Peptides (v.54, #C)

Dynamic changes in dopamine neuron function after DNSP-11 treatment: Effects in vivo and increased ERK 1/2 phosphorylation in vitro by Joshua L. Fuqua; Ofelia M. Littrell; Martin Lundblad; Jadwiga Turchan-Cholewo; Lina G. Abdelmoti; Emilia Galperin; Luke H. Bradley; Wayne A. Cass; Don M. Gash; Greg A. Gerhardt (1-8).
Glial cell-line derived neurotrophic factor (GDNF) has demonstrated robust effects on dopamine (DA) neuron function and survival. A post-translational processing model of the human GDNF proprotein theorizes the formation of smaller, amidated peptide(s) from the proregion that exhibit neurobiological function, including an 11-amino-acid peptide named dopamine neuron stimulating peptide-11 (DNSP-11). A single treatment of DNSP-11 was delivered to the substantia nigra in the rat to investigate effects on DA-neuron function. Four weeks after treatment, potassium (K+) and d-amphetamine evoked DA release were studied in the striatum using microdialysis. There were no significant changes in DA-release after DNSP-11 treatment determined by microdialysis. Dopamine release was further examined in discrete regions of the striatum using high-speed chronoamperometry at 1-, 2-, and 4-weeks after DNSP-11 treatment. Two weeks after DNSP-11 treatment, potassium-evoked DA release was increased in specific subregions of the striatum. However, spontaneous locomotor activity was unchanged by DNSP-11 treatment. In addition, we show that a single treatment of DNSP-11 in the MN9D dopaminergic neuronal cell line results in phosphorylation of ERK1/2, which suggests a novel cellular mechanism responsible for increases in DA function.
Keywords: Dopamine; Glial cell line-derived neurotrophic factor; Parkinson's disease; Striatum; Peptide;

Spider venom contains a very valuable repertoire of natural resources to discover novel components for molecular diversity analyses and therapeutic applications. In this study, HWTX-XI toxins from the spider venom glands of Ornithoctonus huwena which are Kunitz-type toxins (KTTs) and were directly cloned, analyzed and functionally characterized. To date, the HWTX-XI superfamily consists of 38 members deduced from 121 high-quality expressed sequence tags, which is the largest spider KTT superfamily with significant molecular diversity mainly resulted from cDNA tandem repeats as well as focal hypermutation. Among them, HW11c40 and HW11c50 may be intermediate variants between native Kunitz toxins and sub-Kunitz toxins based on evolutionary analyses. In order to elucidate their biological activities, recombinant HW11c4, HW11c24, HW11c27 and HW11c39 were successfully expressed, further purified and functionally characterized. Both HW11c4 and HW11c27 display inhibitory activities against trypsin, chymotrypsin and kallikrein. Moreover, HW11c4 is also an inhibitor relatively specific for Kv1.1 channels. HW11c24 and HW11c39 are found to be inactive on chymotrysin, trypsin, kallikrein, thrombin and ion channels. These findings provide molecular evidence for toxin diversification of the HWTX-XI superfamily and useful molecular templates of serine protease inhibitors and ion channel blockers for the development of potentially clinical applications.
Keywords: Spider toxin; Ornithoctonus huwena; Kunitz-type toxin; Evolution; Trypsin inhibitor; K+ channel blocker;

A glucagon-like peptide-1 analog liraglutide suppresses macrophage foam cell formation and atherosclerosis by Yuko Tashiro; Kengo Sato; Takuya Watanabe; Kyoko Nohtomi; Michishige Terasaki; Masaharu Nagashima; Tsutomu Hirano (19-26).
Macrophage foam cell formation, characterized by cholesterol ester accumulation catalyzed by acyl-CoA:cholesterol acyltransferase 1 (ACAT1), is the hallmark of early atherogenesis. We previously demonstrated the suppressive effects of incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), on the development of atherosclerotic lesions in apolipoprotein E-deficient (apoE−/−) mice. The present study was performed to evaluate the suppressive effects of these incretins and GLP-1 analogs, such as exendin-4 and liraglutide, on human macrophage foam cell formation in vitro and those of liraglutide on atherosclerotic lesion development in apoE−/− mice. We investigated the suppressive effects of GLP-1, GIP, exendin-4, and liraglutide against oxidized low-density lipoprotein (oxLDL)-induced foam cell formation in primary cultured human monocyte-derived macrophages. Seventeen-week-old apoE−/− mice were administered a long-acting GLP-1 analog liraglutide by osmotic mini-pumps for 4 weeks. Aortic atherosclerosis, oxLDL-induced foam cell formation, and related gene expression in exudate peritoneal macrophages were determined in vivo and ex vivo. Receptors for GLP-1 and GIP were expressed at high levels in human aortic smooth muscle cells and monocytes, but at relatively low levels in human macrophages and foam cells. GLP-1, GIP, exendin-4, and liraglutide significantly suppressed oxLDL-induced foam cell formation mainly associated with ACAT1 down-regulation in human monocyte-derived macrophages. The infusion of liraglutide into apoE−/− mice significantly retarded atherosclerotic lesions with monocyte/macrophage infiltration in the aortic wall and suppressed foam cell formation and ACAT1 expression in macrophages. These findings indicate that liraglutide could prevent the development of atherosclerotic lesions by suppressing macrophage foam cell formation mainly associated with ACAT1 down-regulation.
Keywords: Glucagon-like peptide-1; Glucose-dependent insulinotropic polypeptide; Exendin-4; Liraglutide; Macrophage; Atherosclerosis;

Blood levels of adrenomedullin on admission predict outcomes after acute intracerebral hemorrhage by Chuan-Liu Wang; Hai-Yan Lin; Jian-Wei Xu; Fei-Fei Jiang; Ming Yang; Jin-Hua Wang; Xiu-Qing Huang (27-32).
Increased plasma adrenomedullin level has been associated with critical illness. This study aimed to investigate the correlations of plasma adrenomedullin concentration with 3-month clinical outcomes and early neurological deterioration of patients with acute intracerebral hemorrhage. One hundred fourteen patients and 112 healthy controls were recruited. Relationships of plasma adrenomedullin concentrations with early neurological deterioration, 3-month mortality and unfavorable outcome (modified Rankin Scale score >2) were evaluated. Plasma adrenomedullin concentrations were increased in patients than in healthy individuals and were highly associated with National Institutes of Health Stroke Scale scores. A multivariate analysis selected plasma adrenomedullin concentration as an independent predictor for 3-month clinical outcomes and early neurological deterioration. A receiver operating characteristic curve analysis showed plasma adrenomedullin concentration predicted 3-month clinical outcomes and early neurological deterioration with high area under curves. The predictive value of adrenomedullin was similar to that of National Institutes of Health Stroke Scale score. In a combined logistic-regression model, adrenomedullin did not improve the predictive value of National Institutes of Health Stroke Scale score. Thus, elevated plasma adrenomedullin concentration is highly associated with 3-month clinical outcomes and early neurological deterioration of patients with acute intracerebral hemorrhage.
Keywords: Early neurological deterioration; Adrenomedullin; Intracerebral hemorrhage; Outcome; Stroke;

Differential expression and localization of neuropeptide Y peptide in pancreatic islet of diabetic and high fat fed rats by Zheng Ruipan; Meng Xiangzhi; Liu Li; Zhang Ying; Qiao Mingliang; Jing Peng; Liu Jingwei; Zhao Zijun; Gao Yan (33-38).
Neuropeptide Y (NPY) inhibits insulin secretion. Increased numbers of pancreatic islet cells expressing NPY have been observed in type 1 diabetic rats. To understand the functional significance of NPY expression in islet cells, we investigated the effects of high fat feeding and diabetic conditions on the expression and location of NPY expressing cells in normal and diabetic rats. Twenty rats were maintained on either normal chow (ND) or a high fat dietary regimen (HFD) for 4 weeks. In half of each group, type 1 or type 2 diabetes (groups T1DM and T2DM, respectively) was induced by injection of streptozotocin. At 8 weeks rats were euthanized and the pancreases were processed for immunofluorescence labeling (NPY/insulin, NPY/glucagon, NPY/somatostatin, and NPY/pancreatic polypeptide). Compared with the ND group, HFD rats had significantly fewer alpha cells, but beta cells were similar, while T1DM and T2DM rats showed significant increases in the proportions of alpha, delta, and PP cells. Robust increases in NPY-positive islet cells were found in the HFD, T1DM, and T2DM rats compared with ND controls. In ND rats, 99.7% of the NPY-positive cells were PP cells. However, high fat feeding and diabetes resulted in significant increases in NPY-positive delta cells, with concomitant decreases in NPY-positive PP cells. In summary, high-fat feeding and diabetes resulted in changes in the hormonal composition of pancreatic islet and increased number of NPY-expressing islet cells. Under diabetic conditions NPY expression switched from predominantly a characteristic of PP cells to predominantly that of delta cells. This may be a factor in reduced pancreatic hormone secretion during diabetes.
Keywords: Neuropeptide Y; Islet; STZ; High-fat diet; Diabetes mellitus;

Display OmittedInhibition of the enzyme dipeptidyl peptidase (DPP)-IV is one of the strategies used for the treatment of type 2 diabetes. In the present study, pepsin-treated whey protein isolate (WPI) and α-lactalbumin displaying DPP-IV inhibitory activity were fractionated by successive chromatographic steps and the resulting active fractions analyzed for their constituent peptides by liquid chromatography–electrospray ionization-tandem mass spectrometry. Among the identified sequences, 24 peptides derived from α-lactalbumin and 11 from β-lactoglobulin were synthesized and their effects on DPP-IV activity assessed. The most potent fragments, LKPTPEGDL and LKPTPEGDLEIL (IC50  = 45 and 57 μM, respectively), were found to inhibit DPP-IV in an un-competitive manner. Although several of the peptides tested showed some inhibitory activity, only two were as effective as the un-fractionated WPI hydrolysate and none were as potent as the un-fractionated α-lactalbumin hydrolysate. The peptides’ structural features, including length and amino acid composition, were found to impact their inhibitory activity. This study provides new insights on the active components responsible for the DPP-IV inhibitory activity of pepsin-treated whey proteins.
Keywords: α-Lactalbumin; β-Lactoglobulin; Dipeptidyl peptidase-IV inhibitor; Mode of inhibition; Type 2 diabetes;

Plasma somatostatin-like immunoreactivity increases in the plasma of septic patients and rats with systemic inflammatory reaction: Experimental evidence for its sensory origin and protective role by Balazs Suto; Istvan Szitter; Terez Bagoly; Erika Pinter; Janos Szolcsányi; Csaba Loibl; Timea Nemeth; Krisztian Tanczos; Tihamer Molnar; Tamas Leiner; Bianka Varnai; Zsofia Bardonicsek; Zsuzsanna Helyes (49-57).
Alterations of somatostatin-like immunoreactivity (SST-LI) in the plasma of 11 systemic inflammatory response syndrome (SIRS) patients were investigated in correlation with cytokines, adhesion molecules and coagulation markers repeatedly during 4 days. The origin and role of SST were studied in the cecum ligation and puncture (CLP) rat SIRS model. Capsaicin-sensitive peptidergic sensory nerves were defunctionalized by resiniferatoxin (RTX) pretreatment 2 weeks earlier, in a separate group animals were treated with the somatostatin receptor antagonist cyclo-somatostatin (C-SOM). Plasma SST-LI significantly elevated in septic patients compared to healthy volunteers during the whole 4-day period. Significantly decreased Horowitz score showed severe lung injury, increased plasma C-reactive protein and procalcitonin confirmed SIRS. Soluble P-selectin, tissue plasminogen activator and the interleukin 8 and monocyte chemotactic protein-1 significantly increased, interleukin 6 and soluble CD40 ligand did not change, and soluble Vascular Adhesion Molecule-1 decreased. SST-LI significantly increased in rats both in the plasma and the lung 6 h after CLP compared to sham-operation. After RTX pretreatment SST-LI was not altered in intact animals, but the SIRS-induced elevation was absent. Lung MPO activity significantly increased 6 h following CLP compared to sham operation, which was significantly higher both after RTX-desensitization and C-SOM-treatment. Most non-pretreated operated rats survived the 6 h, but 60% of the RTX-pretreated ones died showing a significantly worse survival. This is the first comprehensive study in humans and animal experiments providing evidence that SST is released from the activated peptidergic sensory nerves. It gets into the bloodstream and mediates a potent endogenous protective mechanism.
Keywords: Adhesion molecules; Capsaicin-sensitive sensory nerves; Coagulation factors; Cecum puncture model; Cytokines; Radioimmunoassay; Somatostatin; Cyclo-somatostatin; Systemic inflammatory response syndrome;

Urocortin 3 expression at baseline and during inflammation in the colon: Corticotropin releasing factor receptors cross-talk by Shilpi Mahajan; Min Liao; Paris Barkan; Kazuhiro Takahashi; Aditi Bhargava (58-66).
Urocortins (Ucn1-3), members of the corticotropin-releasing factor (CRF) family of neuropeptides, are emerging as potent immunomodulators. Localized, cellular expression of Ucn1 and Ucn2, but not Ucn3, has been demonstrated during inflammation. Here, we investigated the role of Ucn3 in a rat model of Crohn's colitis and the relative contribution of CRF receptors (CRF1 and CRF2) in regulating Ucn3 expression at baseline and during inflammation. Ucn3 mRNA and peptide were ubiquitously expressed throughout the GI tract in naïve rats. Ucn3 immunoreactivity was seen in epithelial cells and myenteric neurons. On day 1 of colitis, Ucn3 mRNA levels decreased by 80% and did not recover to baseline even by day 9. Next, we ascertained pro- or anti-inflammatory actions of Ucn3 during colitis. Surprisingly, unlike observed anti-inflammatory actions of Ucn1, exogenous Ucn3 did not alter histopathological outcomes during colitis and neither did it alter levels of pro-inflammatory cytokines IL-6 and TNF-α. At baseline, colon-specific knockdown of CRF1, but not CRF2 decreased Ucn3 mRNA by 78%, whereas during colitis, Ucn3 mRNA levels increased after CRF1 knockdown. In cultured cells, co-expression of CRF1  + CRF2 attenuated Ucn3-stimulated intracellular Ca2+ peak by 48% as compared to cells expressing CRF2 alone. Phosphorylation of p38 kinase increased by 250% during colitis and was significantly attenuated after Ucn3 administration. Thus, our results suggest that a balanced and coordinated expression of CRF receptors is required for proper regulation of Ucn3 at baseline and during inflammation.
Keywords: Crohn's colitis; Inflammation; RNAi; Ca2+ signaling; MAPK;

Involvement of adrenomedullin in the attenuation of acute morphine-induced analgesia in rats by Dongmei Wang; Yuanhui Huo; Rémi Quirion; Yanguo Hong (67-70).
Adrenomedullin (AM) is a member of calcitonin gene-related peptide (CGRP) family and a pain-related peptide. We have shown that chronic administration of morphine (20 μg) upregulates AM activity contributing to morphine tolerance. The present study investigated if AM is involved in acute morphine-induced analgesia. Single intrathecal (i.t.) injection of morphine at a dose of 5 μg increased the tail-flick latency (TFL). This analgesic effect was potentiated by the co-administration of the AM receptor antagonist AM22–52 (5 and 10 nmol). Exposure of sensory ganglion culture to morphine increased AM content in the ganglia in concentration (0.33–10 μM)- and time (10–240 min)-dependent manners. However, treatment with morphine (3.3 μM) for 30–240 min did not alter AM mRNA levels in the cultured ganglia. Furthermore, exposure of ganglion cultures to morphine (3.3 μM) for 30–240, but not 10 min induced an increase in AM content in the culture medium. These results reveal that a single morphine treatment potentiates post-translational change and the release of AM in sensory ganglia masking morphine-induced analgesia. Thus, targeting AM and its receptors should be considered as a novel approach to improve the analgesic potency of opiates during their acute use.

CCK-58 elicits both satiety and satiation in rats while CCK-8 elicits only satiation by Joost Overduin; James Gibbs; David E. Cummings; Joseph R. Reeve (71-80).
Reduction of food intake by exogenous cholecystokinin (CCK) has been demonstrated primarily for its short molecular form, CCK-8. Mounting evidence, however, implicates CCK-58 as a major physiologically active CCK form, with different neural and exocrine response profiles than CCK-8. In three studies, we compared meal-pattern effects of intraperitoneal injections CCK-8 vs. CCK-58 in undeprived male Sprague-Dawley rats consuming sweetened condensed milk. In study 1, rats (N  = 10) received CCK-8, CCK-58 (0.45, 0.9, 1.8 and 3.6 nmol/kg) or vehicle before a 4-h test-food presentation. At most doses, both CCK-8 and CCK-58 similarly reduced meal size relative to vehicle. Meal-size reduction prompted a compensatory shortening of the intermeal interval (IMI) after CCK-8, but not after CCK-58, which uniquely increased the satiety ratio (IMI/size of the preceding meal). In the second study, lick patterns were monitored after administration of 0.9 nmol/kg CCK-58, CCK-8 or vehicle. Lick cluster size, lick efficiency and interlick-interval distribution remained unaltered compared to vehicle, implying natural satiation, rather than illness, following both CCK forms. In study 3, threshold satiating doses of the two CCK forms were given at 5 and 30 min after meal termination, respectively. CCK 58, but not CCK-8 increased the intermeal interval and satiety ratio compared to vehicle. In conclusion, while CCK 58 and CCK-8 both stimulate satiation, thereby reducing meal size, CCK-58 consistently exerts a satiety effect, prolonging IMI. Given the physiological prominence of CCK-58, these results suggest that CCK's role in food intake regulation may require re-examination.
Keywords: CCK-58; Intermeal interval; Satiety; Meal pattern; Lick microstructure;

Characterization of a far-red analog of ghrelin for imaging GHS-R in P19-derived cardiomyocytes by Gregory A.F. Douglas; Rebecca McGirr; Carlie L. Charlton; Dov B. Kagan; Lisa M. Hoffman; Leonard G. Luyt; Savita Dhanvantari (81-88).
Ghrelin and its receptor, the growth hormone secretagogue receptor (GHS-R), are expressed in the heart, and may function to promote cardiomyocyte survival, differentiation and contractility. Previously, we had generated a truncated analog of ghrelin conjugated to fluorescein isothiocyanate for the purposes of determining GHS-R expression in situ. We now report the generation and characterization of a far-red ghrelin analog, [Dpr3(octanoyl), Lys19(Cy5)]ghrelin (1–19), and show that it can be used to image changes in GHS-R in developing cardiomyocytes. We also generated the des-acyl analog, des-acyl [Lys19(Cy5)]ghrelin (1–19) and characterized its binding to mouse heart sections. Receptor binding affinity of Cy5-ghrelin as measured in HEK293 cells overexpressing GHS-R1a was within an order of magnitude of that of fluorescein-ghrelin and native human ghrelin, while the des-acyl Cy5-ghrelin did not bind GHS-R1a. Live cell imaging in HEK293/GHS-R1a cells showed cell surface labeling that was displaced by excess ghrelin. Interestingly, Cy5-ghrelin, but not the des-acyl analog, showed concentration-dependent binding in mouse heart tissue sections. We then used Cy5-ghrelin to track GHS-R expression in P19-derived cardiomyocytes. Live cell imaging at different time points after DMSO-induced differentiation showed that GHS-R expression preceded that of the differentiation marker aMHC and tracked with the contractility marker SERCA 2a. Our far-red analog of ghrelin adds to the tools we are developing to map GHS-R in developing and diseased cardiac tissues.
Keywords: Ghrelin; GHS-R; P19 cells; Differentiation; Cardiomyocytes; Fluorescence;

Irisin as a muscle-derived hormone stimulating thermogenesis – A critical update by Tobias Hofmann; Ulf Elbelt; Andreas Stengel (89-100).
The recently described myokine, irisin is cleaved from fibronectin type III domain containing protein 5 (FNDC5) and has been proposed to be secreted upon exercise to promote the browning of beige fat cells in white adipose tissue that results in enhanced thermogenesis and increased energy expenditure. The initial studies suggested irisin as a treatment option for obesity and associated diseases such as type 2 diabetes mellitus and stimulated further research. However, the results of subsequent studies investigating the regulation of irisin by different types of exercise are partly conflicting and effects were only shown in highly selective patient populations so far. Moreover, other parameters like body weight or fat free mass were shown to influence irisin adding more complexity to the mechanisms regulating this hormone. The present review will describe the discovery of irisin, its potential role in adipose tissue-mediated thermogenesis, its regulation by exercise and lastly, discuss current controversies and highlight gaps of knowledge to be filled by future studies.
Keywords: Brown adipose tissue; Exercise; FNDC5; Obesity;

Collagen accumulation is one of the important pathologic changes in the development of pulmonary hypertension. Previous research showed that adrenomedullin (ADM) mitigates the development of pulmonary hypertension. The present study explored the role of ADM in the development of pulmonary artery collagen accumulation induced by high pulmonary blood flow, by investigating the effect of ADM [1.5 μg/(kg h)] subcutaneously administered by mini-osmotic pump on pulmonary hemodynamics, pulmonary vascular structure and pulmonary artery collagen accumulation and synthesis in rats with high pulmonary blood flow induced by aortocaval shunting. The results showed that ADM significantly decreased mean pulmonary artery pressure (mPAP) and the ratio of right ventricular mass to left ventricular plus septal mass [RV/(LV + SP)], attenuated the muscularization of small pulmonary vessels and relative medial thickness (RMT) of pulmonary arteries in rats with high pulmonary blood flow. Meanwhile, ADM ameliorated pulmonary artery collagen deposition represented by a decrease in lung tissue hydroxyproline, collagens I and III content and pulmonary artery collagens I and III expression, reduced collagen synthesis represented by a decrease in lung tissue procollagens I and III mRNA expression. The results suggest that ADM plays a protective role in the development of pulmonary hypertension induced by high blood flow, by inhibiting pulmonary procollagen synthesis and alleviating pulmonary artery collagen accumulation.
Keywords: Pulmonary hypertension; High pulmonary blood flow; Collagen; Adrenomedullin;

CART in the brain of vertebrates: Circuits, functions and evolution by Nishikant K. Subhedar; Kartik T. Nakhate; Manoj A. Upadhya; Dadasaheb M. Kokare (108-130).
Cocaine- and amphetamine-regulated transcript peptide (CART) with its wide distribution in the brain of mammals has been the focus of considerable research in recent years. Last two decades have witnessed a steady rise in the information on the genes that encode this neuropeptide and regulation of its transcription and translation. CART is highly enriched in the hypothalamic nuclei and its relevance to energy homeostasis and neuroendocrine control has been understood in great details. However, the occurrence of this peptide in a range of diverse circuitries for sensory, motor, vegetative, limbic and higher cortical areas has been confounding. Evidence that CART peptide may have role in addiction, pain, reward, learning and memory, cognition, sleep, reproduction and development, modulation of behavior and regulation of autonomic nervous system are accumulating, but an integration has been missing. A steady stream of papers has been pointing at the therapeutic potentials of CART. The current review is an attempt at piecing together the fragments of available information, and seeks meaning out of the CART elements in their anatomical niche. We try to put together the CART containing neuronal circuitries that have been conclusively demonstrated as well as those which have been proposed, but need confirmation. With a view to finding out the evolutionary antecedents, we visit the CART systems in sub-mammalian vertebrates and seek the answer why the system is shaped the way it is. We enquire into the conservation of the CART system and appreciate its functional diversity across the phyla.
Keywords: Neuropeptide CART; Sensory system; Homeostatic mechanism; Neuroendocrine regulation; Reward mechanism; CART system evolution;

Spinal ERK2 activation through δ 2-opioid receptors contributes to nociceptive behavior induced by intrathecal injection of leucine-enkephalin by Takaaki Komatsu; Soh Katsuyama; Hirokazu Mizoguchi; Chikai Sakurada; Minoru Tsuzuki; Shinobu Sakurada; Tsukasa Sakurada (131-139).
Intrathecal (i.t.) injection of leucine-enkephalin (Leu-ENK), co-administered with peptidase inhibitors, phosphoramidon (an endopeptidase 24.11 inhibitor), and bestatin (a general aminopeptidase inhibitor), produced behaviors consisting of the biting and/or licking of the hindpaw and the tail along with hindlimb scratching directed toward the flank, which peaked at 10–15 min after an injection. This characteristic behavior was not observed in mice treated with i.t. Leu-ENK alone. We also investigated the effect of the extracellular signal-regulated kinase (ERK) in spinal processing of nociception induced by i.t. co-administration of Leu-ENK with phospharamidon and bestatin. Western blot analysis of phospho-ERK (pERK) showed a significant increase of pERK2 in the lumbar spinal cord in response to i.t. Leu-ENK co-injected with peptidase inhibitors. The MAP kinase-ERK inhibitor, U0126 dose-dependently attenuated the nociceptive behavior and spinal ERK activation to i.t. Leu-ENK co-injected with peptidase inhibitors. Furthermore, the nociceptive behavior and spinal ERK activation evoked by i.t. Leu-ENK in combination with peptidase inhibitors were inhibited by co-administration of the non-selective δ-opioid receptor antagonist, naltrindole, the selective δ 2-opioid receptor antagonist, naltriben, the non-competitive N-methyl-d-aspartate (NMDA) antagonist, MK-801 or the non-selective nitric oxide synthase inhibitor, l-NAME, the selective nNOS inhibitor, Nω-propyl-l-arginine or the selective iNOS inhibitor, W1400, but not by the selective δ 1-receptor antagonist, BNTX (7-benzylidenenaltrexone). These results suggest that spontaneous nociceptive behaviors produced by i.t. co-administration of Leu-ENK with peptidase inhibitors may be induced by an activation of the glutamate-NO-ERK pathway through the δ 2-opioid receptor in the dorsal spinal cord.
Keywords: Leucine-enkephalin; δ 2-Opioid receptor; Glutamate; Nitric oxide (NO); Nociceptive behavior; Extracellular signal-regulated kinase (ERK); Dorsal spinal cord; Mice;

Orexin-A-induced ERK1/2 activation reverses impaired spatial learning and memory in pentylenetetrazol-kindled rats via OX1R-mediated hippocampal neurogenesis by Xuan Zhao; Rui xue Zhang; Shi Tang; Yan yan Ren; Wei xia Yang; Xiao min Liu; Ji you Tang (140-147).
Epilepsy is characterized by the occurrence of repetitive seizures and can greatly affect a patient's cognition, particularly in terms of learning and memory. Orexin-A is an excitatory neuropeptide produced by the lateral hypothalamus that has been shown to be involved in learning and memory. A reduction in the levels of orexin-A after seizures may underlie the learning and memory impairments induced by epilepsy. Thus, we used pentylenetetrazol (PTZ)-kindled rats to investigate the effects of orexin-A on learning and memory and the involvement of neurogenesis in the dentate gyrus in OX1R-mediated ERK1/2 activation. A Morris water maze test revealed reduced escape latencies, prolonged times in the target quadrant and an increased number of platform crossings in PTZ-kindled rats exposed to orexin-A. These ameliorating effects of orexin-A on spatial learning and memory were attenuated by the intracerebroventricular injection of the OX1R antagonist SB334867 or the ERK1/2 inhibitor U0126. Further studies using bromodeoxyuridine (BrdU) revealed that orexin-A increased the number of BrdU-positive cells, doublecortin (DCX)/BrdU levels and the number of NeuN/BrdU double-positive nuclei in the dentate gyrus of PTZ-kindled rats. However, these effects were inhibited by treatment with SB334867 or U0126. Taken together, these data suggest that orexin-A attenuated the impairment of spatial learning and memory in PTZ-kindled rats and that this attenuation involved neurogenesis in the dentate gyrus via OX1R-mediated ERK1/2 activation.
Keywords: Epilepsy; Orexin-A; Learning and memory; OX1R; ERK1/2; Neurogenesis;

Identification and structural characterization of a new pro-apoptotic cyclic octapeptide cyclosaplin from somatic seedlings of Santalum album L. by Abheepsa Mishra; Samiran S. Gauri; Sourav K. Mukhopadhyay; Soumya Chatterjee; Shibendu S. Das; Santi M. Mandal; Satyahari Dey (148-158).
Small cyclic peptides exhibiting potent biological activity have great potential for anticancer therapy. An antiproliferative cyclic octapeptide, cyclosaplin was purified from somatic seedlings of Santalum album L. (sandalwood) using gel filtration and RP-HPLC separation process. The molecular mass of purified peptide was found to be 858 Da and the sequence was determined by MALDI-ToF-PSD-MS as ‘RLGDGCTR’ (cyclic). The cytotoxic activity of the peptide was tested against human breast cancer (MDA-MB-231) cell line in a dose and time-dependent manner. The purified peptide exhibited significant antiproliferative activity with an IC50 2.06 μg/mL. In a mechanistic approach, apoptosis was observed in differential microscopic studies for peptide treated MDA-MB-231 cells, which was further confirmed by mitochondrial membrane potential, DNA fragmentation assay, cell cycle analysis and caspase 3 activities. The modeling and docking experiments revealed strong affinity (kcal/mol) of peptide toward EGFR and procaspase 3. The co-localization studies revealed that the peptide sensitizes MDA-MB-231 cells by possibly binding to EGFR and induces apoptosis. This unique cyclic octapeptide revealed to be a favorable candidate for development of anticancer agents.
Keywords: Cyclic peptide; Sandalwood; Antiproliferative; Apoptosis; Caspase 3; Cyclosaplin;

Urotensin II (U-II), a novel cyclic peptide, possibly associated with the pathophysiology of osteoarthritis by Bülent Gögebakan; Vedat Uruc; Raif Ozden; Ibrahim Gokhan Duman; Abdullah Erman Yagiz; Hamza Malik Okuyan; Ozgur Aldemir; Yunus Dogramaci; Aydiner Kalaci (159-161).
Synovial fibrosis is one of the main outcomes of osteoarthritis. Some authors have reported that urotensin-II (U-II) may cause pathologic fibrosis in cardiovascular system, lung and liver. However there are no previous reports available in the literature about its relationship with the synovial fibrosis in osteoarthritis. The aim of this study was to compare the U-II levels in knee synovial fluids obtained from osteoarthritic and non-osteoarthritic patients. Two groups were created, the osteoarthritis group and non-osteoarthritic control group. The control group was consisted of patients who underwent arthroscopic surgery for other reasons than cartilage disorders. In the osteoarthritis group all patients had grade 4 primer degenerative osteoarthritis and were treated with total knee arthroplasty. Minimum 1 mL knee synovial fluids were obtained during operation. Levels of U-II were measured by using ELISA kit U-II levels were significantly higher in the osteoarthritic group than that in the control group. No correlation was found between U-II levels and age. In conclusion, the significantly high U-II levels in the knee synovial fluid of osteoarthritic patients supported our hypothesis that “U-II may be associated with the synovial fibrosis in osteoarthritis”.
Keywords: Urotensin-II; Synovial fluid; Osteoarthritis; Fibrosis;

Ghrelin administered spinally increases the blood glucose level in mice by Yun-Beom Sim; Soo-Hyun Park; Sung-Su Kim; Chea-Ha Kim; Su-Jin Kim; Su-Min Lim; Jun-Sub Jung; Hong-Won Suh (162-165).
Ghrelin is known as a regulator of the blood glucose homeostasis and food intake. In the present study, the possible roles of ghrelin located in the spinal cord in the regulation of the blood glucose level were investigated in ICR mice. We found that intrathecal (i.t.) injection with ghrelin (from 1 to 10 μg) caused an elevation of the blood glucose level. In addition, i.t. pretreatment with YIL781 (ghrelin receptor antagonist; from 0.1 to 5 μg) markedly attenuated ghrelin-induced hyperglycemic effect. The plasma insulin level was increased by ghrelin. The enhanced plasma insulin level by ghrelin was reduced by i.t. pretreatment with YIL781. However, i.t. pretreatment with glucagon-like peptide-1 (GLP-1; 5 μg) did not affect the ghrelin-induced hyperglycemia. Furthermore, i.t. administration with ghrelin also elevated the blood glucose level, but in an additive manner, in d-glucose-fed model. Our results suggest that the activation of ghrelin receptors located in the spinal cord plays important roles for the elevation of the blood glucose level.
Keywords: Ghrelin; Blood glucose; Spinal cord; Intrathecal; Insulin; GLP-1;

Trp-Arg-Xaa tripeptides act as uncompetitive-type inhibitors of human dipeptidyl peptidase IV by Vu Thi Tuyet Lan; Keisuke Ito; Sohei Ito; Yasuaki Kawarasaki (166-170).
Human dipeptidyl peptidase IV (hDPPIV, alternative name: CD26) inhibitors provide an effective strategy for the treatment of type 2 diabetes. Recently, our research group discovered a non substrate-mimic inhibitory dipeptide, Trp-Arg, by the systematic analysis of a dipeptide library. In the present study, a tripeptide library Trp-Arg-Xaa (where Xaa represents any amino acid) was analyzed to investigate the interactions of peptidergic inhibitors with hDPPIV. Trp-Arg-Glu showed the highest inhibitory effect toward hDPPIV (Ki  = 130 μM). All of the tested 19 Trp-Arg-Xaa tripeptides showed unique uncompetitive-type inhibition. The inhibition mechanism of Trp-Arg-Xaa is discussed based on the crystal structure of hDPPIV. The information obtained by this study suggests a novel concept for developing hDPPIV inhibitory peptides and drugs.

Functional significance of bioactive peptides derived from soybean by Brij Pal Singh; Shilpa Vij; Subrota Hati (171-179).
Biologically active peptides play an important role in metabolic regulation and modulation. Several studies have shown that during gastrointestinal digestion, food processing and microbial proteolysis of various animals and plant proteins, small peptides can be released which possess biofunctional properties. These peptides are to prove potential health-enhancing nutraceutical for food and pharmaceutical applications. The beneficial health effects of bioactive peptides may be several like antihypertensive, antioxidative, antiobesity, immunomodulatory, antidiabetic, hypocholesterolemic and anticancer. Soybeans, one of the most abundant plant sources of dietary protein, contain 36–56% of protein. Recent studies showed that soy milk, an aqueous extract of soybean, and its fermented product have great biological properties and are a good source of bioactive peptides. This review focuses on bioactive peptides derived from soybean; we illustrate their production and biofunctional attributes.
Keywords: Bioactive peptides; Proteolytic; Biofunctional; Hypocholesterolemic; Lactobacilli;

The orexigenic hormone, ghrelin, is tightly linked to cognition impairment in neurodegenerative disorders. No previous studies have investigated the early ghrelin concentration change in patients with mild traumatic brain injury (mTBI) and it's relationship to cognitive deterioration. This study was performed to investigate the early plasma ghrelin concentrations in patients with mTBI and to explore the relationship between ghrelin and cognitive deterioration. Plasma ghrelin concentrations of 118 adults after acute mTBI were determined by enzyme-linked immunosorbent assay. Forty patients (33.9%) had cognitive deterioration three months after mTBI. Plasma ghrelin levels were significantly lower in mTBI patients with cognitive deterioration than patients without cognitive deterioration (38.8 ± 4.5 pg/mL vs 50.8 ± 7.7 pg/mL, P  < 0.001). Decreased Plasma ghrelin level was identified as an independent predictor for three-month cognitive deterioration after mTBI (odds ratio, 0.746; 95% confidence interval, 0.651–0.856; P  < 0.001). Plasma ghrelin level was negatively associated with serum adrenocorticotrophin hormone level (t  = −6.854, P  < 0.001) and age (t  = −6.112, P  < 0.001). A plasma ghrelin level of 41.6 pg/mL predicted three-month cognitive deterioration after mTBI with the optimal sensitivity (85.9%) and specificity (80.0%) values (area under curve, 0.904; 95% confidence interval, 0.852–0.957; P  < 0.001). The predictive value of ghrelin was bigger than that of serum adrenocorticotrophin hormone level (area under curve, 0.638; 95% confidence interval, 0.536–0.741; P  = 0.014) and age (area under curve, 0.638; 95% confidence interval, 0.536–0.741; P  = 0.014) for three-month cognitive deterioration after mTBI.
Keywords: Ghrelin; Cognitive deterioration; Mild traumatic brain injury;

Endogenous galanin as a novel biomarker to predict gestational diabetes mellitus by Zhenwen Zhang; Chunmei Gu; Penghua Fang; Mingyi Shi; Yan Wang; Yan Peng; Ping Bo; Yan Zhu (186-189).
Although a significantly higher level of plasma galanin was found in patients with gestational diabetes mellitus (GDM) in our previous study, it is unknown whether plasma galanin is biomarker for the prediction of GDM. The present study aims to further evaluate the relationship between endogenous galanin and GDM in pregnant women and to find out the precise mechanism by which galanin plays role in the pathogenesis of GDM. The study registered thirty pregnant women with GDM and thirty pregnant women with normal glucose tolerance (NGT). Demographic and biochemical parameters and fasting venous blood samples of two groups were collected from all cases. Galanin was analyzed by an enzyme-linked immunosorbent assay. Gamma-glutamyl transferase (GGT) was measured by enzymatic methods. The plasma galanin and GGT levels were found higher in GDM compared with NGT (P  < 0.001). In addition, a significant positive correlation was shown between galanin and fasting glucose (P  = 0.049), 1-h glucose (P  = 0.033), body mass index (BMI) (P  < 0.001) and GGT (P  = 0.048) in pregnant women with GDM, whereas there was significant positive correlation between galanin and BMI (P  = 0.030) in NGT group. The plasma galanin and GGT levels are higher in patients with GDM. The plasma galanin levels appear to be related to the changes of blood glucose, BMI and GTT in GDM. The higher level of galanin observed in GDM may represent a adaptation to the rise of glucose, weight, GGT associated with GDM. The higher level of plasma galanin is a novel biomarker for the prediction of GDM.
Keywords: Galanin; GGT; Glucose; Gestational diabetes;