Home About us Contact
|
Home About us Contact | ||
|
|
||
Basal Levels (basal + level)
Kinds of Basal Levels Selected AbstractsGestational diabetes affects platelet behaviour through modified oxidative radical metabolismDIABETIC MEDICINE, Issue 1 2004L. Mazzanti Abstract Aims Patients with Type 1 and Type 2 diabetes mellitus show altered platelet function including decreased nitric oxide synthase (NOS) activity and increased peroxynitrite production. Gestational diabetes mellitus (GDM) is a clinical condition which is ideal for evaluating short-term effects of impaired glucose metabolism, ruling out the possibility that the platelet abnormalities are a consequence of diabetic complications. The aim of the present work was to study NO metabolism in platelets from pregnant women with GDM. The production of peroxides was also studied as it is strongly involved in peroxynitrite formation. Methods Platelet NOS activity and peroxynitrite production, levels of hydroperoxides and thiobarbituric acid reactive substances (TBARS) in platelet membranes in the basal state and after in vitro peroxidative stress with phenylhydrazine were determined in 40 pregnant women with GDM, 40 healthy pregnant women (pregnant controls) of comparable age and gestational age, and 15 healthy non-pregnant women (controls). Results NOS activity was significantly increased in both groups of pregnant women compared with non-pregnant ones, and in GDM women compared with pregnant controls. Production of peroxynitrite was higher in GDM women than in pregnant controls, who also had significantly reduced production compared with non-pregnant women. Basal levels of peroxidation of the platelet membranes evaluated either by hydroperoxide content and TBARS levels or the susceptibility to peroxidation were increased in GDM patients in comparison with both control groups. Conclusions We have shown a modification in platelet NO and peroxynitrite production and an increase in platelet indicators of oxidative stress in GDM women compared with healthy pregnant women which might be at the basis of a cellular dysfunction. [source] Expression of matrix metalloproteinase-1, matrix metalloproteinase-2 and extracellular metalloproteinase inducer in human periodontal ligament cells stimulated with interleukin-1betaJOURNAL OF PERIODONTAL RESEARCH, Issue 6 2009J. Xiang Background and Objectives: Matrix metalloproteinases (MMPs), produced by both infiltrating and resident cells of the periodontium, play important roles in physiologic and pathologic events. Both interleukin-1beta and extracellular MMP inducer can stimulate the expression of MMPs, which in turn leads to breakdown of the periodontium. However, it is currently unknown whether interleukin-1beta up-regulates MMPs through stimulating the expression of extracellular MMP inducer. The aims of this study were to investigate the effect of interleukin-1beta on the expression of MMP-1, MMP-2 and extracellular MMP inducer in human periodontal ligament cells and to evaluate whether the regulation of MMP-1 and MMP-2 by this cytokine occurred through an effect on extracellular MMP inducer expression. Material and Methods: Cultured human periodontal ligament cells were treated with varying concentrations (0.01,10 ng/mL) of interleukin-1beta at for 6, 12 and 24 h. Reverse transcription,polymerase chain reaction, enzyme-linked immunosorbent assay, gelatin zymography and western blotting were performed to measure the mRNA and protein levels of MMP-1, MMP-2 and extracellular MMP inducer. Results: Basal levels of mRNA and protein for MMP-1, MMP-2 and extracellular MMP inducer were detected in untreated human periodontal ligament cells. Interleukin-1beta significantly up-regulated the expression of MMP-1 and MMP-2 mRNA and protein (p < 0.05); however, the levels of mRNA and protein for extracellular MMP inducer were not significantly different (p > 0.05). In the culture medium, the concentration of MMP-1 was also increased significantly, but the concentration of MMP-1 was not related to the concentration of extracellular MMP inducer (R2 = 0.2538, p > 0.05). Conclusion: Interleukin-1beta up-regulated the levels of MMP-1 and MMP-2, but it did not alter the expression of extracellular MMP inducer. Expression of MMP-1 and MMP-2 might be elevated by interleukin-1beta and extracellular MMP inducer via two different signal pathways. [source] ORIGINAL RESEARCH,BASIC SCIENCE: Acute and Repeated Flibanserin Administration in Female Rats Modulates Monoamines Differentially Across Brain Areas: A Microdialysis StudyTHE JOURNAL OF SEXUAL MEDICINE, Issue 5 2010Kelly A. Allers PhD ABSTRACT Introduction., Hypoactive sexual desire disorder (HSDD) is defined as persistent lack of sexual fantasies or desire marked by distress. With a prevalence of 10% it is the most common form of female sexual dysfunction. Recently, the serotonin-1A (5-HT1A) receptor agonist and the serotonin-2A (5-HT2A) receptor antagonist flibanserin were shown to be safe and efficacious in premenopausal women suffering from HSDD in phase III clinical trials. Aim., The current study aims to assess the effect of flibanserin on neurotransmitters serotonin (5-HT), norepinephrine (NE), dopamine (DA), glutamate, and ,-aminobutyric acid (GABA) in brain areas associated with sexual behavior. Methods., Flibanserin was administered to female Wistar rats (280,350 g). Microdialysis probes were stereotactically inserted into the mPFC, NAC, or MPOA, under isoflurane anesthesia. The extracellular levels of neurotransmitters were assessed in freely moving animals, 24 hours after the surgery. Main Outcome Measures., Dialysate levels of DA, NE, and serotonin from medial prefrontal cortex (mPFC), nucleus accumbens (NAC), and hypothalamic medial preoptic area (MPOA) from female rats. Results., Acute flibanserin administration decreased 5-HT and increased NE levels in all tested areas. DA was increased in mPFC and MPOA, but not in the NAC. Basal levels of NE in mPFC and NAC and of DA in mPFC were increased upon repeated flibanserin administration, when compared to vehicle-treated animals. The basal levels of 5-HT were not altered by repeated flibanserin administration, but basal DA and NE levels were increased in the mPFC. Glutamate and GABA levels remained unchanged following either repeated or acute flibanserin treatment. Conclusions., Systemic administration of flibanserin to female rats differentially affects the monoamine systems of the brain. This may be the mechanistic underpinning of flibanserin's therapeutic efficacy in HSDD, as sexual behavior is controlled by an intricate interplay between stimulatory (catecholaminergic) and inhibitory (serotonergic) systems. Allers KA, Dremencov E, Ceci A, Flik G, Ferger B, Cremers TIFH, Ittrich C, and Sommer B. Acute and repeated flibanserin administration in female rats modulates monoamines differentially across brain areas: A microdialysis study. J Sex Med 2010;7:1757,1767. [source] Oestradiol stimulates prolactin secretion in women through oestrogen receptorsCLINICAL ENDOCRINOLOGY, Issue 5 2006A. Garas Summary Objective, To examine the effects of clomiphene and raloxifene on basal and GnRH-induced prolactin (PRL) secretion in postmenopausal women. Design, Postmenopausal women participated in two experimental procedures a month apart. In one experiment they received raloxifene (180 mg/day) (R-Exp) and in the other clomiphene (150 mg/day) (Cl-Exp). In Group 1, the women (n = 8) received raloxifene or clomiphene for 30 days plus oestradiol via skin patches (100 µg/24 h) for the last 10 days. In Group 2, the women (n = 8) received oestradiol for 30 days plus raloxifene (R-Exp) or clomiphene (Cl-Exp) for the last 10 days. The pituitary response to GnRH (100 µg i.v.) was investigated in all women on days 0, 10, 20 and 30 of each experiment. Patients, The study included 16 healthy postmenopausal volunteer women aged 56,60 years. Measurements, Basal levels of PRL and the area under the curve (AUC) of ,PRL response to GnRH were calculated. Results, In Group 1, basal levels of PRL and the area under the curve (AUC) of PRL response to GnRH did not change significantly in both experiments. In Group 2, during both experiments basal levels of PRL and the AUC of PRL increased significantly on days 10 (P < 0·05) and 20 (P < 0·05) as compared to day 0 and then they decreased significantly on day 30 as compared to day 20 (P < 0·05). Conclusions, Our study demonstrates for the first time that raloxifene and clomiphene affect the secretion of PRL in postmenopausal women in a similar manner. It is suggested that oestradiol stimulates the secretion of PRL in women by acting through oestrogen receptors. [source] Chronic inhibition of standing behaviour alters baroreceptor reflex function in ratsACTA PHYSIOLOGICA, Issue 3 2009H. Waki Abstract Aim:, To investigate whether daily orthostatic stress during development is an important factor affecting arterial baroreceptor reflex function, we examined the effect of chronic inhibition of upright standing behaviour on the baroreceptor reflex function in rats. Methods:, Upright standing behaviour was chronically inhibited during the developmental period between 3 and 8 weeks of age in Sprague,Dawley rats and heart rate (HR) and aortic nerve activity in response to increased and decreased mean arterial pressure (MAP) was measured after the treatment period. Results:, The baroreceptor cardiac gain in the rats grown without standing behaviour was significantly lower than the control rats grown in a normal commercial cage (1.0 ± 0.1 beats min,1 mmHg,1 vs. 1.6 ± 0.2 beatsmin,1 mmHg,1, P < 0.05). The range of HR change in the MAP,HR functional curve was also lowered by chronic inhibition of orthostatic behaviour (56.2 ± 5.9 beats min,1) compared with that of the control rats (76.8 ± 6.9 beats min,1, P < 0.05). However the aortic afferent function remained normal after the treatment period, indicating that the attenuated baroreceptor reflex function may be due to other mechanisms involving functional alterations in the cardiovascular centres, efferents and/or peripheral organs. Body weight and adrenal weight were not affected by the inhibition of orthostatic behaviour, suggesting that the animals were not exposed to specific stress by this treatment. Conclusion:, These results indicate that active haemodynamic changes induced by orthostatic behaviour are an important factor for setting the basal level of reflex function during development. Moreover, our experimental model may be useful for studying mechanisms of attenuated baroreceptor reflex observed after exposure to a chronic inactive condition. [source] Losartan decreases vasopressin-mediated cAMP accumulation in the thick ascending limb of the loop of Henle in rats with congestive heart failureACTA PHYSIOLOGICA, Issue 4 2007M. Torp Abstract Introduction:, Vasopressin (AVP) stimulates sodium reabsorption and Na,K,2Cl-cotransporter (NKCC2) protein level in the thick ascending limb (TAL) of Henle's loop in rats. Rats with congestive heart failure (CHF) have increased protein level of NKCC2, which can be normalized by angiotensin II receptor type-1 (AT1) blockade with losartan. Aim:, In this study, we investigated whether CHF rats displayed changes in AVP stimulated cAMP formation in the TAL and examined the role of AT1 receptor blockade on this system. Method:, CHF was induced by ligation of the left anterior descending coronary artery (LAD). SHAM-operated rats were used as controls. Half of the rats were treated with losartan (10 mg kg day,1 i.p.). Results:, CHF rats were characterized by increased left ventricular end diastolic pressure. Measurement of cAMP in isolated outer medullary TAL showed that both basal and AVP (10,6 m) stimulated cAMP levels were significantly increased in CHF rats (25.52 ± 4.49 pmol cAMP ,g,1 protein, P < 0.05) compared to Sham rats (8.13 ± 1.14 pmol cAMP ,g,1 protein), P < 0.05). Losartan significantly reduced the basal level of cAMP in CHF rats (CHF: 12.56 ± 1.93 fmol ,g,1 protein vs. Los-CHF: 7.49 ± 1.08, P < 0.05), but not in Sham rats (SHAM: 4.66 ± 0.59 vs. Los-SHAM: 4.75 ± 0.71). AVP-mediated cAMP accumulation was absent in both groups treated with losartan (Los-SHAM: 4.75 ± 0.71 and Los-CHF: 7.49 ± 1.08). Conclusion:, The results indicate that the increased NKCC2 protein level in the mTAL from CHF rats is associated with increased cAMP accumulation in this segment. Furthermore, the finding that AT1 receptor blockade prevents AVP-mediated cAMP accumulation in both SHAM and CHF rats suggests an interaction between angiotensin II and AVP in regulation of mTAL Na reabsorption. [source] Effects of short-term metformin treatment on insulin sensitivity of blood glucose and free fatty acidsDIABETES OBESITY & METABOLISM, Issue 1 2004S. Iannello Aim:, Based on the known effect of metformin (MET) in improving insulin sensitivity in type 2 diabetes, with the scope to focus the effects on glycaemic and free fatty acids (FFA) levels, we studied the effects of a short-term treatment with this drug in obese subjects and obese patients with diabetes or family history of diabetes (FHD). We used a method to allow us to evaluate the possible difference of insulin sensibility with regard to the insulin action on glycaemia and blood FFA, both in the basal state and during oral glucose tolerance test (OGTT). Methods:, Insulin sensitivity was investigated before and after MET treatment (850 mg bid for 10 days) in seven obese subjects with normal glucose tolerance and without FHD and 13 obese patients with diabetes (n = 7) or FHD (n = 6). By using specifically designed formulae, we calculated four insulin-sensitivity indices (ISI) from basal level (b) and area values (a) (during OGTT) of insulinaemia, glycaemia (gly) or FFA (ffa), namely: ISI (gly)-b, ISI (gly)-a, ISI (ffa)-b and ISI (ffa)-a. Results:, In patients with diabetes or FHD, MET improved ISI (gly)-b (0.79 ± 0.06 vs. 0.59 ± 0.07, p < 0.001) and ISI (gly)-a (0.69 ± 0.09 vs. 0.51 ± 0.07, p < 0.05), whereas only minor changes occurred for ISI (ffa)-b and ISI (ffa)-a. In contrast, in simple obese subjects, MET induced further deterioration of both ISI (gly)-a (0.47 ± 0.07 vs. 0.64 ± 0.10, p < 0.01) and ISI (ffa)-a (0.43 ± 0.07 vs. 0.55 ± 0.08, p < 0.05). Fasting level and total area of lactate were high in the obese patients and were not affected by MET. A statistically significant increase (p < 0.01), however, was observed for the ,decremental' area of lactate in obese subjects with diabetes or FHD, which might probably contribute to the reduction of insulin resistance induced by the drug in these patients. Conclusions:, Although the low number of subjects studied precludes absolute conclusions, data would suggest that MET improved ISI towards glucose but not towards FFA, in the diabetic and ,prediabetic' obese patients, whereas worsened it in the obese subjects without FHD. Therefore, the effects of MET would not be secondary to changes of FFA but rather to a primary action of MET on glucose metabolism. Thus, utilization of MET to treat the insulin resistance in obesity is indicated only in the presence of alterations of glucose metabolism or FHD. [source] Individual differences in the effects of chronic prazosin hydrochloride treatment on hippocampal mineralocorticoid and glucocorticoid receptorsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2007Mohamed Kabbaj Abstract The aim of this study was to investigate the noradrenergic regulation of mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) in high responder (HR) and low responder (LR) male rats, an animal model of individual differences in hypothalamo-pituitary-adrenal axis activity and vulnerability to drugs of abuse. The effects of a chronic treatment with the noradrenergic ,1 antagonist (1-[4-amino-6,7-dimethoxy-2-quinazolinyl]-4-[2-furanylcarbonyl] piperazine) hydrochloride (prazosin) (0.5 mg/kg, i.p., 35 days) were assessed on stress-induced corticosterone (CORT) secretion and on hippocampal MRs and GRs in adrenally intact rats. In order to ascertain whether the effects of chronic prazosin treatment on hippocampal MRs and GRs were direct or indirect, through prazosin-induced CORT secretion, we also assessed the effects of the same treatment on adrenalectomized rats with CORT substitutive therapy. When compared with LR rats, HR rats exhibited a delayed return to the basal level of CORT following acute restraint stress; this was associated with a lower binding of MRs and GRs in HR rats than in LR rats. Chronic prazosin treatment had no effect in HR animals but markedly reduced hippocampal MRs and GRs, and increased stress-induced CORT secretion in LR rats. In LR adrenalectomized rats, prazosin reduced hipppocampal MRs but did not change GRs. Our results provide evidence of a differential regulation by noradrenaline of hippocampal MRs and GRs in HR and LR rats. These data could have clinical implications in terms of individual differences in the resistance to antidepressant treatments and individual differences in drug abuse. [source] Nicotine withdrawal suppresses nicotinic modulation of long-term potentiation induction in the hippocampal CA1 regionEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2006Yoshihiko Yamazaki Abstract We have previously reported that acute and chronic nicotine exposure lower the threshold for long-term potentiation (LTP) induction in the rat hippocampal CA1 region, and acute application of nicotine in the chronic-nicotine-treated hippocampus further reduces the threshold. However, it is unknown how withdrawal from chronic nicotine exposure affects the induction of LTP. Here, we show that, following nicotine withdrawal, the threshold for LTP induction fluctuates before returning to the basal level and acute nicotine is no longer effective in lowering the threshold at 4 days after withdrawal. Chronic nicotine-induced enhancement of N -methyl- d -aspartate receptor responses slowly diminishes and returns to the control level by 8 days of withdrawal. In 4-day-withdrawn hippocampi, there is functional up-regulation of postsynaptic ,7 nicotinic acetylcholine receptors (nAChRs) on interneurons in the stratum radiatum, whereas the release of ,-aminobutyric acid from their terminals is reduced. In both control and chronic nicotine-exposed hippocampi, acute nicotine depresses monosynaptic inhibitory postsynaptic currents recorded in pyramidal cells but has almost no effect at 4 days of withdrawal. The lack of effect is due, at least in part, to the loss of a presynaptic nicotine effect. These withdrawal-induced changes are accompanied by decreases in normal nicotine-induced enhancement of N -methyl- d -aspartate receptor responses, which may be responsible for the lack of acute nicotine-mediated facilitation of LTP induction in 4-day-withdrawn hippocampi. These withdrawal-induced changes may contribute to the cellular basis of unpleasant withdrawal symptoms and, thus, nicotine dependence. [source] Plk3 inhibits pro-apoptotic activity of p73 through physical interaction and phosphorylationGENES TO CELLS, Issue 7 2009Meixiang Sang Plk3, one of Polo-like kinase family members, is involved in the regulation of cell cycle progression and DNA damage response. In this study, we found that Plk3 inhibits pro-apoptotic activity of p73 through physical interaction and phosphorylation. During cisplatin (CDDP)-mediated apoptosis, Plk3 was transcriptionally induced, whereas its protein level was kept at basal level, suggesting that Plk3 might rapidly degrade in response to CDDP. Immunoprecipitation and in vitro pull-down experiments demonstrated that Plk3 interacts with p73. Luciferase reporter assays and RT-PCR experiments revealed that Plk3 inhibits p73-mediated transcriptional activity. Consistent with these results, pro-apoptotic activity of p73 was blocked by Plk3. Additionally, Plk3 decreased the stability of p73. Intriguingly, kinase-deficient Plk3 failed to inhibit p73 function, indicating that kinase activity of Plk3 is required for Plk3-mediated inhibition of p73. Indeed, in vitro kinase reaction showed that NH2 -terminal portion of p73 is phosphorylated by Plk3. In accordance with these observations, knocking down of Plk3 increased the stability of p73 and promoted CDDP-mediated apoptosis in association with up-regulation of p73. Collectively, our present findings suggest that Plk3 plays an important role in the regulation of cell fate determination in response to DNA damage through the inhibition of p73. [source] Ski co-repressor complexes maintain the basal repressed state of the TGF-, target gene, SMAD7, via HDAC3 and PRMT5GENES TO CELLS, Issue 1 2009Takanori Tabata The products encoded by ski and its related gene, sno, (Ski and Sno) act as transcriptional co-repressors and interact with other co-repressors such as N-CoR/SMRT and mSin3A. Ski and Sno mediate transcriptional repression by various repressors, including Mad, Rb and Gli3. Ski/Sno also suppress transcription induced by multiple activators, such as Smads and c-Myb. In particular, the inhibition of TGF-,-induced transcription by binding to Smads is correlated with the oncogenic activity of Ski and Sno. However, the molecular mechanism by which Ski and Sno mediate transcriptional repression remains unknown. In this study, we report the purification and characterization of Ski complexes. The Ski complexes purified from HeLa cells contained histone deacetylase 3 (HDAC3) and protein arginine methyltransferase 5 (PRMT5), in addition to multiple Smad proteins (Smad2, Smad3 and Smad4). Chromatin immunoprecipitation assays indicated that these components of the Ski complexes were localized on the SMAD7 gene promoter, which is the TGF-, target gene, in TGF-,-untreated HepG2 cells. Knockdown of these components using siRNA led to up-regulation of SMAD7 mRNA. These results indicate that Ski complexes serve to maintain a TGF-,-responsive promoter at a repressed basal level via the activities of histone deacetylase and histone arginine methyltransferase. [source] Pancreatic response of rats fed genetically modified soybeanJOURNAL OF APPLIED TOXICOLOGY, Issue 2 2008Javier A. Magaña-Gómez Abstract Mice fed genetically modified (GM) soybean were not affected in nutritional performance, but pancreatic microscopic features were disturbed. The mechanisms for these contradictory findings are unknown. This study analysed the histology of acinar pancreatic cells and the expression of pancreatitis-associated protein (PAP) and trypsinogen mRNA in rats fed GM soy protein. Two bioassays were run, each one with 34 Wistar rats distributed into two groups fed with non-GM or GM-soy protein (18% protein) for 0, 1, 3, 5, 15 and 30 days. Nutritional evaluation, plasma amylase levels, pancreatic histological analysis and quantification of PAP and trypsinogen mRNAs levels using quantitative real-time RT-PCR were done. No differences in nutritional performance among rats fed non-GM and GM diets were found. The GM, but not the non-GM, diet induced zymogen-granule depletion after 15 days feeding, returning to normal levels after 30 days (P < 0.05). Acinar disorganization started as early as 5 days after initiation of the GM diet and it recovered after 30 days. Levels of PAP mRNA significantly increased in the GM diet between day 1 and day 3 and decreased to the basal level by day 15. Trypsinogen mRNA peaked at two different times; at day 1 and at day 15, decreasing to basal levels after 30 days. Plasma amylase levels remained unchanged at all times. This indicates that GM soy protein intake affected pancreas function, evidenced by the early acute PAP mRNA increased levels and pancreas cellular changes followed by recuperation of acinar cells after 30 days. Copyright © 2007 John Wiley & Sons, Ltd. [source] Immunotoxicity of acute acephate exposure in control or IL-1-challenged rats: correlation between the immune cell composition and corticosteroid concentration in bloodJOURNAL OF APPLIED TOXICOLOGY, Issue 5 2002Ashok K. Singh Abstract Corticosterone concentration and the immune cell composition were measured in rats exposed by intraperitoneal (i.p.) injection to different doses (10,500 mg kg,1) of acephate (Ace) and 250 µg kg,1 of interleukin 1 (IL-1), either alone or in combination. Two different combination protocols were used: IL-1 and Ace were administered simultaneously; and IL-1 was injected 60 min after Ace administration (sequential exposure). Ace, in a dose- and time-dependent manner, inhibited blood and brain acetylcholinesterase (AChE) activities, increased blood corticosterone concentrations, suppressed blood CD4, CD8, B cell and monocyte contents and increased blood neutrophil counts. The Ace-induced changes lasted for up to 24 h after Ace exposure. Interleukin 1 increased blood corticosterone concentrations without affecting blood or brain AChE activities. The IL-1-induced corticosterone concentration returned to the basal level within 3,10 h after IL-1 exposure. The CD4, CD8, B cell and monocyte counts increased significantly at 10 min after IL-1 exposure. The cell counts decreased gradually thereafter and returned to the basal level within 30 min after IL-1 exposure. Simultaneous exposure of rats to Ace and IL-1 partially suppressed the IL-1-induced increase in the immune cell counts and decreased the immune cell numbers below the basal values. Sequential injection of Ace and IL-1 blocked the IL-1-induced increase in the immune cell numbers. Thus, Ace exposure would impair the normal distribution of immune cells and deregulate the IL-1 response in rats. This study therefore suggests that Ace would suppress the immune cell numbers in blood, thus decreasing an organism's immunity. Ace exposure occurring concurrent with injury would augment the acute-phase response, which would augment the toxic effects of IL-1 and other cytokines, and Ace exposure occurring prior to the injury would suppress or abolish the initial stimulatory effects of IL-1, which would decrease an organism's ability to combat infection or injury. Copyright © 2002 John Wiley & Sons, Ltd. [source] Craniosynostosis-Associated Gene Nell-1 Is Regulated by Runx2,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2007Thien Truong Abstract We studied the transcriptional regulation of NELL-1, a craniosynostosis-related gene. We identitifed three OSE2 elements in the NELL-1 promoter that are directly bound and transactivated by Runx2. Forced expression of Runx2 induces NELL-1 expression in rat calvarial cells. Introduction: We previously reported the upregulation of NELL-1 in human craniosynostosis and the overexpression of Nell-1 in transgenic animals that induced premature suture closure associated with increased osteoblast differentiation. To study the transcriptional regulation of NELL-1, we analyzed the 5, flanking region of the human NELL-1 gene. We identified three osteoblast specific binding elements 2 (OSE2) sites (A, B, and C) within 2.2 kb upstream of the transcription start site and further studied the functionality of these sites. Materials and Methods: An area of 2.2 kb and a truncated 325 bp, which lacked the three OSE sites, were cloned into a luciferase reporter gene, and co-transfected with Runx2 expression plasmid. The three OSE2 sites were individually mutated and co-transfected with Runx2 expression plasmid into Saos2 cells. Gel shifts and supershifts with Runx2 antibodies were used to determine specific binding to OSE2 sites. CHIP assays were used to study in vivo binding of Runx2 to the Nell-1 promoter. Runx2 expression plasmid was transfected into wildtype and Runx2,/, calvarial cells. Nell-1, osteocalcin, and Runx2 expression levels were measured using RT-PCR. Results: Addition of Runx2 dose-dependently increased the luciferase activity in the human NELL-1 promoter-luciferase p2213. The p325 truncated NELL-1 construct showed significantly lower basal level of activity. Nuclear extract from Saos2 cells formed complexes with site A, B, and C probes and were supershifted with Runx2 antibody. Mutation of sites A, B, and C significantly decreased basal promoter activity. Furthermore, mutation of sites B and C had a blunted response to Runx2, whereas mutation of site A had a lesser effect. Runx2 bound to NELL-1 promoter in vivo. Transfection of Runx2 in rat osteoblasts upregulated Nell-1 and Ocn expression, and in Runx2 null calvarial cells, both Nell-1 and Ocn expression were rescued. Conclusions: Runx2 directly binds to the OSE2 elements and transactivates the human NELL-1 promoter. These results suggest that Nell-1 is likely a downstream target of Runx2. These findings may also extend our understanding of the molecular mechanisms governing the pathogenesis of craniosynostosis. [source] Calcineurin Inhibition Ameliorates Structural, Contractile, and Electrophysiologic Consequences of Postinfarction RemodelingJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 9 2001LILI DENG M.S. Calcineurin Inhibition and Postinfarction Remodeling.Introduction: After myocardial infarction (MI), the heart undergoes an adaptive remodeling process characterized by hypertrophy of the noninfarcted myocardium. Calcineurin, a Ca2+, calmodulin-regulated phosphatase, has been shown to participate in hypertrophic signal transduction. Methods and Results: We investigated the effects of calcineurin inhibition by cyclosporin A on key structural, contractile, and electrophysiologic alterations of post-MI remodeling. Male Sprague-Dawley rats were divided into four groups: (1) sham-operated; (2) sham + cyclosporin A; (3) post-MI (left anterior descending coronary artery ligation); and (4) MI + cyclosporin A. Cyclosporin A (25 mg/kg/day) was initiated 2 days before surgery and continued for 30 days. Hypertrophy was evaluated by echocardiography and by changes in membrane capacitance of isolated myocytes from noninfarcted left ventricle (LV). The effects of cyclosporin A on hemodynamics and cardiac dimensions were investigated, and changes in diastolic function were correlated with changes in protein phosphatase 1 activity and the basal level of phosphorylated phospholamban. The effects of cyclosporin A on Kv4.2/Kv4.3 genes expression and transient outward K + current (Ito) density also were evaluated. One of 12 rats in the post-MI group and 2 of 12 rats in the post-MI + cyclosporin A group died within 48 hours after MI. There were no late deaths in either MI group. There was no evidence of heart failure (lung congestion and/or pleural effusion) in the two groups 4 weeks post-MI. Calcineurin phosphatase activity increased 1.9-fold in post-MI remodeled LV myocardium, and cyclosporin A administration resulted in an 86% decrease in activity. There were statistically significant decreases of LV end-diastolic pressure, LV end-diastolic diameter, and LV relative wall thickness in the post-MI + cyclosporin A group compared with the post-MI group. On the other hand, there was no significant difference in LV end-systolic diameter or peak rate of LV pressure increase between the two post-MI groups. Protein phosphatase 1 activity was elevated by 36% in the post-MI group compared with sham, and this correlated with a 79% decrease in basal level of p16, phospholamban. In the post-MI + cyclosporin A group, the increase in protein phosphatase 1 activity was much less (18% vs 36%; P < 0.05), and the decrease in basal level of p16-phospholamban was markedly ameliorated (20% vs 79%; P < 0.01). The decreases in mRNA levels of Kv4.2 and Kv4.3 and Ito density in the LV of the post-MI + cyclosporin A group were significantly less compared with the post-MI group. Conclusion: Our results show that calcineurin inhibition by cyclosporin A partially ameliorated post-MI remodeled hypertrophy, diastolic dysfunction, decrease in basal level of phosphorylated phospholamban, down-regulation of key K + genes expression, and decrease of K + current, with no adverse effects on systolic function or mortality in the first 4 weeks after MI. [source] Corticosterone induces steroidogenic lesion in cultured adult rat leydig cells by reducing the expression of star protein and steroidogenic enzymesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2008Srinivasan Rengarajan Abstract The present study was designed to investigate the dose-dependent direct effect of corticosterone on adult rat Leydig cell steroidogenesis in vitro. Leydig cells were isolated from the testis of normal adult male albino rats, purified on discontinuous Percoll gradient and plated in culture plates/flasks overnight at 34°C in a CO2 incubator under 95% air and 5% CO2 using DME/F12 medium containing 1% fetal bovine serum. After the attachment of cells, serum-containing medium was removed and cells were exposed to different doses (0, 50, 100, 200, 400, and 800 nM) of corticosterone using serum-free fresh medium for 24 h at 34°C. At the end of exposure period, cells were utilized for assessment of the activities and mRNA expression of steroidogenic enzymes (cytochrome P450 side chain cleavage enzyme, 3,-hydroxysteroid dehydrogenase, 17,-hydroxysteroid dehydrogenase, and cytochrome P450 aromatase) and steroidogenic acute regulatory protein gene expression. Testosterone and estradiol production were also quantified. Activities of cytochrome P450 side chain cleavage enzyme, 3,- and 17,-hydroxysteroid dehydrogenases were declined significantly in a dose-dependent manner after corticosterone exposure, while their mRNA expression were significantly reduced at higher doses of corticosterone exposure. The activity and mRNA expression of cytochrome P450 aromatase registered a significant increase at 100 nM dose of corticosterone whereas at 200,800 nM doses both the activity as well as the mRNA levels was significantly reduced below the basal level. StAR protein gene expression was significantly inhibited by higher doses of corticosterone employed. At all doses employed, corticosterone significantly reduced the production of testosterone by Leydig cells, while estradiol level registered a significant increase at 50 and 100 nM doses but at higher doses, it registered a significant decrease when compared to basal level. It is concluded from the present in vitro study that the molecular mechanism by which corticosterone reduces the production of Leydig cell testosterone is by reducing the activities and mRNA expression of steroidogenic enzymes and steroidogenic acute regulatory protein. J. Cell. Biochem. 103: 1472,1487, 2008. © 2007 Wiley-Liss, Inc. [source] Differential control of apoptosis by DJ-1 in prostate benign and cancer cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2004Yaacov Hod Abstract DJ-1 is a conserved protein reported to be involved in diverse cellular processes ranging from cellular transformation, control of protein,RNA interaction, oxidative stress response to control of male infertility, among several others. Mutations in the human gene have been shown to be associated with an autosomal recessive, early onset Parkinson's disease (PARK7). The present study examines the control of DJ-1 expression in prostatic benign hyperplasia (BPH-1) and cancer (PC-3) cell lines in which DJ-1 abundance differs significantly. We show that while BPH-1 cells exhibit low basal level of DJ-1 expression, stress-inducing agents such as H2O2 and mitomycin C markedly increase the intracellular level of the polypeptide. In contrast, DJ-1 expression is relatively high in PC-3 cells, and incubation with the same cytotoxic drugs does not modulate further the level of the polypeptide. In correlation with the expression of DJ-1, both cytotoxic agents activate the apoptotic pathway in the prostatic benign cells but not in PC-3 cells, which are resistant to their action. We further demonstrate that incubation of BPH-1 cells with TNF-related-apoptosis-inducing-ligand/Apo-2L (TRAIL) also enhances DJ-1 expression and that TRAIL and H2O2 act additively to stimulate DJ-1 accumulation but synergistically in the activation of the apoptotic pathway. Time-course analysis of DJ-1 stimulation shows that while DJ-1 level increases without significant lag in TRAIL-treated cells, there is a delay in H2O2 -treated cells, and that the increase in DJ-1 abundance precedes the activation of apoptosis. Unexpectedly, over-expression of DJ-1 de-sensitizes BPH-1 cells to the action of apoptotic-inducing agents. However, RNA-interference-mediated silencing of DJ-1 expression results in sensitization of PC-3 cells to TRAIL action. These results are consistent with a model in which DJ-1 is involved in the control of cell death in prostate cell lines. DJ-1 appears to play a differential role between cells expressing a low but inducible level of DJ-1 (e.g., BPH-1 cells) and those expressing a high but constitutive level of the polypeptide (e.g., PC-3 cells). © 2004 Wiley-Liss, Inc. [source] Annulus cells release ATP in response to vibratory loading in vitroJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2003Satoru Yamazaki Abstract Mechanical forces regulate the developmental path and phenotype of a variety of tissues and cultured cells. Vibratory loading as a mechanical stimulus occurs in connective tissues due to energy returned from ground reaction forces, as well as a mechanical input from use of motorized tools and vehicles. Structures in the spine may be particularly at risk when exposed to destructive vibratory stimuli. Cells from many tissues respond to mechanical stimuli, such as fluid flow, by increasing intracellular calcium concentration ([Ca2+]ic) and releasing adenosine 5,-triphosphate (ATP), extracellularly, as a mediator to activate signaling pathways. Therefore, we examined whether ATP is released from rabbit (rAN) and human (hAN) intervertebral disc annulus cells in response to vibratory loading. ATP release from annulus cells by vibratory stimulation as well as in control cells was quantitated using a firefly luciferin-luciferase assay. Cultured hAN and rAN cells had a basal level of extracellular ATP ([ATP]ec) in the range of 1,1.5 nM. Vibratory loading of hAN cells stimulated ATP release, reaching a net maximum [ATP] within 10 min of continuous vibration, and shortly thereafter, [ATP] declined and returned to below baseline level. [ATP] in the supernatant fluid of hAN cells was significantly reduced compared to the control level when the cells received vibration for longer than 15 min. In rAN cells, [ATP] was increased in response to vibratory loading, attaining a level significantly greater than that of the control after 30 min of continuous vibration. Results of the current study show that resting annulus cells secrete ATP and maintain a basal [ATP]ec. Annulus cells may use this nucleotide as a signaling messenger in an autocrine/paracrine fashion in response to vibratory loading. Rapid degradation of ATP to ADP may alternatively modulate cellular responses. It is hypothesized that exposure to repetitive, complex vibration regimens may activate signaling pathways that regulate matrix destruction in the disc. As in tendon cells, ATP may block subsequent responses to load and modulate the vibration response. Rabbit annulus cells were used as a readily obtainable source of cells in development of an animal model for testing effects of vibration on the disc. Human cells obtained from discarded surgical specimens were used to correlate responses of animal to human cells. © 2003 Wiley-Liss, Inc. [source] Development of selective tolerance to interleukin-1, by human chondrocytes in vitro,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2002Greta M. Lee Interleukin-1 induces release of NO and PGE2 and production of matrix degrading enzymes in chondrocytes. In osteoarthritis (OA), IL-1 continually, or episodically, acts on chondrocytes in a paracrine and autocrine manner. Human chondrocytes in chondron pellet culture were treated chronically (up to 14 days) with IL-1,. Chondrons from OA articular cartilage were cultured for 3 weeks before treatment with IL-1, (0.05,10 ng/ml) for an additional 2 weeks. Spontaneous release of NO and IL-1, declined over the pretreatment period. In response to IL-1, (0.1 ng/ml), NO and PGE2 release was maximal on Day 2 or 3 and then declined to near basal level by Day 14. Synthesis was recovered by addition of 1 ng/ml IL-1, on Day 11. Expression of inducible nitric oxide synthase (iNOS), detected by immunofluorescence, was elevated on Day 2 and declined through Day 14, which coordinated with the pattern of NO release. On the other hand, IL-1,-induced MMP-13 synthesis was elevated on Day 3, declined on Day 5, and then increased again through Day 14. IL-1, increased glucose consumption and lactate production throughout the treatment. IL-1, stimulated proteoglycan degradation in the early days and inhibited proteoglycan synthesis through Day 14. Chondron pellet cultures from non-OA cartilage released the same amount of NO but produced less PGE2 and MMP-13 in response to IL-1, than OA cultures. Like the OA, IL-1,-induced NO and PGE2 release decreased over time. In conclusion, with prolonged exposure to IL-1,, human chondrocytes develop selective tolerance involving NO and PGE2 release but not MMP-13 production, metabolic activity, or matrix metabolism. © 2002 Wiley-Liss, Inc. [source] Role of protein kinase C-dependent A-kinase anchoring proteins in lysophosphatidic acid-induced cAMP signaling in human diploid fibroblastsAGING CELL, Issue 6 2006Ji-Heon Rhim Summary Previously, we reported that lysophosphatidic acid (LPA)-induced adenosine 3,,5,-cyclic monophosphate (cAMP) production by human diploid fibroblasts depends on the age of the fibroblasts. In this study, we examined the role of A-kinase anchoring proteins (AKAP) in the regulation of LPA-stimulated cAMP production in senescent fibroblasts. We found that levels of protein kinase C (PKC)-dependent AKAPs, such as Gravin and AKAP79, were elevated in senescent cells. Co-immunoprecipitation experiments revealed that Gravin and AKAP79 do not associate with adenylyl cyclase type 2 (AC2) but bind to AC4/6, which interacts with calcium-dependent PKCs ,/, both in young and senescent fibroblasts. When the expression of Gravin and AKAP79 was blocked by small interference RNA transfection, the basal level of cAMP was greatly reduced and the cAMP status after LPA treatment was also reversed. Protein kinase A showed a similar pattern in terms of its basal activity and LPA-dependent modulation. These data suggest that Gravin and to a lesser extent, AKAP79, may play important roles in maintaining the basal AC activity and in coupling the AC systems to inhibitory signals such as Gi, in young cells, and to stimulatory signals such as PKCs in senescent cells. This study also demonstrates that Gravin is especially important for the long-term activation of PKC by LPA in senescent cells. We conclude that LPA-dependent increased level of cAMP in senescent human diploid fibroblasts is associated with increases in Gravin levels resulting in its increased binding with and activation of calcium-dependent PKC ,/, and AC4/6. [source] Basal replication of hepatitis C virus in nude mice harboring human tumorJOURNAL OF MEDICAL VIROLOGY, Issue 3 2002Patrick Labonté Abstract Hepatitis C virus (HCV) can infect and propagate in humans and chimpanzees. Whereas the chimpanzee has been used as an animal model for infection, ethical considerations, conservation, and the prohibitively high cost preclude progress for experimental research on the biology of the virus. The development of a small animal model for HCV infection is thus desirable to facilitate studies on the infectious cycle of the virus and for the evaluation of drugs for the treatment of HCV infections in humans. As an alternative to the chimpanzee model, we have established a model based on ex vivo infection of orthotopically-implanted human hepatocellular carcinoma cells (HCC) in athymic nude mice. The results show that up to 42 days post-infection, HCV RNA was present in the tumor cells as well as in the liver and serum of infected mice. Furthermore, a direct correlation between size of the tumor and the presence of HCV RNA in the liver was observed, which is concordant with the finding that HCV RNA was detectable only in mice harboring human tumor. Immunohistochemistry analysis of infected liver specimens showed cells expressing the HCV encoded NS5B protein. A few mice developed a humoral response against the nonstructural viral proteins, providing further evidence for expression of these proteins during viral infection. In summary, these results suggest that mice harboring orthotopic tumors support a basal level of HCV replication in vivo. J. Med. Virol. 66:312-319, 2002. © 2002 Wiley-Liss, Inc. [source] P,T,t path of the Hercynian low-pressure rocks from the Mandatoriccio complex (Sila Massif, Calabria, Italy): new insights for crustal evolutionJOURNAL OF METAMORPHIC GEOLOGY, Issue 2 2010A. LANGONE Abstract The tectono-metamorphic evolution of the Hercynian intermediate,upper crust outcropping in eastern Sila (Calabria, Italy) has been reconstructed, integrating microstructural analysis, P,T pseudosections, mineral isopleths and geochronological data. The studied rocks belong to a nearly complete crustal section that comprises granulite facies metamorphic rocks at the base and granitoids in the intermediate levels. Clockwise P,T paths have been constrained for metapelites of the basal level of the intermediate,upper crust (Umbriatico area). These rocks show noticeable porphyroblastic textures documenting the progressive change from medium- P metamorphic assemblages (garnet- and staurolite-bearing assemblages) towards low- P/high -T metamorphic assemblages (fibrolite- and cordierite-bearing assemblages). Peak-metamorphic conditions of ,590 °C and 0.35 GPa are estimated by integrating microstructural observations with P,T pseudosections calculated for bulk-rock and reaction-domain compositions. The top level of the intermediate,upper crust (Campana area) recorded only the major heating phase at low- P (,550 °C and 0.25 GPa), as documented by the static growth of biotite spots and of cordierite and andalusite porphyroblasts in metapelites. In situ U,Th,Pb dating of monazite from schists containing low -P/high -T metamorphic assemblages gave a weighted mean U,Pb concordia age of 299 ± 3 Ma, which has been interpreted as the timing of peak metamorphism. In the framework of the whole Hercynian crustal section the peak of low -P/high -T metamorphism in the intermediate-to-upper crust took place concurrently with granulite facies metamorphism in the lower crust and with emplacement of the granitoids in the intermediate levels. In addition, decompression is a distinctive trait of the P,T evolution both in the lower and upper crust. It is proposed that post,collisional extension, together with exhumation, is the most suitable tectonic setting in which magmatic and metamorphic processes can be active simultaneously in different levels of the continental crust. [source] Response of extracelluar zinc in the ventral hippocampus against novelty stressJOURNAL OF NEUROCHEMISTRY, Issue 2 2006Atsushi Takeda Abstract An extensive neuronal activity takes place in the hippocampus during exploratory behavior. However, the role of hippocampal zinc in exploratory behavior is poorly understood. To analyze the response of extracellular zinc in the hippocampus against novelty stress, rats were placed for 50 min in a novel environment once a day for 8 days. Extracellular glutamate in the hippocampus was increased during exploratory behavior on day 1, whereas extracellular zinc was decreased. The same phenomenon was observed during exploratory behavior on day 2 and extracellular zinc had returned to the basal level during exploratory behavior on day 8. To examine the significance of the decrease in extracellular zinc in exploratory activity, exploratory behavior was observed during perfusion with 1 mm CaEDTA, a membrane-impermeable zinc chelator. Locomotor activity in the novel environment was decreased by perfusion with CaEDTA. The decrease in extracellular zinc and the increase in extracellular glutamate in exploratory period were abolished by perfusion with CaEDTA. These results suggest that zinc uptake by hippocampal cells is linked to exploratory activity and is required for the activation of the glutamatergic neurotransmitter system. The zinc uptake may be involved in the response to painless psychological stress or in the cognitive processes. [source] Dependence of Hyperpolarisation-Activated Cyclic Nucleotide-Gated Channel Activity on Basal Cyclic Adenosine Monophosphate Production in Spontaneously Firing GH3 CellsJOURNAL OF NEUROENDOCRINOLOGY, Issue 7 2006K. Kretschmannova Abstract The hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels play a distinct role in the control of membrane excitability in spontaneously active cardiac and neuronal cells. Here, we studied the expression and role of HCN channels in pacemaking activity, Ca2+ signalling, and prolactin secretion in GH3 immortalised pituitary cells. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of mRNA transcripts for HCN2, HCN3 and HCN4 subunits in these cells. A hyperpolarisation of the membrane potential below ,,60 mV elicited a slowly activating voltage-dependent inward current (Ih) in the majority of tested cells, with a half-maximal activation voltage of ,89.9 ± 4.2 mV and with a time constant of 1.4 ± 0.2 s at ,120 mV. The bath application of 1 mM Cs+, a commonly used inorganic blocker of Ih, and 100 µM ZD7288, a specific organic blocker of Ih, inhibited Ih by 90 ± 4.1% and 84.3 ± 1.8%, respectively. Receptor- and nonreceptor-mediated activation of adenylyl and soluble guanylyl cyclase and the addition of a membrane permeable cyclic adenosine monophosphate (cAMP) analogue, 8-Br-cAMP, did not affect Ih. Inhibition of basal adenylyl cyclase activity, but not basal soluble guanylyl cyclase activity, led to a reduction in the peak amplitude and a leftward shift in the activation curve of Ih by 23.7 mV. The inhibition of the current was reversed by stimulation of adenylyl cyclase with forskolin and by the addition of 8-Br-cAMP, but not 8-Br-cGMP. Application of Cs+ had no significant effect on the resting membrane potential or electrical activity, whereas ZD7288 exhibited complex and Ih -independent effects on spontaneous electrical activity, Ca2+ signalling, and prolactin release. These results indicate that HCN channels in GH3 cells are under tonic activation by basal level of cAMP and are not critical for spontaneous firing of action potentials. [source] P2Y receptor-activating nucleotides modulate cellular reactive oxygen species production in dissociated hippocampal astrocytes and neurons in culture independent of parallel cytosolic Ca2+ rise and change in mitochondrial potentialJOURNAL OF NEUROSCIENCE RESEARCH, Issue 15 2007Stefan Kahlert Abstract With mixed cultures of hippocampal astrocytes and neurons, we investigated the influence of nucleotides on cytosolic Ca2+ level, generation of reactive oxygen species (ROS), and mitochondrial potential. We employed ATP and four purine/pyrimidine derivates, which are P2Y receptor subtype-preferring agonists. Stimulation with ATP, a P2Y1/2/4 receptor agonist in rat, caused a large cytosolic Ca2+ increase in astrocytes and a considerably smaller Ca2+ response in neighboring neurons. The P2Y1 receptor antagonist MRS2179 completely blocked the ATP-induced Ca2+ response in astrocytes and neurons. Application of ATP significantly reduced the mitochondrial potential in neurons, which was not inhibited by MRS2179. Interestingly, MRS2179 mediated a mitochondrial depolarization without affecting the cytosolic Ca2+ level. Stimulation with UDP, a P2Y6 receptor agonist; UTP, a P2Y2/4 receptor agonist; 2MeSATP, a P2Y1 receptor agonist; or 2MeSADP, a P2Y1/12/13 receptor agonist, evoked significant Ca2+ responses in astrocytes but small Ca2+ responses in neurons. In astrocytes, there was an inverse relationship between the amplitude of the cytosolic Ca2+ peak and the rate of ROS generation in response to nucleotide application. Activation with UDP resulted in the highest ROS generation that we detected, whereas 2MeSADP and 2MeSATP reduced the ROS generation below the basal level. 2MeSADP and UDP caused mitochondrial depolarization of comparable size. Thus, neither in astrocytes nor in neurons did the degree of mitochondrial depolarization correlate with ROS generation. Nucleotides acting via P2Y receptors can modulate ROS generation of hippocampal neurons without acutely changing the cytosolic Ca2+ level. Thus, ROS might function as a signaling molecule upon nucleotide-induced P2Y receptor activation in brain. © 2007 Wiley-Liss, Inc. [source] Schwann cells express IP prostanoid receptors coupled to an elevation in intracellular cyclic AMP,JOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2007Naser Muja Abstract We have shown previously that prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2) are each produced in an explant model of peripheral nerve injury. We report that IP prostanoid receptor mRNA and protein are present in primary rat Schwann cells. IP prostanoid receptor stimulation using prostacyclin produced an elevation in intracellular cyclic AMP concentration ([cAMP]i) in primary Schwann cells. Peak [cAMP]i was observed between 5,15 min of stimulation followed by a gradual recovery toward basal level. Phosphorylation of cyclic AMP-response element binding protein (CREB) on Ser133 was also detected after IP prostanoid receptor stimulation and CREB phosphorylation was inhibited completely by the protein kinase A inhibitor, H-89. Intracellular calcium levels were not affected by IP prostanoid receptor stimulation. Unlike forskolin, IP prostanoid receptor stimulation did not significantly augment Schwann cell proliferation in response to growth factor treatment. However, IP prostanoid receptor stimulation increased the number of Schwann cells that were able to generate a calcium transient in response to P2 purinergic receptor activation. These findings suggest that signaling via the IP prostanoid receptor may by relevant to Schwann cell biology in vivo. © 2007 Wiley-Liss, Inc. [source] Mild carbon monoxide exposure impairs the developing auditory system of the ratJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2003Douglas S. Webber Abstract The object of this study was to determine if chronic exposure to mild concentrations of CO in air caused changes in the integrity of the inferior colliculus during the most active period of synaptogenesis/auditory development. We examined all subregions of the inferior colliculus (IC) of rats by immunocytochemical approaches after pups were exposed chronically to CO concentrations of, 0, 12.5, 25, and 50 ppm in air starting at Day 8 through 20,22 days of age. Mother-reared pups were compared to the gastrostomy-reared pups with or without CO exposure for basal neural activity, using c-Fos immunoreactivity as a marker. Half the rats were examined at 27 days of age, 5 days after the end of CO exposure, and the other half were examined 50 days later at 75,77 days of age. In the central nucleus of the IC, the number of cells expressing a basal level of c-Fos was decreased significantly in the CO-exposed animals when compared to controls; however, there was little or no difference in the number of cells expressing c-Fos in the other subregions of the IC. We conclude that the central nucleus of the inferior colliculus is affected selectively by mild CO exposure (0.0012% in air) and that this reduction in neuronal activity persists into adulthood. © 2003 Wiley-Liss, Inc. [source] Effect of hyaluronan on osteogenic differentiation of porcine bone marrow stromal cells in vitroJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2008Lijin Zou Abstract Hyaluronan (HA) plays a predominant role in tissue morphogenesis, cell migration, proliferation, and cell differentiation. The aims of the present study were to investigate whether (i) prolonged presence of high concentration (4.0 mg/mL) 800 KDa HA and (ii) pretreatment with HA can modify osteogenic differentiation of pig bone marrow stromal cells (pBMSC). Cell proliferation and mineralization were measured. Expression of differentiation-related genes was evaluated by means of real-time reverse transcription polymerase chain reaction (RT-PCR). HA increased cell proliferation on day 7. HA decreased the basal level of bone-related gene expression and increased the basal level of sox9 marginally during 7-day pretreatment with HA. HA increased calcium deposit on day 21. cbfa1, ALP, and type 1, collagen (Col1) expression was increased when pBMSC were cultivated in osteogenic medium, whereas their expression was decreased in the presence of HA on day 7. On day 14, the addition of HA upregulated cbfa1 and ALP expression compared to osteogenic medium group; there was no significant difference in Col1 expression. At day 21, osteocalcin (OC) expression showed 2.5-fold upregulation over osteogenic medium. These results suggest that exogenous HA stimulates endogenous HA, which together may play a synergetic role in osteogenic differentiation under osteoinducing conditions although gene expression was inhibited at the early stage. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:713,720, 2008 [source] Period 2 Gene Deletion Abolishes ,-Endorphin Neuronal Response to EthanolALCOHOLISM, Issue 9 2010Maria Agapito Background:, Ethanol exposure during early life has been shown to permanently alter the circadian expression of clock regulatory genes and the ,-endorphin precursor proopiomelanocortin (POMC) gene in the hypothalamus. Ethanol also alters the stress- and immune-regulatory functions of ,-endorphin neurons in laboratory rodents. Our aim was to determine whether the circadian clock regulatory Per2 gene modulates the action of ethanol on ,-endorphin neurons in mice. Methods:,Per2 mutant (mPer2Brdml) and wild type (C57BL/6J) mice were used to determine the effect of Per2 mutation on ethanol-regulated ,-endorphin neuronal activity during neonatal period using an in vitro mediobasal hypothalamic (MBH) cell culture model and an in vivo milk formula feeding animal model. The ,-endorphin neuronal activity following acute and chronic ethanol treatments was evaluated by measuring the peptide released from cultured cells or peptide levels in the MBH tissues, using enzyme-linked immunosorbent assay (ELISA). Results:,Per2 mutant mice showed a higher basal level of ,-endorphin release from cultured MBH cells and a moderate increase in the peptide content in the MBH in comparison with control mice. However, unlike wild type mice, Per2 mutant mice showed no stimulatory or inhibitory ,-endorphin-secretory responses to acute and chronic ethanol challenges in vitro. Furthermore, Per2 mutant mice, but not wild type mice, failed to show the stimulatory and inhibitory responses of MBH ,-endorphin levels to acute and chronic ethanol challenges in vivo. Conclusions:, These results suggest for the first time that the Per2 gene may be critically involved in regulating ,-endorphin neuronal function. Furthermore, the data revealed an involvement of the Per2 gene in regulating ,-endorphin neuronal responses to ethanol. [source] In-vitro and in-vivo antioxidant activity of different extracts of the leaves of Clerodendron colebrookianum Walp in the ratJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 12 2003D. Rajlakshmi ABSTRACT The in-vitro antioxidant activities of different concentrations of the water, alcoholic, petroleum ether and ethyl acetate extracts of the dried leaves of Clerodendron colebrookianum Walp, and in-vivo antioxidant activity of the water extract was studied in experimental rat models. The results obtained from in-vitro lipid peroxidation induced by FeSO4 -ascorbate in rat liver homogenate showed a significant inhibition of lipid peroxidation by different extracts of C. colebrookianum leaf. Water extracts at concentrations (w/v) of 1:30, 1:50, 1:200 and 1:1000 showed the strongest inhibitory activity over the other organic extracts, suggesting maximum antioxidant effect. Chronic feeding of the water extract to Wistar albino rats (both sexes, 150,200g) in 1 or 2g kg,1/day doses for 14 days significantly increased the ferric reducing ability of plasma by 19% and 40% on the seventh day, and by 45% and 57% on the fourteenth day of treatment, respectively. Thiobarbituric acid reactive substances (TBARS), as a marker of lipid peroxidation, and some cellular antioxidants (superoxide dismutase, catalase and reduced glutathione) were estimated in heart, liver and kidney. There was a significant reduction in hepatic and renal TBARS with both the doses, without any change in myocardial TBARS. There was no change in the level of antioxidants in heart, liver and kidney, except for the hepatic superoxide dismutase. The findings of this study showed that the leaf extract of C. colebrookianum increased the antioxidant capacity of blood and had an inhibitory effect on the basal level of lipid peroxidation of liver and kidney. This lends scientific support to the therapeutic use of the plant leaves, as claimed by the tribal medicine of North-East India. [source] | ||||||||||||||||||||||||||||||||||