Peptides (v.29, #3)
Announcement - Japan (V).
Editorial Board (CO2).
Anti-hypertensive activity of genetically modified soybean seeds accumulating novokinin by Yuko Yamada; Keito Nishizawa; Megumi Yokoo; Hui Zhao; Kunihiko Onishi; Masayoshi Teraishi; Shigeru Utsumi; Masao Ishimoto; Masaaki Yoshikawa (331-337).
Novokinin (Arg-Pro-Leu-Lys-Pro-Trp), which has been designed based on the structure of ovokinin (2–7), significantly reduces the systolic blood pressure at a dose of 100 μg/kg after oral administration in spontaneously hypertensive rats (SHRs). In this study, we generated a transgenic soybean which accumulates novokinin. A vector encoding a modified β-conglycinin α′ subunit (4novokinin-α′) in which four novokinin sequences have been incorporated by site-directed mutagenesis was introduced into somatic embryos by whisker-mediated gene transformation to produce a transgenic soybean. The 4novokinin-α′ occupied 0.5% of total soluble protein and 5% of the β-conglycinin α′ subunit in the transgenic soybean seeds. Protein extracted from the transgenic soybean reduced systolic blood pressure after single oral administration in SHRs at a dose of 0.15 g/kg. Defatted flour from the transgenic soybean also reduced the systolic blood pressure at a dose of 0.25 g/kg. Thus, the 4novokinin-α′ produced in soybean exhibited an anti-hypertensive activity in SHRs after oral administration.
Keywords: Novokinin; Soybean β-conglycinin; Hypotensive activity; Transgenic soybean; Spontaneously hypertensive rat;
Identification of ACE-inhibitory peptides in salt-free soy sauce that are transportable across caco-2 cell monolayers by Xiao-Lin Zhu; Keisuke Watanabe; Koso Shiraishi; Tatsuro Ueki; Yoshiharu Noda; Toshiro Matsui; Kiyoshi Matsumoto (338-344).
In present study, we aimed to identify angiotensin I-converting enzyme (ACE)-inhibitory peptides from a salt-free soy sauce (SFS), a newly developed antihypertensive seasoning obtained by Aspergillus oryzae fermentation of soybean in the absence of salt, which can be transported through caco-2 cell monolayers. Through an Ussing transport investigation of SFS across caco-2 cell monolayers, three di-peptides, Ala-Phe, Phe-Ile and Ile-Phe, were successfully identified from the SFS as transportable inhibitory peptides. Ala-Phe and Ile-Phe, but not Phe-Ile, exhibited ACE-inhibitory activity with IC50 values of 165.3 μM and 65.8 μM, respectively. Kinetic studies revealed that Ile-Phe (Km: 3.1 mM, P app: 2.4 × 10−6 cm/s) exhibited greater affinity toward the transport compared with Ala-Phe (K m: 48.1 mM, P app: 1.4 × 10−6 cm/s) and Phe-Ile (K m: 12.7 mM, P app: 1.4 × 10−6 cm/s).
Keywords: Peptide; Absorption; Salt-free soy sauce; Caco-2; Angiotensin I-converting enzyme;
Met-Arg-Trp derived from Rubisco lowers blood pressure via prostaglandin D2-dependent vasorelaxation in spontaneously hypertensive rats by Hui Zhao; Hachiro Usui; Kousaku Ohinata; Masaaki Yoshikawa (345-349).
Met-Arg-Trp (MRW) has been isolated as an inhibitor for angiotensin I-converting enzyme (ACE) from a pepsin–pancreatin digest of spinach ribulose bisphosphate carboxylase/oxygenase (Rubisco) (IC50 = 0.6 μM). It has been reported that hypotensive activity of ACE-inhibitory peptides derived from food proteins are weakened in spontaneously hypertensive rats older than 25 weeks (old SHR). However, MRW reduced blood pressure after oral administration at a dose of 5 mg/kg in old SHR as well as in younger SHR. MRW exhibited vasorelaxing activity above 1 μM in isolated mesenteric artery from adult and old SHR. The vasorelaxing activity of MRW was blocked by indomethacin and BW A868C, a cyclooxygenase inhibitor and an antagonist for DP1 receptor, respectively. However, N G-nitro-l-arginine methyl ester, an inhibitor for nitric oxide synthase, had no effect on the relaxation. The hypotensive activity of MRW was also blocked by indomethacin and BW A868C, respectively, in adult and old SHR. Taken together, the vasorelaxing and hypotensive activities of MRW may be mediated by prostaglandin D2 and the DP1 receptor. These findings suggest that the hypotensive activity of MRW is mainly caused by vasorelaxation rather than by ACE-inhibition.
Keywords: Rubisco; ACE-inhibitory peptide; Vasorelaxation; PGD2; DP1 receptor;
A novel small antifungal peptide from Bacillus strain B-TL2 isolated from tobacco stems by Beibei Zhang; Chengjian Xie; Xingyong Yang (350-355).
A novel small antifungal peptide produced by a Bacillus strain B-TL2 isolated from tobacco stems was purified. The purification procedure consisted of ammonium sulfate precipitation, cation exchange chromatography on CM-Sepharose Fast Flow column and reverse-phase HPLC on SOURCE 5RPC column. After the final isolation step, one peptide with antifungal activity, designated as BTL, was obtained. The molecular mass of the purified BTL was determined as 2500 Da and 2237.7 Da by SDS-PAGE and matrix-assisted laser desorption/ionization time of flight mass spectrometry, respectively. The N-amino acid sequence of BTL was determined to be NH2-KQQLATEAESAGPIL, which shows relatively low identity to other antimicrobial peptides from bacteria. The peptide exhibited strong inhibitory activity against mycelial growth of Bipolaris maydis, Alternaria brassicae, Aspergillus niger, Cercospora personata. The purified BTL displayed thermostability, almost retaining 100% activity at 100 °C for 15 min.
Keywords: Bacillus B-TL2; Purification; Antimicrobial peptide; Thermostability; Tobacco;
A selective fluorescence reaction for peptides and chromatographic analysis by Tsutomu Kabashima; Zhiqiang Yu; Chenhong Tang; Yoshiki Nakagawa; Kyosuke Okumura; Takayuki Shibata; Jianzhong Lu; Masaaki Kai (356-363).
A novel and selective fluorescence reaction is proposed for the quantitative determination of peptides by reversed-phase liquid chromatography (RPLC). A single fluorescent product was formed when a peptide was heated at 120 °C for 20 min in a neutral aqueous medium (pH 7.0) containing catechol, sodium periodate, and sodium borate. The fluorescent products of four peptides such as Leu-Gly, Ala-Leu-Gly, Tyr-Gly-Gly-Phe-Leu, and Leu-Leu-Leu were easily separated on a reversed-phase column by gradient elution of methanol in a mobile phase containing sodium borate (pH 7.0), and then quantitatively detected by fluorimetry. The lower limits (S/N = 3) of the detection for the tested peptides were 0.5–1.0 pmol per an injection volume (40 μl). In addition, the fluorescent products of phenylalanine amide and Leu-Leu-Leu were identified by electrospray ionization-time of flight-mass spectrometry (ESI-TOF/MS) for the elucidation of their chemical structures.
Keywords: Peptide; Fluorescence reaction; Catechol; Borate; Periodate; Liquid chromatography;
Effect and mechanisms underlying scorpion toxin action from Androctonus australis garzonii on atrial natriuretic peptide in rat atria: An in vitro study by Hayet Soualmia; Fekri Abroug; Yasmina Djeridane (364-368).
Scorpion envenomation is considered public health problem in Northern African countries. The mechanisms of cardiac dysfunction following scorpion envenomation are not fully understood. This study examined the effect and mechanisms underlying scorpion toxin action from Androctonus australis garzonii on atrial natriuretic peptide (ANP) release from rat atrium using in vitro organ perifusion. Male Sprague Dawley rats were used in this study. Three experiments were conducted. In experiment 1, atrial tissues were exposed either to Krebs-bicarbonate buffer medium (control) or to scorpion toxin (10−8 M to 10−6 M). In experiment 2, animals were chemically sympathectomized with a single intraperitoneal injection of 6-hydroxydopamine (6-OHDOPA) at a dose of 40 μg/g 24 h before sacrifice. Vehicle-treated rats served as control. Atrial tissues were collected and perifused in the presence of 10−6 M scorpion toxin. In experiment 3, atrial tissues were exposed to 10−6 M scorpion toxin either in the absence or presence of 10−6 M propranolol (a β-adrenoceptor blocker), or 10−6 M tetrodotoxin (a sodium channel blocker). ANP levels released in the perifusion medium were determined by radioimmunoassay. The scorpion toxin at 10−6 M induced a significant (p < 0.01) increase (106%) in ANP levels. This effect was decreased (20%) by 6-OHDOPA. Propranolol and tetrodotoxin significantly (p < 0.01) inhibited 55% and 60%, respectively, the toxin-induced ANP release. The data show that the North African scorpion toxin from Androctonus australis garzonii increases the ANP release in rat atrium through stimulation of sympathetic cardiac nerves and sodium channels activation.
Keywords: Atrial natriuretic peptide; Scorpion toxin; 6-Hydroxydopamine; Rat; Heart;
A novel serine protease inhibitor from Bungarus fasciatus venom by Jia Lu; Hailong Yang; Haining Yu; Weikai Gao; Ren Lai; Jingze Liu; Xingcai Liang (369-374).
By Sephadex G-50 gel filtration, cation-exchange CM-Sephadex C-25 chromatography and reversed phase high-performance liquid chromatography (HPLC), a novel serine protease inhibitor named bungaruskunin was purified and characterized from venom of Bungarus fasciatus. Its cDNA was also cloned from the cDNA library of B. fasciatus venomous glands. The predicted precursor is composed of 83 amino acid (aa) residues including a 24-aa signal peptide and a 59-aa mature bungaruskunin. Bungaruskunin showed maximal similarity (64%) with the predicted serine protease inhibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin, chymotrypsin, and elastase. By screening the cDNA library, two new B chains of beta-bungarotoxin are also identified. The overall structures of bungaruskunin and beta-bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.
Keywords: Snake venom; Serine protease inhibitor; Kunitz; Bungarus fasciatus;
Probing the conformation and dynamics of allatostatin neuropeptides: A structural model for functional differences by Monimoy Banerjee; Eric Meyerowitz; Chengdong Huang; Smita Mohanty (375-385).
Allatostatins are a family of related neuropeptides that play an important role in development, reproduction, and digestion in insects. The cockroach Diploptera punctata has 13 allatostatin neuropeptides, with pleiotropic functions, two of which are: inhibition of juvenile hormone (JH) production and inhibition of gut muscle contraction. In this study, the conformation and dynamics of D. punctata allatostatin 5 (Dippu-AST 5) and allatostatin 8 (Dippu-AST 8) are investigated by CD, NMR, and molecular dynamics simulations. These peptides contain eight and nine residues, respectively, and the identical six-residue C-terminal motif. Yet Dippu-AST 5 and Dippu-AST 8 affect juvenile hormone production and hindgut contraction with different potencies. Dippu-AST 5 is one of the most potent inhibitors of juvenile hormone production and one of the least potent inhibitors of gut contraction, whereas Dippu-AST 8 has the opposite potencies with respect to these tissues. From the NMR structure, it is clear that Dippu-AST 5 has a 310 helix involving three of its residues and a “γ” turn at the end of its C-terminal motif. In contrast Dippu-AST 8 has an open “π” turn among five of its central residues. In addition, the orientation preferences within the membrane of the two peptides were simulated. Our simulation results show that the C-terminal segment of Dippu-AST 5 orients in the membrane surface with an average angle of 17.5°, whereas Dippu-AST 8 orients with an average angle of 5.1°. Taken together, from the structures and orientation preferences of these peptides within the membrane, it appears that these peptides may interact with the receptor very differently.
Keywords: Allatostatins; Nuclear magnetic resonance; Neuropeptides; 310 helix; γ turn; π turn;
Identification of a tachykinin-related peptide with orexigenic properties in the German cockroach by Núria Pascual; José L. Maestro; Cristina Chiva; David Andreu; Xavier Bellés (386-392).
A number of evidences suggest that tachykinin-related peptides (TRPs) of insects can stimulate food consumption after being released from the midgut to the hemolymph. The idea of the present work has been to test this hypothesis in the anautogenous cockroach Blattella germanica. First, we have identified the peptide LemTRP-1 (APSGFLGVR-NH2) from brain extracts, by means of an ELISA developed with a polyclonal antibody against this peptide. ELISA studies have also shown that, whereas brain LemTRP-1 levels were fairly constant, midgut levels increase to a maximum on day 3 after adult emergence, falling thereafter until the end of the gonadotrophic cycle. Interestingly, maximum values of food consumption are concomitant with the decrease of LemTRP-1 immunoreactivity in the midgut. Furthermore, starvation decreases LemTRP-1 immunoreactivity in midgut, whereas in the hemolymph it increases. Finally, injection of synthetic LemTRP-1 to adult females significantly stimulates food consumption. The whole observations suggest that LemTRP-1 is released from the midgut to the hemolymph when sustained food consumption is required to maintain vitellogenesis at the highest levels, and that LemTRP-1 in the hemolymph stimulates food consumption in these days.
Keywords: Tachykinin; Blattella germanica; Food consumption; Myotropic peptide;
Skin bradykinin-related peptides (BRPs) and their biosynthetic precursors (kininogens): Comparisons between various taxa of Chinese and North American ranid frogs by YeeTing Sin; Mei Zhou; Wei Chen; Lei Wang; Tianbao Chen; Brian Walker; Chris Shaw (393-403).
Bradykinins and related peptides (BRPs) occur in the defensive skin secretions of many amphibians. Here we report the structures of BRPs and their corresponding biosynthetic precursor cDNAs from the Chinese brown frog, Rana chensinensis, and the North American leopard frog, Lithobates pipiens. R. chensinensis skin contained four transcripts each encoding a different kininogen whose organizations and spectrum of encoded BRPs were similar to those reported for the pickerel frog, Lithobates palustris. In contrast, from L. pipiens, a single skin kininogen was cloned whose structural organization and spectrum of mature BRPs were similar to those reported for the Chinese piebald odorous frog, Huia schmackeri. These data also implied that the endogenous precursor processing proteases in each species pair have identical site-directed specificities, which in part may be dictated by the primary structures of encoded BRPs. Thus the spectra of skin BRPs and the organization of their biosynthetic precursors are not consistent with recent taxonomy. The natural selective pressures that mould the primary structures of amphibian skin secretion peptides are thought to be related to the spectrum of predators encountered within their habitats. Thus similarities and differences in skin bradykinins may be reflective of predator spectra rather than indicative of species relatedness.
Keywords: Amphibian; Skin; Bradykinin; Precursor; Kininogen; Cloning;
Enalapril treatment corrects the reduced response to bradykinin in diabetes increasing the B2 protein expression by Viviani Milan Ferreira Rastelli; Maria Aparecida Oliveira; Rosangela dos Santos; Rita de Cássia Tostes Passaglia; Dorothy Nigro; Maria Helena Catelli de Carvalho; Zuleica Bruno Fortes (404-411).
Considering the growing importance of the interaction between components of kallikrein–kinin and renin–angiotensin systems in physiological and pathological processes, particularly in diabetes mellitus, the aim of the present study was to investigate the effect of enalapril on the reduced response of bradykinin and on the interaction between angiotensin-(1–7) (Ang-(1–7)) and bradykinin (BK), important components of these systems, in an insulin-resistance model of diabetes. For the above purpose, the response of mesenteric arterioles of anesthetized neonatal streptozotocin-induced (n-STZ) diabetic and control rats was evaluated using intravital microscopy. In n-STZ diabetic rats, enalapril treatment restored the reduced response to BK but not the potentiation of BK by Ang-(1–7) present in non-diabetic rats. The restorative effect of enalapril was observed at a dose that did not correct the altered parameters induced by diabetes such as hyperglycemia, glicosuria, insulin resistance but did reduce the high blood pressure levels of n-SZT diabetic rats. There was no difference in mRNA and protein expressions of B1 and B2 kinin receptor subtypes between n-STZ diabetic and control rats. Enalapril treatment increased the B2 kinin receptor expression. From our data, we conclude that in diabetes enalapril corrects the impaired BK response probably by increasing the expression of B2 receptors. The lack of potentiation of BK by Ang-(1–7) is not corrected by this agent.
Keywords: Type 2 diabetes; Bradykinin; Angiotensin-(1–7); Enalapril;
Hypotensive activity of novokinin, a potent analogue of ovokinin(2–7), is mediated by angiotensin AT2 receptor and prostaglandin IP receptor by Yuko Yamada; Daiki Yamauchi; Hachiro Usui; Hui Zhao; Megumi Yokoo; Kousaku Ohinata; Masaru Iwai; Masatsugu Horiuchi; Masaaki Yoshikawa (412-418).
Novokinin (Arg-Pro-Leu-Lys-Pro-Trp) is a potent hypotensive peptide previously designed based on the structure of ovokinin(2–7) (Arg-Ala-Asp-His-Pro-Phe), a vasorelaxing and hypotensive peptide derived from ovalbumin. Novokinin exhibited an affinity for the angiotensin AT2 receptor (Ki = 7.35 μM). Novokinin significantly lowered systolic blood pressure at a dose of 0.03 and 0.1 mg/kg after intravenous and oral administration, respectively, in spontaneously hypertensive rats (SHRs), and the hypotensive activity was blocked by PD123319, an antagonist of the AT2 receptor. Novokinin lowered blood pressure in C57BL/6J mice after oral administration at a dose of 50 mg/kg. However, in AT2 receptor-deficient mice, novokinin did not reduce blood pressure. These results demonstrate that the hypotensive activity of novokinin is mediated by the AT2 receptor. The hypotensive activity of novokinin in SHRs was completely blocked by indomethacin and CAY10441, an inhibitor of cyclooxygenase and an antagonist of the prostaglandin IP receptor, respectively. These suggest that the hypotensive activity is mediated by prostacyclin and the IP receptor downstream of the AT2 receptor.
Keywords: Novokinin; Ovokinin(2–7); Angiotensin; AT2 receptor; Prostacyclin; IP receptor; Nitric oxide; Blood pressure; Spontaneously hypertensive rat;
Endothelin-1 increases expression of cyclooxygenase-2 and production of interlukin-8 in hunan pulmonary epithelial cells by Hong Peng; Ping Chen; Ying Cai; Yan Chen; Qing-hua Wu; Yun Li; Rui Zhou; Xiang Fang (419-424).
Inducible cyclooxygenase (COX-2) and inflammatory cytokines play important roles in inflammatory processes of chronic obstructive pulmonary disease (COPD). Endothelin-1 (ET-1) might be also involved in the pathophysilogical processes in COPD. In the present study, we determined whether ET-1 could regulate the expression of COX-2 and alter the production of interleukin-8 (IL-8) in human pulmonary epithelial cells (A549). Induced sputum samples were collected from 13 stable COPD patients and 14 healthy subjects. The COX-2 protein, ET-1, PGE2 and IL-8 in these sputum samples were analyzed. A549 cells were incubated with ET-1 in the presence or absence of celecoxib, a selective COX-2 inhibitor. The expression of COX-2 protein in the cell and the amounts of PGE2 and IL-8 in the medium were measured. The levels of COX-2 protein, ET-1, PGE2 and IL-8 were significantly increased in induced sputum from COPD patients when compared to healthy subjects. ET-1 increased the expression of COX-2 protein, as well as the production of PGE2 in A549 cells. Increased production of PGE2 was inhibited by celecoxib. ET-1 also increased the production of IL-8. Interestingly, ET-1-induced production of IL-8 was also inhibited by celecoxib. These findings indicate that ET-1 plays important roles in regulating COX-2 expression and production of IL-8 in A549 cells. ET-1 mediated production of IL-8 is likely through a COX-2-dependent mechanism.
Keywords: Cyclooxygenase-2; Chronic obstructive pulmonary disease; Endothelin-1; Prostaglandin E2; Interleukin-8;
Role of carnosine in preventing thioacetamide-induced liver injury in the rat by Güldal Mehmetçik; Gül Özdemirler; Necla Koçak-Toker; Uğur Çevikbaş; Müjdat Uysal (425-429).
Carnosine (β-alanyl-l-histidine) is a dipeptide with antioxidant properties. Free radicals are involved in the pathogenesis of acute liver injury induced by thioacetamide (TAA). In this study, we investigated the effect of carnosine treatment on TAA-induced oxidative stress and hepatotoxicity. Rats were injected intraperitoneally with TAA (500 mg/kg) and carnosine (250 mg/kg, intraperitoneal) was co-administered with TAA. All animals were killed 24 h after injections. TAA administration resulted in hepatic necrosis, significant increases in plasma transaminase activities as well as hepatic lipid peroxide levels. In addition, hepatic antioxidant system was found to be depressed following TAA administration. When carnosine was co-administered with TAA in rats, plasma transaminase activities were found to approach to normal values in rats. Histological findings also suggested that carnosine has preventive effect on TAA-induced hepatic necrosis. Carnosine treatment caused significant decreases in lipid peroxide levels in TAA-treated rats without any changes in enzymatic and non-enzymatic antioxidants except vitamin E in the liver of rats. Our findings indicate that carnosine, in vivo may have a preventive effect on TAA-induced oxidative stress and hepatotoxicity by acting as an non-enzymatic antioxidant itself.
Keywords: Carnosine; Thioacetamide; Liver injury; Oxidative stress; Rats;
Cyclo-glycyl-glutamine inhibits ethanol intake in P and Sprague–Dawley rats by Garth E. Resch; C. Wayne Simpson (430-439).
Peptide inhibitors of ethanol consumption have shown promise. The purpose of this study was to test the cyclized form of the opioid-derived dipeptide, glycyl-l-glutamine to reduce ethanol consumption after either peripheral injections or site-specific injections into the nucleus accumbens (NAC) of high drinking and low drinking rats. Following I.P. cyclo-glycyl-glutamine (c-GQ), the data show a mean decrease in ethanol intake of 34.4% in P rats, and 39.4% in Sprague–Dawley rats at doses between 5 and 25 mg/kg. The data show that peripherally administered c-GQ is effective in reducing ethanol consumption in both high (P) and low (SD) drinking strains of rats and suggests a therapeutic potential.
Keywords: Cyclo-glycyl-glutamine; P rat; Sprague–Dawley rat; Alcohol intake;
Effects of selective modulation of the central melanocortin-3-receptor on food intake and hypothalamic POMC expression by Michelle Lee; Andrea Kim; Irene M. Conwell; Victor Hruby; Alexander Mayorov; Minying Cai; Sharon L. Wardlaw (440-447).
Hypothalamic POMC neurons regulate energy balance via interactions with brain melanocortin receptors (MC-Rs). POMC neurons express the MC3-R which can function as an inhibitory autoreceptor in vitro. We now demonstrate that central activation of MC3-R with ICV infusion of the specific MC3-R agonist, [d-Trp8]-γ-MSH, transiently suppresses hypothalamic Pomc expression and stimulates food intake in rats. Conversely, we also show that ICV infusion of a low dose of a selective MC3-R antagonist causes a transient decrease in feeding and weight gain. These data support a functional inhibitory role for the MC3-R on POMC neurons that leads to changes in food intake.
Keywords: POMC; MSH; Melanocortin-3-receptor; Feeding;
Dose dependent effects of ghrelin on pentylenetetrazole-induced oxidative stress in a rat seizure model by Basra Deniz Obay; Ezel Taşdemir; Cemıl Tümer; Hakkı Murat Bilgin; Mukadder Atmaca (448-455).
It has been suggested that free oxygen radicals play a role in the genesis of epilepsy and in post-seizure neuronal death. The aim of this study was to investigate the dose dependent effect of ghrelin on pentylenetetrazole (PTZ)-induced oxidative stress in a rat seizure model. For this purpose, the ghrelin groups were treated with intraperitoneal injections of ghrelin at doses of 20, 40, 60 and 80 μg/kg before the PTZ injection. Superoxide dismutase (SOD) and catalase (CAT) activities, and reduced glutathione (GSH) and thiobarbituric acid-reactive substance (TBARS) levels were measured in erythrocytes, liver and brain tissue. TBARS, the indicator of lipid peroxidation, was significantly increased in erythrocytes, liver and brain tissue, while antioxidant enzyme activities and glutathione levels were significantly decreased in PTZ injected rats. Ghrelin pretreatment prevented lipid peroxidation and the reduction in antioxidant enzyme activities and GSH levels against PTZ-induced oxidative stress in a dose dependent manner. The present data indicates that PTZ at a convulsive dose induces an oxidative stress response by depleting the antioxidant defense systems and increasing lipid peroxidation in the erythrocytes, liver and brain of rats. Ghrelin pretreatment diminished oxidative stress and prevented the decrease in antioxidant enzyme activities, and thus may reduce neuronal death in the brain during seizures. However, further studies are needed in order to confirm our hypothesis.
Keywords: Ghrelin; Epilepsy; Brain; Liver; Pentylenetetrazole; Oxidative stress;
Effects of estradiol on regulation of corticotropin-releasing factor gene and interleukin-6 production via estrogen receptor type β in hypothalamic 4B cells by Eriko Ogura; Kazunori Kageyama; Komaki Hanada; John Kasckow; Toshihiro Suda (456-464).
Corticotropin-releasing factor (CRF) is produced in the hypothalamic paraventricular nucleus (PVN) in response to stress and stimulates the release of adrenocorticotropic hormone in the corticotrophs. Estrogens acting centrally are able to modulate the stress responses. In fact, direct estrogenic regulation of CRF gene expression has been demonstrated in various tissues. However, the mechanisms responsible for the actions of estrogens on CRF regulation in the PVN remain undetermined. We investigated whether estradiol (E2) contributes to the regulation of CRF gene and promoter activity in hypothalamic 4B cells. Furthermore, the involvement of E2 in the regulation of interleukin (IL)-6 and its role in hypothalamic 4B cells was explored. We demonstrated the dominant expression of estrogen receptor type β (ERβ) and found that a physiologically relevant dose of E2 and an ERβ agonist stimulated CRF gene transcription in hypothalamic 4B cells. E2 stimulated IL-6 transcriptional activity via ERβ, and subsequently the levels of IL-6 mRNA and protein. We also found that treatment with IL-6 significantly reduced cell viability. Thus, these data suggest the important effects of E2 on the regulation of CRF gene and IL-6 production via ERβ in hypothalamic 4B cells.
Keywords: Estradiol; Corticotropin-releasing factor; Interleukin-6; Gene transcription; Hypothalamus; Stress;
Synergistic activation of the human adrenomedullin gene promoter by Sp1 and AP-2α by Yan Zhang; Yin Li; Shigeki Shibahara; Kazuhiro Takahashi (465-472).
Adrenomedullin (AM) is a potent vasodilator peptide, which is ubiquitously expressed and has various biological actions, such as proliferative action and anti-oxidative stress action. AM expression is induced by various stresses, such as hypoxia and inflammatory cytokines, and during cell differentiation. The human AM gene promoter region (−70/−29) contains binding sites for stimulatory protein 1 (Sp1) and activator protein-2α (AP-2α), and has been shown to be important for the AM gene expression during cell differentiation to macrophages or adipocytes. We here show that Sp1 and AP-2α synergistically activate the AM gene promoter. Co-transfection of the reporter plasmid containing the AM promoter region (−103/−29) with Sp1 and AP-2α expression plasmids showed that Sp1 and AP-2α synergistically increased the promoter activity in HeLa cells. Sp1 or AP-2α alone caused only small increases in the promoter activity. EMSA showed that Sp1 bound to the promoter region (−70/−29), whereas AP-2α bound to a more upstream promoter region (−103/−71). Thus, the synergistic activation of the human AM gene promoter by Sp1 and AP-2α may be mediated by the binding of Sp1 to the promoter region (−70/−29) and the interaction with AP-2α, which binds to the promoter region (−103/−71).
Keywords: Adrenomedullin; AP-2; Transcription; Sp1;
A novel cryptic peptide derived from the rat neuropeptide FF precursor reverses antinociception and conditioned place preference induced by morphine by Tomasz Dylag; Agnieszka Pachuta; Hana Raoof; Jolanta Kotlinska; Jerzy Silberring (473-478).
Neuropeptide FF (NPFF) precursors from different species contain at least three known neuropeptides, i.e. FF (FLFQPQRF-NH2), AF (AGEGLSSPFWSLAAPQR-NH2) and SF (SLAAPQRF-NH2). We demonstrate that the rat NPFF precursor contains another bioactive sequence, NAWGPWSKEQLSPQA, spanning between positions 85 and 99. Synthetic NPFF precursor (85–99) (10 and 20 nmol, i.c.v.) blocked the expression of conditioned place preference induced by morphine (5 mg/kg, s.c.). This peptide alone (10 and 20 nmol, i.c.v.) had no influence on the baseline latency of a nociceptive reaction but reversed the antinociceptive activity of morphine (5 mg/kg, s.c.) in the tail-immersion test in rats. These data suggest the existence of a novel bioactive cryptic peptide within an already known NPFF precursor.
Keywords: NPFF precursor (85–99); Morphine; Pain; Conditioned place preference; Processing;
Generation of PEGylated VPAC1-selective antagonists that inhibit proliferation of a lung cancer cell line by Clark Q. Pan; Sarah Hamren; Steve Roczniak; Irene Tom; Mary DeRome (479-486).
Vasoactive intestinal peptide (VIP) binds to two receptors, VPAC1 and VPAC2. Non-selective VIP antagonists have been shown to inhibit human cancer cell proliferation and reduce tumor growth in mice. Many human cancers over-express VPAC1 but not VPAC2. We show that VPAC1-selective antagonists can inhibit human cancer cell proliferation and identify five positions in the VPAC1-selective antagonist PG 97-269 that may be responsible for VPAC1 selectivity. Position 16 appears to be particularly critical for selectivity, as demonstrated in the replacement of Arg16 of PG 97-269 with the native VIP amino acid; this single change results in greatly reduced VPAC1 binding and selectivity. Finally, we show that site-specific conjugation with a 22 kDa polyethylene glycol (PEG) at the C-terminus of VPAC1-selective antagonists further improves VPAC1-selective binding and has minimal effect on antagonistic activity. Our studies have further solidified VPAC1 as a cancer target and offer the possibility of generating highly potent VPAC1-selective antagonists with minimal number of mutations to reduce the risk of immunogenicity and potentially prolonged duration of action to allow more efficient treatment regimen.
Keywords: VPAC; VIP; PG 97-269; JV-1-53;
A single circuit-resistance exercise has no effect on plasma obestatin levels in female college students by Abbass Ghanbari-Niaki; Marziyeh Saghebjoo; Fatemeh Rahbarizadeh; Mehdi Hedayati; Hamid Rajabi (487-490).
Obestatin is a 23 amino acid peptide recently isolated from rat stomach that is encoded by the same gene as ghrelin. Obestatin has opposite action to ghrelin on food intake and plays a role in energy balance. The purpose of the present study was to investigate the effect of a circuit-resistance exercise (9 exercises, 25 s per exercise, at 40, 60, 80% of 1RM) at different intensities on plasma obestatin and growth hormone (GH). Twenty volunteer females were randomly divided into four; 40, 60, 80%, combined (40 + 80 + 80%) loads groups (COL). Blood samples were collected before and immediately following the exercise protocol. Changes in plasma obestatin levels were not significant within and between groups. Plasma GH concentrations were significantly higher in high and COL groups, respectively. The data indicate that although circuit-resistance exercise resulted in a significant change in GH levels, it had no effect on plasma obestatin levels.
Cholinergic regulation of neuropeptide Y synthesis and release in human neuroblastoma cells by Elena Dozio; Massimiliano Ruscica; Daniel Feltrin; Marcella Motta; Paolo Magni (491-495).
The biosynthesis and release of neuropeptide Y (NPY) is regulated by several factors. Here, the effect of the muscarinic agonist carbachol on NPY biosynthesis and release was analyzed utilizing the SH-SY5Y human neuroblastoma cell line. We observed that: (a) carbachol moderately increased the post-translational cleavage of proNPY to NPY; (b) carbachol treatment stimulated NPY accumulation into the medium in a time- and dose-related manner; (c) protein kinase C activation is involved in carbachol-mediated NPY synthesis/release (>6 h). In conclusion, the present observations support the hypothesis that muscarinic receptor activation regulates the biosynthesis and secretion of NPY.