; Spectroscopic information; Disulfide peptide synthesis; Structure–activity relationship;

Characterization of the novel antifungal protein PgAFP and the encoding gene of Penicillium chrysogenum by Andrea Rodríguez-Martín; Raquel Acosta; Susan Liddell; Félix Núñez; Mª José Benito; Miguel A. Asensio (541-547).
The strain RP42C from Penicillium chrysogenum produces a small protein PgAFP that inhibits the growth of some toxigenic molds. The molecular mass of the protein determined by electrospray ionization mass spectrometry (ESI-MS) was 6 494 Da. PgAFP showed a cationic character with an estimated pI value of 9.22. Upon chemical and enzymatic treatments of PgAFP, no evidence for N- or O-glycosylations was obtained. Five partial sequences of PgAFP were obtained by Edman degradation and by ESI-MS/MS after trypsin and chymotrypsin digestions. Using degenerate primers from these peptide sequences, a segment of 70 bp was amplified by PCR from pgafp gene. 5′- and 3′-ends of pgafp were obtained by RACE-PCR with gene-specific primers designed from the 70 bp segment. The complete pgafp sequence of 404 bp was obtained using primers designed from 5′- and 3′-ends. Comparison of genomic and cDNA sequences revealed a 279 bp coding region interrupted by two introns of 63 and 62 bp. The precursor of the antifungal protein consists of 92 amino acids and appears to be processed to the mature 58 amino acids PgAFP. The deduced amino acid sequence of the mature protein shares 79% identity to the antifungal protein Anafp from Aspergillus niger. PgAFP is a new protein that belongs to the group of small, cysteine-rich, and basic proteins with antifungal activity produced by ascomycetes. Given that P. chrysogenum is regarded as safe mold commonly found in foods, PgAFP may be useful to prevent growth of toxigenic molds in food and agricultural products.
Keywords: Antifungal protein; Penicillium chrysogenum; Glycosylation; Toxigenic molds; RACE-PCR; Preproprotein;

Antimicrobial peptides from the skin secretions of the South-East Asian frog Hylarana erythraea (Ranidae) by Nadia Al-Ghaferi; Jolanta Kolodziejek; Norbert Nowotny; Laurent Coquet; Thierry Jouenne; Jérôme Leprince; Hubert Vaudry; Jay. D. King; J. Michael Conlon (548-554).
Peptidomic analysis of norepinephrine-stimulated skin secretions of the South-East Asian frog Hylarana erythraea (formerly Rana erythraea partim) has led to the identification of multiple peptides with antimicrobial activity. Structural characterization of the peptides demonstrated that they belong to the brevinin-1 (3), brevinin-2 (2), esculentin-2 (4), and temporin (1) families. The values in parentheses indicate the number of paralogs. In addition, a peptide (GVIKSVLKGVAKTVALG ML.NH2) was isolated that shows some structural similarity to the brevinin-2-related peptides (B2RP) previously isolated from North American frogs of the genus Lithobates. A synthetic replicate of the species B2RP showed broad-spectrum growth inhibitory activity against reference strains of Escherichia coli (MIC = 12.5 μM), Staphylococcus aureus (MIC = 12.5 μM) and Candida albicans (MIC = 50 μM) and was active against multidrug-resistant clinical isolates of Acetinobacter baumannii (MIC in the range 6–12.5 μM). The hemolytic activity of the peptide was relatively low (LC50  = 280 μM). Phylogenetic analysis based upon the amino acid sequences of 47 brevinin-2 peptides from 17 Asian species belonging to the family Ranidae provides support for the placement of H. erythraea in the genus Hylarana.
Keywords: Frog skin; Antimicrobial peptide; Brevinin; Esculentin-2; Temporin; Hylarana;

Milk versus caseinophosphopeptides added to fruit beverage: Resistance and release from simulated gastrointestinal digestion by María José García-Nebot; Amparo Alegría; Reyes Barberá; María del Mar Contreras; Isidra Recio (555-561).
The influence of simulated gastrointestinal digestion on caseinophosphopeptides (CPPs) formation in milk-based fruit beverage was evaluated, together with resistance of a pool of CPPs added to fruit beverage. In milk-based fruit beverage, four CPPs were identified that can be justified by their presence in raw milk or due to processing. When it was subjected to simulated gastrointestinal digestion, 10 CPPs were identified, and only 1 presented the cluster (SpSpSpEE) (3 phosphoseryl group followed by 2 glutamic acid residues), which corresponded to αs2-CN(1–19)4P. CPPs added to fruit beverage are resistant to simulated gastrointestinal digestion, and 16 CPPs were identified originating from the fragmentation of added CPPs, and with a greater presence of the cluster compared with CPPs originating from milk-based fruit beverage. This could justify the use of CPPs as functional ingredients, and offer a good alternative to milk-based fruit beverage for improving mineral bioavailability.
Keywords: Caseinophosphopeptides; Simulated gastrointestinal digestion; Fruit beverage; High-performance liquid chromatography; Mass spectrometry;

Improvement of cathepsin S detection using a designed FRET peptide based on putative natural substrates by Marcela Oliveira; Ricardo J.S. Torquato; Marcio F.M. Alves; Maria A. Juliano; Dieter Brömme; Nilana M.T. Barros; Adriana K. Carmona (562-567).
Cathepsin S is a lysosomal cysteine peptidase of the papain superfamily which is implicated in physiological and pathological states. The enzyme is highly expressed in antigen presenting cells and is thought to play an important role in the processing of the major histocompatibility complex (MHC) class II-associated invariant chain. In pathological processes, cathepsin S is associated with Alzheimer's disease, atherosclerosis and obesity and can be regarded as a potential target in related disorders. However, due to the broad substrate specificities of the lysosomal cathepsins, the specific detection of cathepsin S is difficult when other cathepsins are present. In an attempt to distinguish cathepsin S from other cathepsins we synthesized and tested fluorescence resonance energy transfer (FRET) peptides derived from two of its putative natural substrates, namely insulin β-chain and class II-associated invariant chain (CLIP). The influence of ionic strength on the catalytic activity and the enzyme stability in neutral pH was also analyzed. Using data gathered from our study we developed a selective substrate for cathepsin S and establish the assay conditions to differentiate the enzyme from cathepsins L, B, V and K. The peptide Abz-LEQ-EDDnp (Abz =  ortho-aminobenzoic acid; EDDnp = N-[2,4-dinitrophenyl]ethylenediamine]) in 50 mM sodium phosphate buffer, pH 7.4, containing 1 M NaCl was hydrolyzed by cathepsin S with k cat/K m value of 3585 mM−1  s−1, and was resistant to hydrolysis by cathepsins L, V, K and B. Thus, we developed a sensitive and selective cathepsins S substrate that permits continuous measurement of the enzymatic activity even in crude tissue extracts.
Keywords: Lysosomal cathepsins; Cathepsin S; FRET peptides; Continuous assay; Selective substrate;

In vitro and in vivo antitumor effects of novel actinomycin D analogs with amino acid substituted in the cyclic depsipeptides by Bang-zhi Zhang; Kai-rong Wang; Jie-xi Yan; Wei Zhang; Jing-jing Song; Jing-man Ni; Rui Wang (568-573).
The actinomycin D (AMD) analogs in which the d-valine residues (the second amino acid residue in the cyclic depsipeptide of AMD) and the N-methyl-l-valine residues (the fifth amino acid residue in the cyclic depsipeptide of AMD) were replaced with d-Phe or l- and d-forms N-methylvalines, N-methylisoleucine, N-methylleucine, N-methylphenylalanine, N-methylalanine, and sarcosine were synthesized. The antimicrobial activity and cytotoxic activities of these compounds in vitro were investigated. The results showed that most d-valine substituted analogs had much lower antimicrobial activity and cytotoxic activities in vitro than AMD itself, but three N-methyl-l-valine substituted analogs had comparable or even more remarkable cytotoxic activities in vitro than AMD. Acute toxicities and antitumor effects of the N-methyl-l-valine substituted analogs in mice were also examined. The result showed that the acute toxicity of compound 4 l-methylleucine5-AMD analog is comparable to AMD itself and that of compound 3(l-Methylisoleucine5-AMD analog) is slightly more toxic, about 1.25-fold than AMD. However, the acute toxicity of compound 5 d-methylleucine5-AMD analog is about 2-fold lower than AMD. This suggested that the N-methyl-d-amino acid replacement in the cyclic ring might play a vital role in their decreased acute toxicities, and perhaps the N-methyl-d-leucine substituent is more favorable, though there may be a slight loss of antitumor activity. This finding may be helpful for the design and development of more potent antitumor agents together with low acute toxicity, and suggests that the N-methyl-d-leucine substituent has the potential to be used as antitumor drug lead.
Keywords: Actinomycin D; Analogs; Amino acid; Cytotoxic activity; Acute toxicity; Antitumor;

SecretP: A new method for predicting mammalian secreted proteins by Lezheng Yu; Yanzhi Guo; Zheng Zhang; Yizhou Li; Menglong Li; Gongbing Li; Wenjia Xiong; Yuhong Zeng (574-578).
Keywords: Auto covariance; Classically secreted proteins; Non-classically secreted proteins; Pseudo-amino acid composition; Support vector machine;

Functional characterization of two human receptor activity-modifying protein 3 variants by Richard J. Bailey; Joshua W.I. Bradley; David R. Poyner; Dan L. Rathbone; Debbie L. Hay (579-584).
Adrenomedullin (AM) and amylin are involved in angiogenesis/lymphangiogenesis and glucose homeostasis/food intake, respectively. They activate receptor activity-modifying protein (RAMP)/G protein-coupled receptor (GPCR) complexes. RAMP3 with the calcitonin receptor-like receptor (CLR) forms the AM2 receptor, whereas when paired with the calcitonin receptor AMY3 receptors are formed. RAMP3 interacts with other GPCRs although the consequences of these interactions are poorly understood. Therefore, variations in the RAMP3 sequence, such as single nucleotide polymorphisms or mutations could be relevant to human health. Variants of RAMP3 have been identified. In particular, analysis of AK222469 (Homo sapiens mRNA for receptor (calcitonin) activity-modifying protein 3 precursor variant) revealed several nucleotide differences, three of which encoded amino acid changes (Cys40Trp, Phe100Ser, Leu147Pro). Trp56Arg RAMP3 is a polymorphic variant of human RAMP3 at a conserved amino acid position. To determine their function we used wild-type (WT) human RAMP3 as a template for introducing amino acid mutations. Mutant or WT RAMP3 function was determined in Cos-7 cells with CLR or the calcitonin receptor (CT(a)). Cys40Trp/Phe100Ser/Leu147Pro RAMP3 was functionally compromised, with reduced AM and amylin potency at the respective AM2 and AMY3(a) receptor complexes. Cys40Trp and Phe100Ser mutations contributed to this phenotype, unlike Leu147Pro. Reduced cell-surface expression of mutant receptor complexes probably explains the functional data. In contrast, Trp56Arg RAMP3 was WT in phenotype. This study provides insight into the role of these residues in RAMP3. The existence of AK222469 in the human population has implications for the function of RAMP3/GPCR complexes, particularly AM and amylin receptors.
Keywords: Adrenomedullin; Amylin; Receptor activity-modifying protein; RAMP3;

Inhibition of CD26/DPP IV attenuates ischemia/reperfusion injury in orthotopic mouse lung transplants: The pivotal role of vasoactive intestinal peptide by Wolfgang Jungraithmayr; Ingrid De Meester; Veerle Matheeussen; Ilhan Inci; Koen Augustyns; Simon Scharpé; Walter Weder; Stephan Korom (585-591).
The T cell activation Ag CD26/dipeptidylpeptidase IV (DPP IV) combines co-stimulatory and enzymatic properties. Catalytically, it functions as an exopeptidase, modulating biological activity of key chemokines and peptides. Here we investigated the effect of organ-specific inhibition of DPP IV catalytic activity on ischemia/reperfusion injury after extended ischemia in the mouse model of orthotopic single lung transplantation. C57BL/6 mice were syngeneically, transplanted, grafts were perfused and stored in Perfadex with (treated) or without (control) a DPP IV enzymatic activity inhibitor (AB192). Transplantation was performed after 18 h cold ischemia time; following 2-h reperfusion, grafts were analyzed for oxygenation, thiobarbituric acid-reactive substances, histomorphology, and immunohistochemistry was performed for leukocyte Ag 6, myeloperoxidase, hemoxygenase 1, vasoactive intestinal protein (VIP), and real-time PCR for VIP. Treatment with the DPP IV inhibitor AB192 resulted in significant improvement of gas exchange, less lipid oxidation, preservation of parenchymal ultrastructure, reduced neutrophil infiltration, reduced myeloperoxidase expression, increased hemoxygenase 1 expression, pronounced expression of VIP in alveolar macrophages and increased mRNA expression of VIP. Inhibition of intragraft DPP IV catalytic activity with AB192 strikingly ameliorates ischemia/reperfusion injury after extended ischemia. Furthermore, preservation of endogenous intragraft VIP levels correlate with maintaining lung function and structural integrity.
Keywords: CD26/dipeptidylpeptidase IV; Ischemia-reperfusion injury; Mouse lung transplantation; Vasoactive intestinal peptide (VIP);

Pituitary adenylate cyclase-activating polypeptide ameliorates cisplatin-induced acute kidney injury by Min Li; Saravanan Balamuthusamy; Altaf M. Khan; Jerome L. Maderdrut; Eric E. Simon; Vecihi Batuman (592-602).
Cisplatin nephrotoxicity involves DNA damage, proinflammatory responses and apoptosis/necrosis of renal proximal tubular epithelial cells. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to protect kidneys from ischemic injury and light chain-induced damage by modulating inflammation. Confluent monolayer of HK-2 human renal cells were exposed to 50 μM cisplatin in the presence or absence of either PACAP38 or p53 siRNA. Mice injected with cisplatin were also treated with PACAP38 daily for 3 days. The damage to HK-2 cells caused by cisplatin involved the activation of p53, caspase-7, and poly (ADP-ribose) polymerase-1 (PARP-1). PACAP38 prevented the decrease in the apurinic/apyrimidinic endonuclease-1 by suppressing p53 activation and blocked the cleavage of caspase-7 and PARP-1 in cisplatin-exposed cells. PACAP also markedly inhibited cisplatin-induced apoptotic tubule cell death. Exposure to cisplatin significantly suppressed the expression of fibronectin and collagens I and IV, and altered the integrin repertoire of human renal tubule cells, while PACAP partially reversed the reduction of fibronectin, collagen IV, and the integrin subunits in cells exposed to cisplatin. Experiments with PACAP receptor antagonists and siRNA silencing of p53 showed that the renoprotection with PACAP was mediated by the PAC1 receptor and through both p53-dependent and -independent suppression of apoptosis. PACAP was renoprotective in vivo and prevented the rise in blood urea nitrogen and creatinine in mice treated with cisplatin. These results suggest that p53 plays a pivotal role in decreased integrin-mediated extracellular matrix component expression in cisplatin-induced tubule cell apoptosis, and reveal a novel aspect of PACAP-mediated renoprotection.
Keywords: Apoptosis; Cancer chemotherapy; Inflammation; p53; Renoprotection;

Vasoactive intestinal peptide (VIP) receptor expression in monocyte-derived macrophages from COPD patients by Bernhard Burian; Angela Storka; Beatrice A. Marzluf; Yong-Cheng Yen; Christopher Lambers; Bruno Robibaro; Karin Vonbank; Wilhelm Mosgoeller; Ventzislav Petkov (603-608).
Vasoactive intestinal peptide (VIP) is one of the most abundant molecules found in the respiratory tract. Due to its anti-inflammatory and bronchodilatatory properties, it has been proposed as a novel treatment for chronic obstructive pulmonary disease (COPD). The actions of VIP are mediated via three different G-protein-coupled receptors (VPAC1, VPAC2 and PAC1) which are expressed in the respiratory tract and on immunocompetent cells including macrophages. Alveolar macrophages (AM) are key players in the pathogenesis of COPD and contribute to the severity and progression of the disease. While VPAC1 has been reported to be elevated in subepithelial cells in smokers with chronic bronchitis, little is known about VPAC expression of AM in COPD patients. AM from COPD patients show a strong VPAC1 expression which exceeds VPAC2. A similar receptor expression pattern was also observed in lipopolysaccharide (LPS)-activated monocyte-derived macrophages (MDM) from healthy volunteers and COPD patients. VIP has been shown to down-regulate interleukin 8 (IL-8) secretion significantly in MDM after LPS stimulation. The response to VIP was similar in MDM from COPD patients and healthy volunteers. Our results indicate that VPAC1 up-regulation in macrophages is a common mechanism in response to acute and chronic pro-inflammatory stimuli. Although VPAC1 up-regulation is dominant, both receptor subtypes are necessary for optimal anti-inflammatory signaling. The high VPAC1 expression in AM may reflect the chronic pro-inflammatory environment found in the lung of COPD patients. Treatment with VIP may help to decrease the chronic inflammation in the lung of COPD patients.
Keywords: Anti-inflammatory therapy; Macrophages; Vasoactive intestinal peptide; Interleukin 8;

Short-term angiotensin-1 receptor antagonism in type 2 diabetic Goto–Kakizaki rats normalizes endothelin-1-induced mesenteric artery contraction by Takayuki Matsumoto; Keiko Ishida; Kumiko Taguchi; Tsuneo Kobayashi; Katsuo Kamata (609-617).
Endothelin (ET)-1 and angiotensin II (Ang II) are likely candidates for a key role in diabetic vascular complications. We demonstrated previously that an enhanced ET-1-induced contraction is present in mesenteric arteries from Goto–Kakizaki (GK) rats at the chronic stage of type 2 diabetes. Here, we investigated whether short-term treatment of such rats with losartan, an angiotensin type 1 receptor antagonist, might normalize the ET-1-induced contraction. In mesenteric arteries from GK rats at the chronic stage (34–38 weeks) (vs. those from age-matched control Wistar rats): (1) the ET-1-induced contraction was enhanced, (2) the levels of ET-1 and Ang II were increased, (3) ET-1-stimulated ERK2 phosphorylation was increased, and (4) the ACh-induced endothelium-dependent relaxation was reduced. Mesenteric arteries isolated from such GK rats following treatment with losartan (25 mg/kg/day for 2 weeks) exhibited reduced ET-1- and Ang II-induced contractions, suppressed ET-1-stimulated ERK phosphorylation, and increased ACh-induced relaxation, while the rats exhibited normalized plasma NO metabolism and their mesenteric arteries exhibited increased basal NO formation. However, such losartan treatment did not alter the increased levels of ET-1 and Ang II seen in GK mesenteric arteries. Our data suggest that within the timescale studied here, losartan normalizes ET-1-induced mesenteric artery contraction through a suppression of ERK activities and/or by normalizing endothelial function.
Keywords: Angiotensin II; Diabetes; Endothelin-1; ERK; GK rat; Losartan; Mesenteric artery;

Neurochemicals that stimulate food foraging and hoarding in Siberian hamsters are becoming more apparent, but we do not know if cessation of these behaviors is due to waning of excitatory stimuli and/or the advent of inhibitory factors. Cholecystokinin (CCK) may be such an inhibitory factor as it is the prototypic gastrointestinal satiety peptide and is physiologically important in decreasing food intake in several species including Siberian hamsters. Systemic injection of CCK-33 in laboratory rats decreases food intake, doing so to a greater extent than CCK-8. We found minimal effects of CCK-8 on food foraging and hoarding previously in Siberian hamsters, but have not tested CCK-33. Therefore, we asked: Does CCK-33 decrease normal levels or food deprivation-induced increases in food foraging, hoarding and intake? Hamsters were housed in a wheel running-based foraging system with simulated burrows to test the effects of peripheral injections of CCK-33 (13.2, 26.4, or 52.8 μg/kg body mass), with or without a preceding 56 h food deprivation. The highest dose of CCK-33 caused large baseline reductions in all three behaviors for the 1st hour post-injection compared with saline; in addition, the intermediate CCK-33 dose was sufficient to curtail food intake and foraging during the 1st hour. In food-deprived hamsters, we used a 52.8 μg/kg body mass dose of CCK-33 which decreased food intake, hoarding, and foraging almost completely compared with saline controls for 1 h. Therefore, CCK-33 appears to be a potent inhibitor of food intake, hoarding, and foraging in Siberian hamsters.
Keywords: Appetitive behavior; Consummatory behavior; Injection; Satiety;

Effect of somatostatin analog on high-fat diet-induced metabolic syndrome: Involvement of reactive oxygen species by Wu Li; Yong-Hui Shi; Rui-li Yang; Jue Cui; Ying Xiao; Bin Wang; Guo-Wei Le (625-629).
Oxidative stress plays an important role in overnutrition-induced metabolic syndrome. Somatostatin (SST) inhibits a wide variety of physiologic functions in the gastrointestinal tract, which may in turn control the levels of reactive oxygen species (ROS) derived from ingestion of macronutrients. In this study, the involvement of SST in the progression of metabolic syndrome in response to a high-fat diet (HFD) was investigated. Male C57BL/6 mice were fed either a normal diet (4.89% fat) or a high-fat diet (21.45% fat) for 4 weeks. The SST analog octreotide (20 μg/kg/day) was then administered intraperitoneally to half of the HFD mice throughout the 10-day experimental period. Body weight, adipose tissue weight, gastric acidity, total bile acid, and lipase activity were measured. Plasma lipid, glucose, insulin, SST, the levels of ROS and GSH/GSSG, and lipid peroxidation in the stomach, small intestine, pancreas, and liver were also evaluated. Following HFD intake for 38 days, a decrease in the plasma levels of SST and GSH/GSSG ratio was observed, while there was an increase in body weight, adipose tissue weight, plasma glucose, triglyceride, and levels of ROS and lipid peroxidation of the stomach, small intestine, pancreas, and liver. However, simultaneous administration of SST analog octreotide to HFD-fed mice significantly reduced ROS production of the digestive system and resulted in the improvement of all the aforesaid adverse changes, suggesting the involvement of SST in the progression of HFD-induced metabolic syndrome.
Keywords: Somatostatin; Metabolic syndrome; Reactive oxygen species; High-fat diet;

Hexarelin suppresses high lipid diet and vitamin D3-induced atherosclerosis in the rat by Jinjiang Pang; Qihua Xu; Xiangbin Xu; Hongchao Yin; Rongkun Xu; Shu Guo; Wei Hao; Luya Wang; Chen Chen; Ji-Min Cao (630-638).
Growth hormone-releasing peptides (GHRP) and ghrelin are synthetic and natural ligands of growth hormone secretagogue receptor (GHSR) respectively and are shown to exert protective actions on cardiac dysfunction. Because ghrelin has been reported to inhibit proinflammatory responses in human endothelium and GHSR has been identified in blood vessels, we hypothesized that GHRP could alleviate the development of atherosclerosis (As). Atherosclearosis was induced by a short period (4 days) of vitamin D3 and chronic (three months) intragastric feeding of high fat emulsion (containing 0.5% propylthiouracil) in adult SD rats. Some As rats received chronic hexarelin (a variant of GHRP) injection (SC BID, 30 days) and normal rats received placebo as control. Significant atherosclerosis developed in animals fed with the emulsion. Serum total cholesterol and LDL-c increased, and HDL-c and aortic nitric oxide (NO) decreased significantly in As group. Hexarelin suppressed the formation of atherosclerotic plaques and neointima, partially reversed serum HDL-c/LDL-c ratio and increased the levels of serum NO and aortic mRNAs of eNOS, GHSR and CD36 in As rats. Hexarelin also decreased [3H]-TdR incorporation in cultured vascular smooth muscle cell (VSMC) and calcium sedimentation in aortic wall. Furthermore, foam cell formation induced by ox-LDL was decreased by hexarelin. In conclusion, hexarelin suppresses high lipid diet and vitamin D3-induced atherosclerosis in rats, possibly through upregulating HDL-c/LDL-c ratio, vascular NO production and downregulating the VSMC proliferation, aortic calcium sedimentation and foam cell formation. These novel anti-atherosclerotic actions of hexarelin suggest that the peptide might have a clinical potential in treating atherosclerosis.
Keywords: Growth hormone secretagogues; Atherosclerosis; Foam cell; Nitric oxide; Calcification;

Intermittent MTII application evokes repeated anorexia and robust fat and weight loss by Yi Zhang; Renata Collazo; Yongxin Gao; Gang Li; Philip J. Scarpace (639-643).
Central melanocortins (MC) evoke potent but transient anorectic responses with tachyphylaxis developing within days. We hypothesized that intermittent therapy using the MC analog, melanotan II (MTII), would minimize the tachyphylaxis and enhance the long-term efficacy of MTII treatment. F344/BN rats were infused with MTII or vehicle into the lateral ventricle by mini pump for 14 days. Half the MTII-infused rats were then given vehicle (MTII-On/Off), while the remaining received fresh MTII (MTII-On) for 10 days. Finally, pumps in both groups were replaced with ones containing fresh MTII for an additional 6 days. The first MTII application induced a 30% food reduction that attenuated within 5 days. Reapplication of MTII in MTII-On/Off rats, after the off period, invoked a new and equally robust anorectic response while continuation of MTII supplement in the MTII-On group did not change food intake from the control level. Body weights decreased similarly in both MTII groups at termination (day 30). Hypothalamic MC3 receptor, AgRP, and POMC expressions were unchanged, but MC4 receptor expression was diminished by 25%, and adiposity reduced by 80% in both MTII groups. Acetyl-CoA carboxylase 1 phosphorylation was elevated in perirenal fat by over 10 fold with either MTII treatment. In conclusion, intermittent MTII treatment preserves anorectic responses but does not prevent tachyphylaxis, whereas constant MTII application blunts further food response after the initial tachyphylaxis. Either form of MTII administration results in significant weight and adiposity reductions, involving perhaps fatty acid oxidation within specific adipose tissues.
Keywords: Melanocortin activation; MTII; ACC1; Fatty acid synthesis; Tachyphylaxis;

Isolation of peptide ligands that interact specifically with human glioma cells by Ivy A.W. Ho; Kam M. Hui; Paula Y.P. Lam (644-650).
Poor prognosis of high grade gliomas coupled with the difficulty of widespread delivery of therapeutic agents prompted the search into new molecular targets. Our aim is to isolate glioma-specific peptide sequences that can be used for targeted delivery of therapeutic drugs and imaging tracer to accurately demarcate tumor volume as a response to therapy. Herein, we describe the isolation and characterization of a glioma-specific peptide sequence, GL1, that interact exclusively with human glioma cells lines and primary glioma cells derived from human biopsy in vitro. Further analysis showed that the receptors for GL1 were located on the external side of the plasma membrane, where the GL1 peptides could bind stably up to a period of 180 min. More importantly, GL1 phages home specifically to human glioma xenograft when administered through tail vein, a phenomenon that was not observed when non-specific phages were used as control. Taken together, our results confirmed that GL1 could represent a novel peptide that target to tumor of glial origins, and could potentially be used as a targeting moiety for the conjugation of therapeutic drugs or diagnostic imaging radiolabels.
Keywords: Glioma; Phage display; Vector targeting; Glioma-specific;

Protein synthesis dependent effects of kinins on astrocyte prostaglandin synthesis by Talia Filipovich-Rimon; Sigal Fleisher-Berkovich (651-656).
It has been shown that kinins and their receptors are over expressed in the brain under pathophysiological conditions such as inflammation. However, little is known about the possible role of kinins, and especially bradykinin in brain inflammation. Although kinins are thought to have immediate effects, peptides may also exert longer and protein synthesis dependent actions. To evaluate this possibility, we assessed the regulation of prostaglandin E2 synthesis after 15 h bradykinin or Lys-des-Arg9-bradykinin (B1 receptor agonist) treatment in rat neonatal astrocytes. Bradykinin, dose dependently stimulated basal and lipopolysaccharide-induced prostaglandin E2 production, whereas exposure of astrocytes to the B1 receptor agonist decreased both basal and lipopolysaccharide-induced prostaglandin E2 release in a dose-dependent manner. These kinin effects on PGE2 synthesis were completely abrogated by actinomycin-D and cycloheximide, suggesting de novo synthesis of proteins. Bradykinin also increased cyclooxygenase-2 protein levels about 2-fold, while the B1 receptor agonist decreased cyclooxygenase-2 protein expression. There was no change in cyclooxygenase-1 protein levels after treatment with either of the kinins. Our data suggest a delayed feedback regulatory mechanism of kinins on astrocyte inflammation, whereby astrocyte prostaglandin synthesis is initially enhanced by bradykinin (B2) and eventually blocked by kinin breakdown product, acting on B1 receptors. At least part of this presumed feedback loop could be mediated by de novo protein synthesis of cyclooxygenase-2.
Keywords: Bradykinin; B1 receptor agonist; Astrocytes; Prostaglandins; Lipopolysaccharide;

Comparison of pituitary–adrenal responsiveness between insulin tolerance test and growth hormone-releasing peptide-2 test: A pilot study by Toshiko Kano; Hitoshi Sugihara; Mariko Sudo; Mototsugu Nagao; Taro Harada; Akira Ishizaki; Yasushi Nakajima; Kyouko Tanimura; Fumitaka Okajima; Hideki Tamura; Shinya Ishii; Tamotsu Shibasaki; Shinichi Oikawa (657-661).
Insulin tolerance test (ITT) is the gold standard for assessing the hypothalamic–pituitary–adrenal (HPA) function. GH-releasing peptide (GHRP)-2, which has a strong GH-stimulating activity, is useful for diagnosing GH deficiency as well as ITT. Additionally, GHRP-2 is also known to activate HPA axis. There have been no comparative studies of pituitary–adrenal responsiveness between GHRP-2 test and ITT in patients with hypothalamic/pituitary disease. To assess whether GHRP-2 test could be an alternative to ITT for diagnosing HPA axis failure, both ITT and GHRP-2 test were performed in 15 patients suspected of hypopituitarism. A 100 μg dose of GHRP-2 was administered intravenously and plasma ACTH and serum cortisol concentrations were measured. In ITT, a peak cortisol value over 18 μg/dl is considered normal. Nine patients were diagnosed as HPA axis failure by ITT. Their median peak cortisol in GHRP-2 test was 11.4 μg/ml. In 6 patients diagnosed as normal HPA axis status by ITT, their median peak cortisol in response to GHRP-2 test was 21.4 μg/dl, significantly higher (p  = 0.0032) than seen in patients diagnosed as HPA axis failure. There was a strong correlation between the peak cortisol in GHRP-2 test and ITT (r  = 0.817; p  < 0.0001). When the cut-off value for the peak cortisol in GHRP-2 test was set to 13–14 μg/dl for diagnosing HPA axis failure, the specificity and sensitivity were 100% and 88.9%, respectively. Although further studies that include normal subjects are needed, these preliminary results suggest the possibility that GHRP-2 test may be an alternative to ITT for assessing HPA axis function.
Keywords: HPA axis; Insulin tolerance test; GHRP-2;

Early-life stress is a key predisposing factor to the development of functional gastrointestinal (GI) disorders. Thus, changes in stress-related molecular substrates which influence colonic function may be important in understanding the pathophysiology of such disorders. Activation of peripheral corticotropin-releasing factor (CRF) receptors is thought to be important in the maintenance of GI function homeostasis. Therefore, immunofluorescent and Western blotting techniques were utilized to investigate colonic expression of CRF receptors in the maternal separation (MS) model as compared to non-separated (NS) rats. Receptor expression was also assessed following exposure to two different acute stressors, the open field (OF) and colorectal distension (CRD). Immunofluorescent dual-labeling demonstrated increased activation of both CRFR1 (MS: 79.6 ± 4.4% vs. NS: 43.8 ± 6.8%, p  < 0.001) and CRFR2 (MS: 65.9 ± 3.2% vs. NS: 51.6 ± 5.8%, p  < 0.05) positive cells in MS rats. Protein expression of CRFR1 and CRFR2 in the proximal colon was similar under baseline conditions and not affected by exposure to an OF stressor in either cohort. In contrast, distal CRFR1 and CRFR2 levels were higher in MS rats but were significantly reduced post OF stress. Moreover, decreases in expression of CRFR1 in the proximal and distal colon of NS rats following exposure to CRD were blunted in MS rats. CRD also caused an increase in the functional isoform of CRFR2 in the distal colon of MS rats with no effect in NS colons. This study demonstrates that acute stressors alter colonic CRF receptor expression in a manner that is determined by the underlying stress sensitivity of the subject.
Keywords: Corticotropin-releasing factor receptors; Irritable bowel syndrome; Stress; Maternal separation;

NPY Y2 receptor agonist PYY(3-36) inhibits diarrhea by reducing intestinal fluid secretion and slowing colonic transit in mice by Ryuichi Moriya; Takashi Shirakura; Hiroyasu Hirose; Tetsuya Kanno; Jun Suzuki; Akio Kanatani (671-675).
Peptide YY (PYY)(3-36), a neuropeptide Y (NPY) Y2 receptor agonist, is a powerful inhibitor of intestinal secretion. Based on this anti-secretory effect, NPY Y2 receptor agonists may be useful as novel anti-diarrheal agents, but anti-diarrheal efficacy has yet to be determined. We therefore examined the anti-diarrheal efficacy of PYY(3-36) and a selective Y2 receptor agonist, N-acetyl-[Leu28, Leu31]-NPY(24-36), in experimental mouse models of diarrhea. Intraperitoneal administration of PYY(3-36) (0.01–1 mg/kg) and N-acetyl-[Leu28, Leu31]-NPY(24-36) (10 mg/kg) significantly inhibited diarrhea (increase in wet fecal weight and diarrhea score) induced by dimethyl-prostaglandin E2, 5-hydroxytryptamine, and castor oil. Anti-diarrheal activities of PYY(3-36) and N-acetyl-[Leu28, Leu31]-NPY(24-36) were comparable to the effects of loperamide (1 mg/kg), a widely used anti-diarrheal drug. To clarify the anti-diarrheal mechanisms of NPY Y2 receptor agonists, we investigated the effects of PYY(3-36) and N-acetyl-[Leu28, Leu31]-NPY(24-36) on intestinal fluid secretion and colonic transit. PYY(3-36) (1 mg/kg) and N-acetyl-[Leu28, Leu31]-NPY(24-36) (10 mg/kg) significantly reduced dimethyl-prostaglandin E2-induced intestinal fluid accumulation in conscious mice, suggesting that NPY Y2 receptor agonists inhibit diarrhea, at least in part, by reducing intestinal secretion. In addition, PYY(3-36) (0.01–1 mg/kg) and N-acetyl-[Leu28, Leu31]-NPY(24-36) (10 mg/kg) potently inhibited normal fecal output, suggesting that NPY Y2 receptor activation inhibits colonic motor function and NPY Y2 receptor agonists inhibit diarrhea partly by slowing colonic transit. These results indicate that NPY Y2 receptor agonists inhibit diarrhea in mice by not only reducing intestinal fluid secretion, but also slowing colonic transit, and illustrate the therapeutic potential of NPY Y2 receptor agonists as effective treatments for diarrhea.
Keywords: Peptide YY(3-36); NPY Y2; Diarrhea;

Expression of the spexin gene in the rat adrenal gland and evidences suggesting that spexin inhibits adrenocortical cell proliferation by Marcin Rucinski; Andrea Porzionato; Agnieszka Ziolkowska; Marta Szyszka; Veronica Macchi; Raffaele De Caro; Ludwik K. Malendowicz (676-682).
Spexin (SPX, also called NPQ) is a recently identified, highly conserved peptide which is processed and secreted. We analysed the SPX gene and its protein product in the rat adrenal gland to ascertain whether SPX is involved in the regulation of corticosteroid secretion of and growth of adrenocortical cells. In adult rat adrenal glands the highest levels of SPX mRNA were present in the glomerulosa (ZG) and fasciculate/reticularis (ZF/R) zones. High SPX gene expression levels were found in freshly isolated adult rat ZG and ZF/R cells. In cultured adrenocortical cells the levels of SPX mRNA were lower than in freshly isolated cells. SPX mRNA expression levels were found to be 2–3 times higher during days 90–540 of postnatal development than found during days 2–45. Prolonged ACTH administration lowered and dexamethasone increased adrenal SPX mRNA levels in vivo. Adrenal enucleation produced a significant linear increase in SPX mRNA levels, with the highest value occurring at day 8 after surgery, with control values taken on day 30 after enucleation. Immunohistochemistry revealed SPX-like immunoreactivity in the entire cortex of the adult male rat and in enucleation-induced regenerating cortex. A concentration of 10–6 M SPX peptide stimulated basal aldosterone secretion by freshly isolated ZG. In prolonged exposure of adrenocortical cell primary cultures to SPX (10–6 M) resulted in a small increase in corticosterone secretion and a notable decrease in BrdU incorporation. The results suggest the direct involvement of SPX in the regulation of adrenocortical cell proliferation; however, the mechanism of action remains unknown.
Keywords: Spexin; NPQ; Gene expression; Rat; Adrenal cortex; Proliferation; Steroid;

Pressor and tachycardic responses to intrathecal administration of neuropeptide FF in anesthetized rats by Quan Fang; Ning Li; Tian-nan Jiang; Qian Liu; Yu-lin Li; Rui Wang (683-688).
Neuropeptide FF (NPFF) belongs to a neuropeptide family including two precursors (pro-NPFFA and pro-NPFFB) and two receptors (NPFF1 and NPFF2). NPFF and NPFF receptor mRNAs have been reported to be highly expressed and localized in the rat and human spinal cord. In the present study, the i.t. action of NPFF system on blood pressure and heart rate were examined using NPFF and two related agonists, NPVF and dNPA, which exhibit highest selectivities for NPFF1 and NPFF2 receptors, respectively. In urethane-anesthetized rats, NPFF and related peptides (5–40 nmol, i.t.) produced significant pressor and tachycardic responses at the spinal cord level. These effects were dose-dependent and similar with respect to time-course for the three peptides. Furthermore, i.t. injection of RF9 (20 nmol), a selective NPFF antagonist, significantly antagonized the cardiovascular responses to 20 nmol NPFF and related peptides (i.t.). Moreover, pretreatment of the rats with α-adrenoceptor antagonist phentolamine (1 mg/kg, i.v.) significantly reduced the pressor effects of NPFF. Nevertheless, pretreatment with muscarinic receptor and adrenoceptor antagonists (i.v.) could block the tachycardic effects induced by NPFF. Collectively, our results suggested that i.t. administration of NPFF and related peptides increased MAP and HR which were possibly mediated by the activation of both NPFF1 and NPFF2 receptors in the rat spinal cord. In addition, our results showed that the muscarinic receptor and adrenoceptor participated in the tachycardic response to i.t. NPFF, while α-adrenoceptor played an important role in the regulation of pressor effect of NPFF.
Keywords: Neuropeptide FF (NPFF); MAP (mean arterial pressure); HR (heart rate); i.t. (intrathecal); Rat;

In our previous study, Endokinin A/B (EKA/B, the common C-terminal decapeptide in Endokinin A and Endokinin B) was found to induce analgesic effect at high dose and nociception at low dose, while Endokinin C/D (EKC/D, the common C-terminal duodecapeptide in Endokinin C and Endokinin D) has analgesic effect only. So in this study an attempt was undertaken to investigate the interaction of EKA/B and EKC/D with Endomorphin-1 (EM-1) on antinociceptive effect at supraspinal level. Results showed that the antinociceptive effect of EM-1 was enhanced by high dose of EKA/B and abolished by low dose of EKA/B, while EKC/D could only enhance the analgesic effect. Mechanism studies showed that EKA/B blocked the antinociception of EM-1 by activating neurokinin-1 receptor (NK1), whose specific antagonist, SR140333B could fully block EKA/B-induced attenuation on the analgesic response of EM-1. Surprisingly, EKC/D could also block the same EKA/B-induced attenuation. Taken together, the different effects of EKA/B and EKC/D on the antinociception of EM-1 may pave the way for a new strategy on investigating the interaction between tachykinins and opioids on pain modulation.
Keywords: Tachykinin; Endokinin; Endomorphin-1; Antinociception; SR140333B;

The ventrolateral periaqueductal gray (vlPAG) is a major site of opioid analgesic action and a key locus for the development of morphine tolerance. Previous experimental evidence supports the hypothesis that the brain synthesizes and secretes neuropeptides, which act as a part of the homeostatic system to attenuate the effects of morphine and endogenous opioid peptides. Among the known antiopioid peptides, nociceptin/orphanin FQ (N/OFQ) has been shown to inhibit various opioid effects, especially analgesia. The present study investigated the effect of NOP receptor blockade on the tolerance to morphine antinociception in the vlPAG. Systemic morphine (10 mg/kg s.c. twice per day) induced an antinociceptive effect that diminished significantly on the third day when tolerance developed, as quantified by the tail flick and the hot plate tests. Intra vlPAG (i.vlPAG) administration of the NOP receptor antagonist (±)–J 113397 restored the opioid's analgesic effect. When (±)–J 113397 was administered beginning the first day preceding each morphine administration, tolerance did not develop, but it appeared if the NOP antagonist had been suspended. These data suggest that the N/OFQ in the vlPAG may play a key role in opioid-induced antinociceptive tolerance.
Keywords: Nociceptin/orphanin FQ; Morphine; Tolerance; vlPAG; Analgesia;

Arginine vasopressin induces rat caudate nucleus releasing acetylcholine to participate in pain modulation by Da-Xin Wang; Jun Yang; Zhi-Xin Gu; Chao-You Song; Wen-Yan Liu; Jing Zhang; Xue-Ping Li; Hui Li; Gen Wang; Cai Song; Bao-Cheng Lin (701-705).
A lot of studies have pointed that acetylcholine (Ach), a classic neurotransmitter can regulate pain process in the caudate nucleus (CdN). Our previous report has proven that arginine vasopressin (AVP) effects on pain modulation in the CdN. The communication was designed to investigate the interaction between AVP and Ach in the rat CdN during the pain process. AVP concentration was determined by radioimmunoassay (RIA) and Ach concentration by high performance liquid chromatography (HPLC). The results showed that pain stimulation increased both AVP and Ach concentrations in the CdN perfusion liquid; AVP increased Ach concentration in the CdN perfusion liquid, while AVP receptor antagonist including d(CH2)5Tyr(Me)AVP (V1 receptor antagonist) and d(CH2)5[D-Ile2, Ile4, Ala-NH2 9]AVP (V2 receptor antagonist) decreased Ach concentration in the CdN perfusion liquid. The data indicated that the analgesic effect of AVP might be involved in the Ach system in the CdN.
Keywords: Arginine vasopressin; Acetylcholine; Caudate nucleus; Pain modulation;

Central but not systemic inhibition of inducible nitric oxide synthase modulates oxytocin release during endotoxemic shock by Angelita Maria Stabile; Viviana Moreto; José Antunes-Rodrigues; Evelin Capellari Carnio (706-711).
Previous studies have shown that immunological challenges as lipopolysaccharide (LPS) administration increases plasma oxytocin (OT) concentration. Nitric oxide (NO), a free radical gas directly related to the immune system has been implicated in the central modulation of neuroendocrine adaptive responses to immunological stress. This study aimed to test the hypothesis that the NO pathway participates in the control of OT release induced by LPS injection. For this purpose, adult male Wistar rats received bolus intravenous (i.v.) injection of LPS, preceded or not by i.v. or intracerebroventricular (i.c.v.) injections of aminoguanidine (AG), a selective inducible nitric oxide synthase (iNOS) inhibitor. Rats were decapitated after 2, 4 and 6 h of treatment, for measurement of OT by radioimmunoassay. In a separate set of experiments, mean arterial pressure (MAP) and heart rate (HR) were measured every 15 min over 6 h, using a polygraph. These studies revealed that LPS reduced MAP and increased HR at 4 and 6 h post-injection. LPS significantly increased plasma OT concentration at 2 and 4 h post-injection. Pre-treatment with i.c.v. AG further increased plasma OT concentration and attenuated the LPS-induced decrease in MAP, however, i.v. AG failed to show similar effects. Thus, iNOS pathway may activate a central inhibitory control mechanism that attenuates OT secretion during endotoxemic shock.
Keywords: Blood pressure; Lipopolysaccharide; Nitric oxide; Oxytocin; Septic shock;

Electrophysiological effects of neuropeptide S on rat ventromedial hypothalamic neurons in vitro by Keitaro Yoshida; Juhyon Kim; Kazuki Nakajima; Yutaka Oomura; Matthew J. Wayner; Kazuo Sasaki (712-719).
The newly identified neuropeptide S (NPS) is a ligand for a previously orphan G protein-coupled GPR 154 receptor, now named the NPS receptor (NPSR). Previous studies have shown that NPS induces hyperlocomotion, increases arousal and suppresses anxiety-like behaviors via NPSR. Although NPS also inhibits food intake, nothing is known about the neuronal mechanisms underlying this action. Anatomical studies show that NPSRs are expressed abundantly in the dorsomedial part of the ventromedial hypothalamic nucleus (VMH), a satiety center for food intake. Hence, we examined the electrophysiological effects of NPS on rat VMH neurons in vitro. NPS predominantly depolarized the VMH neurons, and the effects were postsynaptic and dose-dependent. Membrane resistance was significantly decreased during the depolarization, suggesting an opening of some ionic channels. The NPS-induced depolarization was significantly attenuated in Ca2+-free, NiCl2-containing and mibefradil-containing TTX ACSFs, but it did not disappear. The NPS-induced depolarization was also attenuated in low-Na+ TTX ACSF, and completely abolished in Ca2+-free/low-Na+ TTX ACSF. Pretreatment with 30 μM KB-R7943, an inhibitor of forward-mode Na+/Ca2+ exchanger, did not have any significant effect on the NPS-induced depolarization in Ca2+-free TTX ACSF. These results suggest that NPS depolarizes VMH neurons via activations of R- and T-type Ca2+ channels and nonselective cation channels, and that VMH neurons might be involved in the cellular process through which NPS participates in the regulation of food intake and energy homeostasis.
Keywords: NPS; Ventromedial hypothalamus; Feeding suppression; Ca2+ channel; Nonselective cation channel; Depolarization;

Plasma nociceptin/orphanin FQ levels in response to the hyperventilation test in healthy subjects by Fiorella Fontana; Carmine Pizzi; Pasquale Bernardi; Emilio Merlo Pich; Andrea Bedini; Santi Spampinato (720-722).
In vitro and in vivo studies demonstrated that nociceptin/orphanin FQ inhibits norepinephrine release, while the effects of norepinephrine on nociceptin/orphanin FQ release remain unknown. Previous studies in healthy and hypertensive subjects showed that prolonged and forced hyperventilation induces different blood pressure (BP) responses depending on changes in plasma catecholamine levels. We investigated whether the effects of hyperventilation on the sympatho-adrenergic system involve nociceptin/orphanin FQ release. Fifty-six healthy subjects (26 females, mean age 63 ± 2 and 30 males, mean age 63 ± 3) underwent the hyperventilation test. A hierarchical cluster analysis based on BP response to hyperventilation identified three groups of subjects: group 1 (n  = 20) with a decrease in BP, norepinephrine (1311.1 ± 45.5 fmol/ml versus 900.0 ± 55.3 fmol/ml, P  < 0.01) and nociceptin/orphanin FQ (13.0 ± 0.7 pg/ml versus 7.9 ± 0.8 pg/ml, P  < 0.01), group 2 (n  = 18) without any change in BP and norepinephrine (1133.0 ± 31.5 fmol/ml versus 1176.0 ± 44.6 fmol/ml), with a decrease in nociceptin/orphanin FQ (12.5 ± 3.2 pg/ml versus 7.4 ± 0.6 pg/ml, P  < 0.01) and group 3 (n  = 18) with an increase in BP, norepinephrine (1216.7 ± 50.9 fmol/ml versus 1666.7 ± 44.9 fmol/ml, P  < 0.01) and nociceptin/orphanin FQ values (11.5 ± 1.6 pg/ml versus 19.9 ± 1.5 pg/ml, P  < 0.01). Norepinephrine changes in response to hyperventilation in groups 1 and 3 were directly (P  < 0.01) correlated with those of nociceptin/orphanin FQ. Our results showed that vigorous and prolonged hyperventilation changes plasma nociceptin/orphanin FQ levels due to the direct effects of hypocapnic alkalosis or to different sympatho-adrenergic system responses.
Keywords: Nociceptin/orphanin FQ; Hyperventilation; Norepinephrine;

Biological activities and molecular interactions of the C-terminal residue of thrombospondin-4, an epitome of acidic amphipathic peptides by Luis F. Congote; Gulzhakhan Sadvakassova; Monica C. Dobocan; Marcos R. DiFalco; Leonid Kriazhev (723-735).
C21, the C-terminal residue of thrombospondin-4 (TSP-4), was identified as a peptide growth factor during an investigation concerning erythropoietin-dependent, erythroid stimulating factors of endothelial origin. It is active in cultures of several human hematopoietic stem cells, skin fibroblasts and kidney epithelial cells and stimulates red cell formation in anemic mice. A method of affinity chromatography in the presence of high concentrations of Triton X-100, previously developed for identifying proteins associated with the TSP-1 receptor CD47, was utilized for the detection of C21 binding molecules and their detergent-resistant, associated partners. These experiments helped to delineate two different mechanisms of C21 action, which are compatible with its cell proliferating activity. As a cell matrix peptide, C21 binds to the osteopontin receptor CD44 and could act as an osteopontin antagonist, preventing the inhibition of primitive hematopoietic stem cell proliferation. TSP-1, another matrix protein, binds to C21 and could indirectly act as an antagonist, by shunting C21–CD44 interactions. The second mechanism is a direct effect of C21 on cell proliferation. The extremely rapid internalization and nuclear localization of the peptide could be explained by CD44-mediated internalization, followed by a microtubule-mediated transport towards the nucleus, or, eventually, direct membrane insertion. These alternative hypotheses are supported by previously observed membrane insertion of similar synthetic and viral acidic amphipathic peptides, the presence of microtubule-associated protein 1B (MAP1B) and dynactin in the triton-soluble complexes associated with C21 and the presence in such complexes of dual compartment proteins for nuclei and plasma membranes, such as MAP1B, AHNAK and CD44.
Keywords: Cell penetrating peptide; DNA replication; Hepatitis C virus; Integrins; NS5A; Papillomavirus; Posttranscriptional regulation; RNA recognition motifs; ROD1; STAT3; Transcriptional activator E2;

Behavioral effects of neuropeptides in rodent models of depression and anxiety by Susan Rotzinger; David A. Lovejoy; Laura A. Tan (736-756).
In recent years, studies have advocated neuropeptide systems as modulators for the behavioral states found in mood disorders such as depression and anxiety disorders. Neuropeptides have been tested in traditional animal models and screening procedures that have been validated by known antidepressants and anxiolytics. However, it has become clear that although these tests are very useful, neuropeptides have distinct behavioral effects and dose-dependent characteristics, and therefore, use of these tests with neuropeptides must be done with an understanding of their unique characteristics. This review will focus on the behavioral actions of neuropeptides and their synthetic analogs, particularly in studies utilizing various preclinical tests of depression and anxiety. Specifically, the following neuropeptide systems will be reviewed: corticotropin-releasing factor (CRF), urocortin (Ucn), teneurin C-terminal associated peptide (TCAP), neuropeptide Y (NPY), arginine vasopressin (AVP), oxytocin, the Tyr-MIF-1 family, cholecystokinin (CCK), galanin, and substance P. These neuropeptide systems each have a unique role in the regulation of stress-like behavior, and therefore provide intriguing therapeutic targets for mood disorder treatment.
Keywords: Depression; Anxiety; Stress; Animal models; Corticotropin-releasing factor; Urocortin; Teneurin; Neuropeptide Y; Vasopressin; Oxytocin; Tyr-MIF-1; Cholecystokinin; Galanin; Substance P; Receptor antagonists;

The blood-brain barrier (BBB) is a single uninterrupted barrier that in the brain capillaries is located at the endothelial cells and in the circumventricular organs, such as the choroid plexuses (CP) and median eminence (ME), is displaced to specialized ependymal cells. How do hypothalamic hormones reach the portal circulation without making the BBB leaky? The ME milieu is open to the portal vessels, while it is closed to the cerebrospinal fluid (CSF) and to the arcuate nucleus. The cell body and most of the axons of neurons projecting to the ME are localized in areas protected by the BBB, while the axon terminals are localized in the BBB-free area of the ME. This design implies a complex organization of the intercellular space of the median basal hypothalamus. The privacy of the ME milieu implies that those neurons projecting to this area would not be under the influence of compounds leaking from the portal capillaries, unless receptors for such compounds are located at the axon terminal. Amazingly, the arcuate nucleus also has its private milieu that is closed to all adjacent neural structures and open to the infundibular recess. The absence of multiciliated cells in this recess should result in a slow CSF flow at this level. This whole arrangement should facilitate the arrival of CSF signal to the arcuate nucleus. This review will show how peripheral hormones can reach hypothalamic targets without making the BBB leaky.
Keywords: Blood-brain barrier; Tightness; Hypothalamus; Arcuate nucleus; Cerebrospinal fluid; Choroid plexuses; Peripheral hormones;