Peptides (v.31, #2)

Expression of human β-defensin-2 in intratumoral vascular endothelium and in endothelial cells induced by transforming growth factor β by Hameem I. Kawsar; Santosh K. Ghosh; Stanley A. Hirsch; Henry B. Koon; Aaron Weinberg; Ge Jin (195-201).
Human β-defensin-2 (hBD-2) is a small cationic peptide originally identified from psoriatic skin lesions as an antimicrobial agent of the innate immune system. The expression of hBD-2 is believed to be induced exclusively in epithelial cells by microbial components and certain proinflammatory cytokines, such as interleukin-1β (IL-1β). In this study, we report, for the first time, that hBD-2 is expressed in vascular endothelial cells associated with oral squamous cell carcinoma (OSCC) and Kaposi's sarcoma lesions, but not in that of normal stroma. Expression of hBD-2 in vascular endothelial cells was further substantiated by in vitro experiments using cultured human umbilical vein endothelial cells (HUVECs). Transforming growth factor β1 (TGFβ1) and IL-1β, two well-known tumorigenic inflammatory mediators, induce hBD-2 transcript and peptide expression in HUVECs. However, TGFβ1 does not stimulate hBD-2 expression in oral epithelial cells. In addition, proinflammatory cytokines and microbial reagents do not induce the expression of hBD-1 and hBD-3 in HUVECs. Since hBD-2 has been shown to modulate migration, proliferation, and tube formation of HUVECs in vitro and participate in immune cell trafficking, its expression in vascular endothelial cells located within malignant lesions may play a role in tumor angiogenesis and cancer metastasis.
Keywords: Oral squamous cell carcinoma (OSCC); β-Defensin; HUVEC; hBD-2; Angiogenesis; Kaposi's sarcoma; TGFβ;

Antibody purification technology is of particular industrial importance due to the rapidly increasing use of antibodies in protein purification, diagnostic and therapeutic applications. Such purification has mostly relied on affinity chromatography using Protein A or Protein G as affinity ligands. Several synthetic ligands have also been developed to overcome the disadvantages associated with protein affinity ligands, which include high cost, low stability and possible contamination if the proteins have been expressed in bacteria. In the present study, a convenient selection method for new peptides binding to the IgG Fc domain was developed. The method includes the construction of a DNA library fused to the 5′-position of the eGFP gene expressed from a constitutive vector, expression of the library in Escherichia coli, fluorescence-based screening, and determination of the antibody-binding affinities of selected peptides using surface plasmon resonance. With this method, five novel peptides were identified as new affinity ligands for the IgG Fc domain, and the binding affinities were appropriate for antibody purification. This method is a convenient alternative to phage or bacterial surface display and can be used in the routine biochemistry laboratory.
Keywords: Peptide library; Dot-blot assay; IgG-affinity ligand; IgG Fc-binding peptide; SPR;

Characterization of a novel LFRFamide neuropeptide in the cephalopod Sepia officinalis by Céline Zatylny-Gaudin; Benoit Bernay; Bruno Zanuttini; Jérôme Leprince; Hubert Vaudry; Joël Henry (207-214).
From a single LC–MS/MS analysis, a new C-terminally extended RFamide neuropeptide was characterized in Sepia officinalis. The experimental strategy was based on the specific neutral loss associated with RFamide breakdown. Mass losses of 17 Da (C-terminally amide) and 320 Da (RFamide) have been observed for three known peaks of m/z 581.7 (FLRFamide), 599.8 (FMRFamide), 1096.3 (ALSGDAFLRFamide) and one unknown of m/z 752.8. The primary sequence of the peptide of m/z 752.8 was GNLFRFamide. MS/MS analyses revealed that this novel neuropeptide, called sepFRF1, is largely distributed in the central nervous system of cuttlefish of both sexes. Probably transported in the visceral nerve from the subesophageal mass (the peptide was not detected in the hemolymph), this neuropeptide targeted the rectum in agreement with its peripheral distribution. From concentrations as low as 10−9  M, sepFRF1 increased the frequency, tonus and amplitude of rectal contractions. SepFRF1 is the first RFamide peptide identified in Sepia officinalis that is not derived from the FaRPs precursor. SepFRF1 could belong to a RFamide subfamily identified in gastropods and may be involved in feeding behavior.
Keywords: Marine mollusk; Invertebrate; Regulatory peptide; RFamide; Myotropin; Feeding; Mass spectrometry;

Pharmacological characterization of the mouse NPFF2 receptor by Franck Talmont; Lionel Moulédous; Laura Piedra-Garcia; Martine Schmitt; Frédéric Bihel; Jean-Jacques Bourguignon; Jean-Marie Zajac; Catherine Mollereau (215-220).
This study presents the binding and functional properties of the mouse NPFF2 (mNPFF2) receptor, in comparison with its human counterpart (hNPFF2). Binding experiments were performed by using the NPFF2 selective radioligand [3H]-EYF in membranes from CHO cells transfected with mouse and human NPFF2 receptors and compared to membranes from mouse olfactory bulb, the brain region expressing the highest density of NPFF2 receptors in mouse. mNPFF2 receptors exhibited a high affinity (Kd = 0.2–0.4 nM) for [3H]-EYF, comparable to that of hNPFF2 receptors. Also, the binding selectivity profile of mNPFF2 receptors was comparable to that of hNPFF2 receptors, except for three ligands (NPSF, NPVF, RF9) that were about tenfold more potent and active on mouse receptors than on human receptors. In particular, compared to hNPFF2 receptors, mNPFF2 receptors were less discriminative towards the proNPFFB-derived peptide. This suggests some species-related differences in the binding properties of NPFF2 receptors that could have repercussion when evaluating the pharmacological properties of drugs in vivo.
Keywords: Neuropeptide FF; Receptor; Binding; Species comparison;

Central locomotor and cognitive effects of a NPFF receptor agonist in mouse by Alexandre Betourne; Virginie Marty; Johnatan Ceccom; Hélène Halley; Jean-Michel Lassalle; Jean-Marie Zajac; Bernard Frances; Lionel Mouledous (221-226).
NPFF receptors are expressed in several brain regions directly or indirectly involved in cognition and behavior. However, the cognitive effects of the NPFF system have been poorly studied. Therefore, the aim of our study was to analyze the effects of i.c.v. injections of 1DMe, a stable agonist of NPFF receptors, on behavioral and cognitive performances in C57BL/6J mice. We measured locomotor activity, and an open field with objects was used to estimate the ability of mice to react to spatial changes and to measure short-term retention of information. The Morris navigation task was used to evaluate the acquisition, as well as long-term retention of a hippocampo-dependant spatial memory with a distributed training procedure. Finally, 1DMe was tested in a contextual fear conditioning paradigm to study its effect on long-term memory of contextual information acquired in a single training session. Altogether, our results demonstrate a small but complex influence of the NPFF system on mouse behavior. 1DMe injected i.c.v. induces a delayed hyperlocomotion and mildly impairs both short-term and long-term spatial memory processing without affecting contextual fear memory.
Keywords: Neuropeptide FF; Locomotion; Cognition; Memory; Morris water maze; Fear conditioning;

Protease-activated receptor 2 (PAR2) expression is up-regulated during vascular injury associated with edema. PAR2 and bradykinin subtype 2 receptor (B2) expression and function were assessed in relation to hypertensive encephalopathy (HE) and cerebral hemorrhage (CH) in middle cerebral arteries (MCA) of Kyoto Wistar stroke-prone spontaneously hypertensive rats (SHRsp). Before stroke, bradykinin and PAR2 activation by 2-furoyl-leucine-isoleucine-glycine-arginine-leucine-ornithine-amide (2Fly) produced endothelium-dependent vasodilation that was inhibited by K+ depolarization, carbenoxolone, and the blockade of intermediate (IKCa) plus small (SKCa) and (in the case of bradykinin) smooth muscle (SM) large conductance (BKCa) calcium-activated K+ channels. Responses were not altered by Nω-nitro-l-arginine methyl ester, indomethacin, 17-octadecynoic acid or Ba2+  + ouabain. We concluded that vasodilation to 2Fly or bradykinin was not mediated by NO, cyclooxygenases, arachidonic acid-metabolizing cytochrome P450s or SM Kir channels + Na+/K+ ATPase activation. Vasodilation likely involved the spread of endothelial hyperpolarization (generated by IKCa  + SKCa) through myoendothelial junctions and in some cases SM BKCa activation. SHRsp with HE or CH had MCA that could not constrict to pressure and did not vasodilate to bradykinin. Their responses to 2Fly remained unaltered. The patterns and densities of PAR2 and B2 immunoreactivity in frozen MCA sections were not altered with stroke. MCA function remained normal in SHRsp subjected to dietary manipulations that prevented stroke without altering hypertension. Despite the presence of vascular injury, edema, inflammation and the loss of endothelium-dependent bradykinin vasodilation we found no evidence that PAR2 expression or vascular function was altered in MCA after stroke.
Keywords: Endothelium; Peptides; Hemorrhage; SHRsp; Myogenic response; Nitric oxide;

Bradykinin increases the secretion and expression of endothelin-1 through kinin B2 receptors in melanoma cells by Tsugunobu Andoh; Ahmad Akira; Ikuo Saiki; Yasushi Kuraishi (238-241).
The present study was conducted to determine whether bradykinin would affect the secretion and expression of endothelin-1 (ET-1) in B16-BL6 melanoma cells. Bradykinin administered to cultured melanoma cells increased preproET-1 mRNA level and the secretion of ET-1. Although kinin B1 and B2 receptor mRNAs are expressed in the melanoma cells, the increase of preproET-1 mRNA expression and the secretion of ET-1 were inhibited by kinin B2, but not by B1, receptor antagonist. These results suggest that bradykinin regulates the secretion and biosynthesis of ET-1 through kinin B2 receptor in tumor cells, especially melanoma cells.
Keywords: Bradykinin; Kinin B2 receptor; Endothelin-1; Melanoma cell;

Differential expression of new splice variants of the neurotensin receptor 1 gene in human prostate cancer cell lines by Teresa A. Almeida; Yurena Rodriguez; Mariano Hernández; Ricardo Reyes; Aixa R. Bello (242-247).
Neurotensin is a neuroendocrine peptide acting as a trophic factor in a variety of cells in vivo but it can also function as an autocrine growth factor in human prostate cancer cells in vitro. In addition, the high-affinity G protein-coupled NT receptor (NTS1) is overexpressed in prostate cancer cell lines. Increasing evidence argues for a direct correlation between specific alternative splice variants and cancer. We detected four splice variants of the NTS1 receptor in human prostate cancer cell lines. These isoforms include one or more exons skipping as well as an alternative 5′ splice donor site and are expressed in the late-stage androgen independent prostate cancer cell lines PC3 and DU145, but not in the early-stage androgen-sensitive LNCaP or in normal prostate tissue, which only express the normal transcript. This result shows new splice variants of NTS1 for the first time. The differential expression observed among prostate cancer cell lines and normal prostate tissue opens the interesting possibility of a new role of NT/NTS1 pathway in prostate cancer.
Keywords: Neurotensin; Neurotensin receptor 1; RT-PCR; Alternative splicing; Cancer;

Controlled in situ preparation of Aβ(1–42) oligomers from the isopeptide “iso-Aβ(1–42)”, physicochemical and biological characterization by Zsolt Bozso; Botond Penke; Dóra Simon; Ilona Laczkó; Gábor Juhász; Viktor Szegedi; Ágnes Kasza; Katalin Soós; Anasztázia Hetényi; Edit Wéber; Hajnalka Tóháti; Mária Csete; Márta Zarándi; Lívia Fülöp (248-256).
β-Amyloid (Aβ) peptides play a crucial role in the pathology of the neurodegeneration in Alzheimer's disease (AD). Biological experiments (both in vitro and animal model studies of AD) require synthetic Aβ peptides of standard quality, aggregation grade, neurotoxicity and water solubility. The synthesis of Aβ peptides has been difficult, owing to their hydrophobic character, poor solubility and high tendency for aggregation. Recently an isopeptide precursor (iso-Aβ(1–42)) was synthesized by Fmoc-chemistry and transformed at neutral pH to Aβ(1–42) by ON acyl migration in a short period of time. We prepared the same precursor peptide using Boc-chemistry and studied the transformation to Aβ(1–42) by acyl migration. The peptide conformation and aggregation processes were studied by several methods (circular dichroism, atomic force and transmission electron microscopy, dynamic light scattering). The biological activity of the synthetic Aβ(1–42) was measured by ex vivo (long-term potentiation studies in rat hippocampal slices) and in vivo experiments (spatial learning of rats). It was proven that ON acyl migration of the precursor isopeptide results in a water soluble oligomeric mixture of neurotoxic Aβ(1–42). These oligomers are formed in situ just before the biological experiments and their aggregation grade could be standardized.
Keywords: β-Amyloid; Oligomers; Aggregation; Alzheimer's disease; Isopeptide;

Novel insight in distribution of nesfatin-1 and phospho-mTOR in the arcuate nucleus of the hypothalamus of rats by Tobias Inhoff; Andreas Stengel; Lisa Peter; Miriam Goebel; Yvette Taché; Norbert Bannert; Bertram Wiedenmann; Burghard F. Klapp; Hubert Mönnikes; Peter Kobelt (257-262).
Recently, two proteins have been localized in the arcuate nucleus (ARC) and implicated in the regulation of food intake: the serine–threonine-kinase mammalian target of rapamycin (mTOR) as part of the TOR signaling complex 1 (TORC1), and nesfatin-1 derived from the precursor protein nucleobindin2. However, the exact cell types are not well described. Therefore, we performed double-labeling studies for NPY, CART, nesfatin-1 and pmTOR in the ARC. In this study, we showed that nesfatin-1 is not only intracellularly co-localized with cocaine- and amphetamine-regulated transcript (CART) peptide as reported before, but also with phospho-mTOR (pmTOR) and neuropeptide Y (NPY) in ARC neurons. Quantification revealed that 59 ± 5% of the pmTOR-immunoreactive (ir) neurons were immunoreactive for nesfatin-1. Moreover, double labeling for nesfatin-1 and NPY exhibited that 19 ± 5% of the NPY positive cells were also immunoreactive for nesfatin-1. Furthermore, we could also confirm results from previous studies, showing that the majority of nesfatin-1 neurons are also positive for CART peptide, whereas most of the pmTOR is co-localized with NPY and only to a lesser extent with CART.
Keywords: Arcuate nucleus; Rat; Nesfatin-1; Phospho-mTOR; CART; NPY;

Abdominal surgery activates nesfatin-1 immunoreactive brain nuclei in rats by Andreas Stengel; Miriam Goebel; Lixin Wang; Yvette Taché (263-270).
Abdominal surgery-induced postoperative gastric ileus is well established to induce Fos expression in specific brain nuclei in rats within 2-h after surgery. However, the phenotype of activated neurons has not been thoroughly characterized. Nesfatin-1 was recently discovered in the rat hypothalamus as a new anorexigenic peptide that also inhibits gastric emptying and is widely distributed in rat brain autonomic nuclei suggesting an involvement in stress responses. Therefore, we investigated whether abdominal surgery activates nesfatin-1-immunoreactive (ir) neurons in the rat brain. Two hours after abdominal surgery with cecal palpation under short isoflurane anesthesia or anesthesia alone, rats were transcardially perfused and brains processed for double immunohistochemical labeling of Fos and nesfatin-1. Abdominal surgery, compared to anesthesia alone, induced Fos expression in neurons of the supraoptic nucleus (SON), paraventricular nucleus (PVN), locus coeruleus (LC), Edinger-Westphal nucleus (EW), rostral raphe pallidus (rRPa), nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM). Double Fos/nesfatin-1 labeling showed that of the activated cells, 99% were nesfatin-1-immunoreactive in the SON, 91% in the LC, 82% in the rRPa, 74% in the EW and VLM, 71% in the anterior parvicellular PVN, 47% in the lateral magnocellular PVN, 41% in the medial magnocellular PVN, 14% in the NTS and 9% in the medial parvicellular PVN. These data established nesfatin-1 immunoreactive neurons in specific nuclei of the hypothalamus and brainstem as part of the neuronal response to abdominal surgery and suggest a possible implication of nesfatin-1 in the alterations of food intake and gastric transit associated with such a stressor.
Keywords: Abdominal surgery; Fos; Hypothalamus; NUCB2; Postoperative ileus; Rat brain;

Estrogens induce visfatin expression in 3T3-L1 cells by Jingyu Zhou; Edward R. Seidel (271-274).
Visfatin is a 56 kDa protein that is overexpressed in pregnant women. Like insulin, 2 nM visfatin induced GLUT 4 translocation from the cytosolic fraction to the membrane in 3T3-L1 cells. We have previously reported that visfatin induces glucose uptake into 3T3-L1 cells. These two actions define visfatin as an insulinomimetic. Three estrogens are elevated in pregnancy. Estradiol, the predominant estrogen, estriol, produced by the fetal liver and the pro-estrogen progesterone are all higher during pregnancy than in nonparous women. 3T3-L1 cells were treated with 150 ng/ml estriol, 16 ng/ml estradiol or 190 ng/ml progesterone to reflect the circulating concentrations of these steroids during pregnancy. Estriol treatment produced a 2.5-fold increase in visfatin gene expression. Estradiol and progesterone had small but insignificant effects on visfatin gene expression. In a second experiment, cells were treated with a combination of all three steroids together at the same concentrations listed above. The combination treatment produced a 13-fold increase in visfatin gene expression. These data suggest that the estriol, estradiol and progesterone exert a synergistic effect on visfatin gene expression. Taken together these data suggest that visfatin may play a physiological role during pregnancy. Since visfatin potently and efficaciously induced GLUT 4 translocation in a cell culture model, any hypothetical role for visfatin in pregnancy should include the possibility that it may play a role in maternal/fetal glucose metabolism or distribution. Two possibilities present: either maternal visfatin is overexpressed as a protective response in the pregnant female to compensate for the insulin resistance that often accompanies pregnancy or the excess visfatin is a compensatory response to ensure adequate glucose delivery to the growing fetus.
Keywords: Estradiol; Progesterone; Estriol; Pregnancy; GLUT 4; Insulin;

Maturation of kisspeptinergic neurons coincides with puberty onset in male rats by Agnete H. Bentsen; Laura Ansel; Valerie Simonneaux; Manuel Tena-Sempere; Anders Juul; Jens D. Mikkelsen (275-283).
Kisspeptins, derived from the Kiss1 gene play a central role in activation of the hypothalamo-pituitary gonadal (HPG) axis via stimulation of GnRH neurons. Both Kiss1 and Kiss1R (receptor) mRNA levels are found to be low in pre-pubertal rats, but whether an increase in kisspeptin and/or its receptor is the primary component in the initiation of puberty and where in the hypothalamus regulation of the kisspeptin/Kiss1R system occurs is unresolved. Using immunohistochemistry and in situ hybridization, we analyzed the level of Kiss1 mRNA and kisspeptin-immunoreactivity in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus of male rats along pubertal development. Neurons expressing Kiss1 mRNA were first detected at PND15, but increased significantly around puberty, and declined again in the adult rat. While virtually no immunoreactive cell bodies were detectable in the AVPV at any age, numerous kisspeptin-positive neurons in the arcuate nucleus were detected in the adult rat. Increasing doses of kisspeptin-54 given peripherally to male rats at PND15, 30, 45, and 60 evoked roughly similar effects, as revealed by the induction of c-Fos in the pituitary and secretion of LH and testosterone. These results show that both Kiss1 mRNA and the peptide increase in arcuate nucleus along pubertal maturation. Since kisspeptin signaling is potentially functional, even for peripheral activation, and well before the kisspeptin neuronal system is fully matured, our data support that the regulation of kisspeptin synthesis and release are key events in puberty onset in the male rat.
Keywords: Kisspeptin; Hypothalamus; In situ hybridization; Puberty; Arcuate nucleus; Ontogeny; Kiss1; Luteinizing hormone; Development;

Isolation, identification and biological activity of gastrin-releasing peptide 1–46 (oGRP1–46), the primary GRP gene-derived peptide product of the pregnant ovine endometrium by A.S. Giraud; C. Dumesny; J.C. Whitley; L.M. Parker; I. Jennings; B. Kemp; T.W. Moody; V. Sancho; R.T. Jensen; A. Shulkes (284-290).
We have previously demonstrated that pregnant ovine endometrium expresses the gastrin-releasing peptide (GRP) gene at a high level following conceptus implantation. Here we report the isolation, characterization and biological activity of ovine GRP1–46, the primary product of this gene in the pregnant endometrium. Full thickness 125–140-day pregnant sheep uterus (term is 145 day) was homogenized in 80% acetonitrile/2% trifluoroacetic acid (1:7 ACN/TFA), concentrated on reverse-phase C18 cartridges and chromatographed successively on gel filtration (Sephadex G-50) and reverse-phase HPLC (C18 μBondapak). Purification was monitored by RIA. Purified GRP peptide was analysed by mass spectrometry giving a major mass ion at 4963 which corresponds exactly to GRP1–46. Other mass ions from pro-GRP did not contain a biologically active N-terminus or antigenic determinant. Proteolytic cleavage of pro-GRP to give rise to GRP1–46 would require preferential cleavage at the Glu–Glu bond by a Glu-C2-like enzyme, rather than the trypsin-like and C-terminal amidation enzymes (PAM) that produce GRP18–27 and GRP1–27 in other tissues. GRP1–46 was synthesized and receptor binding and biological activity tested on a range of rodent and human cell lines that express GRP-related receptors GRPR, NMBR and BRS3. GRP1–46 bound GRPR and NMBR with low affinity, and mobilized inositol phosphate in cell lines expressing the GRPR and NMBR, but not BRS-3. This study describes a new processed product of the GRP gene, GRP1–46, which is highly expressed in the pregnant sheep endometrium and which acts as a weak agonist at the GRPR and NMBR.
Keywords: Ovine; Gastrin-releasing peptide; Pregnancy; Endometrium;

Decreased gastric body mucosa obestatin expression in overweight and obese patients by Xin-Yuan Gao; Hong-Yu Kuang; Xiao-Min Liu; Zhi-Bin Ma (291-296).
Obestatin is a recently discovered gastrointestinal hormone. It might play a role in the pathophysiology of obesity. We tried to investigate the expression of obestatin in gastric body mucosa in overweight (BMI ≥ 24 kg/m2)/obese (BMI ≥ 28 kg/m2) patients. Thirty overweight/obese patients and 20 healthy controls were included in the study. Biopsy specimens of gastric mucosa were obtained from the middle body of the greater curvature. Obestatin expression in gastric mucosa was evaluated by immunohistochemistry. Fasting plasma obestatin levels were measured by radioimmunoassay. The number of obestatin-positive cells in gastric body mucosa was significantly lower in overweight and obese patients than that in healthy subjects. The plasma concentrations of obestatin were also decreased in overweight and obese patients. There was a positive correlation between the numbers of obestatin-positive cells in the gastric body mucosa and circulating obestatin levels. The results indicate that overweight and obese subjects have a reduction in the number of obestatin-positive cells in gastric body mucosa.
Keywords: Obestatin; Overweight; Obesity; Immunohistochemistry;

Plasma ghrelin and obestatin levels are increased in spontaneously hypertensive rats by Zhao-Feng Li; Zhi-Fu Guo; Jiang Cao; Jian-Qiang Hu; Xian-Xian Zhao; Rong-Liang Xu; Xin-Miao Huang; Yong-Wen Qin; Xing Zheng (297-300).
Obestatin, encoded by the same gene as ghrelin, was first described as a physiological opponent of ghrelin. We investigated fasting plasma ghrelin and obestatin levels in spontaneously hypertensive rats and Wistar-Kyoto rats. We found that ghrelin levels, obestatin levels and the ratio of ghrelin to obestatin were significantly higher in spontaneously hypertensive rats than Wistar-Kyoto rats. Systolic blood pressure and diastolic blood pressure were positively correlated; however, heart period and baroreflex sensitivity were negatively correlated with ghrelin levels. Systolic blood pressure was positively correlated, whereas baroreflex sensitivity was negatively correlated with obestatin levels. In addition, systolic blood pressure was a significantly independent variable of ghrelin levels, obestatin levels, and the ghrelin to obestatin ratio in a multiple regression analysis. Our data suggests that there is a disturbance of ghrelin and obestatin in the circulation of spontaneously hypertensive rats and the ghrelin/obestatin system might play a role in blood pressure regulation.
Keywords: Ghrelin; Obestatin; Blood pressure; Spontaneously hypertensive rats;

Ontogeny of acylated ghrelin degradation in the rat by Hehong Ni; Pallavi Walia; Jean-Pierre Chanoine (301-306).
Ghrelin circulates as acylated (AG) and unacylated (or desacyl) ghrelin (UAG). We aimed at clarifying the effect of age and sex on plasma deacylation and degradation of AG in vivo and in vitro in the rat. In vivo, we compared AG and UAG concentrations following administration of 1 μg AG intraperitoneally in rat neonates during the first 3 h of life. AG administration caused a 2–3 times increase in plasma AG concentrations contrasting with a ≈1000 times increase in UAG concentrations suggesting rapid deacylation of AG into UAG. In vitro, we demonstrated that AG degradation was greater in the fetus (97% over 30 min) and decreased progressively to 57% in adult animals (P  < 0.001). Carboxylesterase and butyrylcholinesterase activities were determined during the fetal (day 21 of pregnancy) and postnatal period (days 1, 6, 13, 21 and 28) and in the adult rat and were found to increase with age (P  < 0.001). While inhibition of carboxylesterase and butyrylcholinesterase did not affect AG deacylation, serine protease inhibitors decreased AG degradation in the adult rat (from 59% to 23%) and, to a lesser extent, in the rat neonate (from 92% to 57%) by reducing both deacylation and degradation into non-UAG metabolites. Our data suggest that degradation of AG into UAG and non-UAG metabolites is much faster in the fetus and in the rat neonate compared to the adult. We speculate that this process allows for fine tuning of the physiological effects of both AG and UAG.
Keywords: Acylated ghrelin; Unacylated ghrelin; Ontogeny; Protease inhibitor; Carboxylesterase; Butyrylcholinesterase;

Inhibition of Foxo1 mediates protective effects of ghrelin against lipotoxicity in MIN6 pancreatic β-cells by Wei Wang; Ying Liu; Ying Chen; Cuiping Cao; Ying Xiang; Dan Zhang; Lingling Han; Hong Zhao; Guoliang Liu (307-314).
Ghrelin is a 28-amino-acid peptide secreted predominantly by X/A-like cells of the gastric fundus. Ghrelin increases pancreatic β-cell proliferation and survival via sequential activation of phosphatidylinositol-3 kinase (PI3K) and Akt. The transcription regulator Foxo1 is a prominent effector of PI3K/Akt; when it is inhibited, pancreatic β-cells are protected against fatty-acid-induced apoptosis. We investigated the role of Foxo1 in the protective effect of ghrelin under lipotoxic conditions in the MIN6 pancreatic β-cell line. Results showed that ghrelin promoted cell proliferation and attenuated palmitate-induced apoptosis in cultured MIN6 cells. Nuclear exclusion of Foxo1 was necessary for the function of ghrelin. Treatment of MIN6 cells with palmitate and ghrelin-induced rapid nuclear exclusion and phosphorylation of Foxo1. Unlike the JNK inhibitor SP600125, Akt inhibitor IV blocked the anti-lipotoxic effect of ghrelin and stimulated Foxo1 nuclear translocation. In addition, treatment with ghrelin combined with SP600125 showed a synergistic antiapoptotic effect in palmitate-treated MIN6 cells. Ghrelin also inhibited the endoplasmic reticulum stress pathway of apoptosis in MIN6 cells, decreased expression of cytoplasmic triglyceride, and downregulated gene expression of Bcl-2-associated X (BAX), sterol-response element-binding protein 1c (SREBP1c), and C/EBP homologous protein (CHOP-10). These findings suggest that ghrelin protects pancreatic β-cells from lipotoxicity by inhibiting the nuclear translocation of Foxo1.
Keywords: Ghrelin; Lipotoxicity; MIN6 cells; Transcription factor Foxo1; Endoplasmic reticulum stress; Diabetes;

The combination of neurokinin-1 and galanin receptor antagonists ameliorates caerulein-induced acute pancreatitis in mice by Savio G. Barreto; Colin J. Carati; Ann C. Schloithe; James Toouli; Gino T.P. Saccone (315-321).
Both galanin and substance P have been separately implicated in the pathogenesis of acute pancreatitis. We compared the efficacy of the combination of the galanin antagonist galantide and the neurokinin-1 receptor antagonist L703,606 with that of either alone in the treatment of acute pancreatitis. Acute pancreatitis was induced in mice with 7-hourly caerulein injections. Galantide was co-administered with each caerulein injection commencing with the first injection (prophylactic) or 2 h after the first injection (therapeutic). L703,606 was administered either 30 min before (prophylactic), or 2 h after the first caerulein injection (therapeutic). Combination of the two agents was also administered. Control groups received galantide, L703,606, or saline, without caerulein. Pancreata were harvested for histological examination and estimation of myeloperoxidase activity. Plasma amylase activity was measured. Prophylactic and therapeutic administration of galantide reduced the hyperamylasemia by 37% and 30% respectively whereas only prophylactic L703,606 reduced hyperamylasemia (by 34%). Prophylactic administration of the combined antagonists reduced the hyperamylasemia by 44%. In contrast, therapeutic administration of the combination significantly increased plasma amylase levels by 27%. The plasma amylase activity in the control groups was similar to basal levels. Prophylactic and therapeutic administration of either antagonist or the combination significantly reduced myeloperoxidase activity. Galantide and L703,606 individually, and in combination, significantly reduced the acute pancreatitis-induced necrosis score. The administration of the combined antagonists does not offer any further benefit as compared to galantide alone. An interaction between neurokinin-1 and galanin receptors may occur to modulate amylase secretion.
Keywords: Substance P; Neurogenic inflammation; Prophylactic; Therapeutic;

Little is known about CRF expression and regulation in the rat colon compared to the brain. We investigated CRF gene expression, cellular location, and regulation by endotoxin and corticosterone in the male rat colon at 6 h after intraperitoneal (ip) injection. CRF mRNA level, detected by reverse transcription-polymerase chain reaction (RT-PCR) was 1.3-fold higher in the distal than proximal colon and 3.4-fold higher in the proximal colonic submucosa plus muscle layers than in mucosa. CRF immunoreactivity was located in the epithelia, lamina propria and crypts, and co-localized with tryptophan hydroxylase, a marker for enterochromaffin (EC) cells, and in enteric neurons. Lipopolysaccharide (LPS, 100 μg/kg, ip) increased defecation by 2.9-fold and upregulated CRF mRNA by 2.5-fold in the proximal and 1.1-fold in the distal colon while there was no change induced by corticosterone as monitored by quantitative PCR. LPS-induced increased CRF mRNA expression occurred in the submucosa plus muscle layers (1.5-fold) and the mucosa of proximal colon (0.9-fold). LPS increased significantly CRF immunoreactivity in the submucosal and myenteric plexuses of proximal and distal colon compared to saline groups. These results indicate that in rats, CRF is expressed in both proximal and distal colon and more prominently in enteric neurons of the submucosa plus muscle layers and subject to upregulation at the gene and protein levels by LPS through corticosteroid independent pathways. These data suggests that colonic CRF may be part of the local effector limb of the CRF1 receptor mediated colonic alterations induced by acute stress.
Keywords: Corticotropin releasing factor; Enterochromaffin cells; Enteric nervous system; Lipopolysaccharide; Colon; Stress; Defecation; Corticosterone;

Opioid peptides derived from food proteins suppress aggregation and promote reactivation of partly unfolded stressed proteins by N.V. Artemova; Z.M. Bumagina; A.S. Kasakov; V.V. Shubin; B.Ya. Gurvits (332-338).
A new view of the opioid peptides is presented. The potential of small peptides derived from precursor food proteins, to bind to partly unfolded stressed proteins to prevent their irreversible aggregation and inactivation has been demonstrated in various in vitro test systems: dithiothreitol-induced aggregation of alpha-lactalbumin (LA), heat-induced aggregation of alcohol dehydrogenase (ADH), and aggregation and inactivation of bovine erythrocyte carbonic anhydrase (CA) in the process of its refolding after removal of stress conditions. Using dynamic light scattering (DLS), turbidimetry, fluorescence, and circular dichroism measurements protective effects of the synthetic opioid peptides: exorphin C from wheat gluten (Tyr-Pro-Ile-Ser-Leu), rubiscolin-5 from spinach ribulose-bisphosphate-carboxylase/oxygenase (Rubisco) (Tyr-Pro-Leu-Asp-Leu), and hemorphin-6 from bovine hemoglobin (Tyr-Pro-Trp-Thr-Gln-Arg) have been revealed. We have demonstrated the concentration-dependent suppression of light scattering intensity of aggregates of LA and ADH in the presence of the peptides, the population of nanoparticles with higher hydrodynamic radii being shifted to the lower ones, accompanied by an increase in the lag period of aggregation. The presence of the peptides in the refolding solution was shown to assist reactivation of CA and enhance the yield of the CA soluble protein. The results suggest that bioactive food protein fragments may be regarded as exogenous supplements to the endogenous defense mechanisms of the human organism under stress conditions.
Keywords: Protein aggregation; Amorphous aggregates; Opioid peptides; Exorphins; Peptide–protein interaction; Dynamic light scattering;

Synthesis and biological evaluation of cyclic endomorphin-2 analogs by Renata Perlikowska; Jean Claude do-Rego; Aurore Cravezic; Jakub Fichna; Anna Wyrebska; Geza Toth; Anna Janecka (339-345).
In our previous paper we reported synthesis and biological activity of two cyclic analogs of endomorphin-2 (EM-2): Tyr-c(Lys-Phe-Phe-Asp)-NH2 and Tyr-c(Asp-Phe-Phe-Lys)-NH2, achieved by making an amid bond between Lys and Asp side-chains. The first analog did not bind to the μ-opioid receptor, the affinity of the second one was very low. In the present study, we describe the synthesis of four novel cyclic analogs of similar structure, but with d-amino acids in position 2 (d-Lys or d-Asp). All new analogs displayed high affinity for the μ-opioid receptor, were much more stable than EM-2 in rat brain homogenate and showed remarkable antinociceptive activity after intracerebroventricular (i.c.v.) administration. Analgesic effect of the most potent cyclic analog, Tyr-c(d-Lys-Phe-Phe-Asp)NH2 was much stronger and longer lasting than that of EM-2. This analog elicited analgesia also after peripheral administration and this effect was reversed by concomitant i.c.v. injection of the μ-opioid antagonist, β-funaltrexamine, which indicated that antinociception was mediated by the μ-opioid receptor in the brain. Central action of the cyclic analog gives evidence that it was able to cross the blood–brain barrier, most likely due to the increased lipophilicity. Our results demonstrate that cyclization might be a promising strategy to enhance bioavailability of peptides and may serve a role in the development of novel endomorphin analogs with increased therapeutic potential.
Keywords: Binding studies; μ-, δ-Opioid receptor; Hot-plate test; Solid phase peptide synthesis;

Diabetes-associated changes and role of Nɛ-(carboxymethyl)lysine in big ET-1-induced coronary vasoconstriction by Takayuki Matsumoto; Yuta Ozawa; Kumiko Taguchi; Tsuneo Kobayashi; Katsuo Kamata (346-353).
Using perfused hearts from streptozotocin-induced long-term diabetic rats, we studied the coronary vasoconstrictor effect of the endothelin-1 (ET-1) precursor big ET-1 and also whether this response was modulated by Nɛ-(carboxymethyl)lysine (CML; a representative advanced glycation end product that is implicated in the pathogenesis of diabetic vasculopathy). The big ET-1-induced vasoconstriction (a) developed more rapidly (i.e., was greater in the first 30 min) in the diabetic group than in the age-matched controls, and (b) in each group was largely suppressed by phosphoramidon [nonselective endothelin-converting enzyme (ECE)/neutral endopeptidase (NEP) inhibitor] or CGS35066 (selective ECE inhibitor), but not by thiorphan (selective NEP inhibitor). The ET-1 release occurring after treatment with big ET-1, which was greater in diabetic coronary arteries than in the controls, was reduced by CGS35066. The dose–response curve for ET-1 was shifted to the left in the diabetics, so that at some lower doses of ET-1 the vasoconstriction was greater than in the controls. CML enhanced big ET-1- or ET-1-induced vasoconstriction in the controls, but not in the diabetics. Finally, the plasma level of CML was higher in diabetic than in control rats. These findings suggest (a) that the increased responsiveness to big ET-1 shown by diabetic coronary arteries may be attributable both to a more rapid conversion of big ET-1 to ET-1 (by ECE), allowing it to exert its contractile activity, and to an increased vascular sensitivity to ET-1, and (b) that CML may be at least partly responsible for the diabetes-associated enhancement of big ET-1-mediated coronary vasoconstriction.
Keywords: Big ET-1; CML; Coronary artery; Diabetes; Endothelin; Perfused heart;

Circulating levels of stress associated peptide Urocortin in heart failure patients by D. Gruson; S.A. Ahn; J.M. Ketelslegers; M.F. Rousseau (354-356).
The available data suggest that Urocortin (UCN), a cardioprotective and vasoactive peptide known from fish neuroendocrinology, is involved in cardiac regulation. The aim of this study was to determine UCN plasma concentrations in patients with heart failure (HF). Plasma concentrations of UCN, measured in 42 fully treated HF patients. UCN, were determined using a specific ELISA assay after acidic extraction with Sep-Pak C18 columns. Circulating levels of other neurohormones Nt-proBNP, Nt-proANP and Big ET-1 were also determined. Reference values were obtained from 20 healthy age- and sex-matched subjects. In comparison with controls, UCN plasma concentrations (geometric mean [95% CI]) were significantly increased in HF patients (88 pmol/L [75–105] vs 46 [39–54], p  < 0.001). As expected, the other neurohormones were also significantly increased in HF patients (Nt-proBNP: 3501 pg/ml [2356–5202] vs 35 [24–51], Nt-proANP: 5498 pg/ml [4336–6971] vs 324 [255–411] and Big ET-1: 15.8 pg/ml [13.6–18.4] vs 5.9 [5.2–6.8]; p  < 0.01 for all vs controls). No significant correlation was observed between UCN and the other HF biomarkers. Our results demonstrate that plasma concentrations of UCN are significantly increased in patients with HF and that UCN may participate in the neurohumoral response of HF.

Numerous peptides released from endocrine cells in the intestinal mucosa were established early on to be involved in the physiological regulation of food intake with a prominent role in termination of food ingestion when nutrients pass along the intestinal tract. Recently, peptides released from X/A-like endocrine cells of the gastric oxyntic mucosa were recognized as additional key players in the regulation of feeding and energy expenditure. Gastric X/A-like cells release the octanoylated peptide, ghrelin, the only known peripherally produced hormone stimulating food intake through interaction with growth hormone secretagogue 1a receptor (GHS-R1a). Additionally, non-octanoylated (des-acyl) ghrelin present in the circulation at higher levels than ghrelin is currently discussed as potential modulator of food intake by opposing ghrelin's action independent from GHS-R1a although the functional significance remains to be established. Obestatin, a ghrelin-associated peptide was initially reported as anorexigenic modulator of ghrelin's orexigenic action. However, subsequent reports did not support this contention. Interesting is the recent identification of nesfatin-1, a peptide derived from the nucleobindin2 gene prominently expressed in gastric X/A-like cells in different vesicles than ghrelin. Circulating nesfatin-1 levels vary with metabolic state and peripheral or central injection inhibits dark phase feeding in rodents. Overall, these data point to an important role of gastric X/A-like cells in food intake regulation through the expression of the orexigenic peptide ghrelin along with des-acyl ghrelin and nesfatin-1 capable of reducing food intake upon exogenous injection although their mechanisms of action and functional significance remain to be established.
Keywords: Body weight; Endocrine cell; Food intake; Ghrelin; Nesfatin-1; NUCB2; Obesity; Stomach;