Home About us Contact
|
Home About us Contact | ||
|
|
||
Dependently
Kinds of Dependently Selected AbstractsEstimation of endogenous adenosine activity at adenosine receptors in guinea-pig ileum using a new pharmacological methodACTA PHYSIOLOGICA, Issue 2 2010K. F. Nilsson Abstract Aim:, Adenosine modulates neurotransmission and in the intestine adenosine is continuously released both from nerves and from smooth muscle. The main effect is modulation of contractile activity by inhibition of neurotransmitter release and by direct smooth muscle relaxation. Estimation of adenosine concentration at the receptors is difficult due to metabolic inactivation. We hypothesized that endogenous adenosine concentrations can be calculated by using adenosine receptor antagonist and agonist and dose ratio (DR) equations. Methods:, Plexus-containing guinea-pig ileum longitudinal smooth muscle preparations were made to contract intermittently by electrical field stimulation in organ baths. Schild plot regressions were constructed with 2-chloroadenosine (agonist) and 8-(p -sulfophenyl)theophylline (8-PST; antagonist). In separate experiments the reversing or enhancing effect of 8-PST and the inhibiting effect of 2-chloroadenosine (CADO) were analysed in the absence or presence of an adenosine uptake inhibitor (dilazep), and nucleoside overflow was measured by HPLC. Results:, Using the obtained DR, baseline adenosine concentration was calculated to 28 nm expressed as CADO activity, which increased dose dependently after addition of 10,6 m dilazep to 150 nm (P < 0.05). HPLC measurements yielded a lower fractional increment (80%) in adenosine during dilazep, than found in the pharmacological determination (440%). Conclusion:, Endogenous adenosine is an important modulator of intestinal neuro-effector activity, operating in the linear part of the dose,response curve. Other adenosine-like agonists might contribute to neuromodulation and the derived formulas can be used to calculate endogenous agonist activity, which is markedly affected by nucleoside uptake inhibition. The method described should be suitable for other endogenous signalling molecules in many biological systems. [source] Exenatide: a review from pharmacology to clinical practiceDIABETES OBESITY & METABOLISM, Issue 6 2009R. Gentilella Background:, Exenatide is an incretin mimetic that activates glucagon-like-peptide-1 receptors. It blunts the postprandial rise of plasma glucose by increasing glucose-dependent insulin secretion, suppressing inappropriately high glucagon secretion and delaying gastric emptying. Methods:, In seven clinical trials performed in 2845 adult patients with type 2 diabetes mellitus who were inadequately controlled by a sulphonylurea and/or metformin (glycosylated haemoglobin, HbA1c ,11%), or by thiazolidinediones (with or without metformin) and treated for periods from 16 weeks to 3 years, exenatide (5 ,g b.i.d. s.c. for the first 4 weeks of treatment and 10 ,g b.i.d. s.c. thereafter) reduced HbA1c, fasting and postprandial glucose, and body weight dose dependently, and was similar to insulin glargine and biphasic insulin aspart in reducing HbA1c. Body weight diminished with exenatide, whereas it increased with both insulin preparations. Positive effects on the lipid profile and a reduction in C-reactive protein were also recorded with exenatide. Treatment extensions up to 3 years showed that benefits were maintained in the long term. Adverse events were usually mild to moderate in intensity, and generally the frequency decreased with continued therapy. The most common was nausea (whose incidence may be reduced by gradual dose escalation from 5 ,g b.i.d. to 10 ,g b.i.d.), vomiting, diarrhoea, headache and hypoglycaemia (almost exclusively in patients treated with a sulphonylurea). Results and conclusions:, Exenatide is a new, promising therapeutic option for type 2 diabetic patients inadequately controlled by oral agents, before insulin therapy, offering the added benefits of body weight reduction and tight postprandial glucose control. [source] Perinatal exposure to bisphenol-A changes N -methyl- D -aspartate receptor expression in the hippocampus of male rat offspringENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 1 2010Xiao-Hong Xu Abstract Bisphenol-A (BPA) is one of the most common environmental endocrine disrupters with mixed estrogen agonist/antagonist properties. The toxicity of BPA has been extensively evaluated in a variety of tests in rodents, including developmental and reproductive toxicity, and carcinogenicity. The objective of the present study is to evaluate whether or not perinatal maternal exposure to BPA at 0.05, 0.5, 5, 50, and 200 mg/kg/d affects N -methyl- D -aspartate (NMDA) receptor (NMDAR) subunits NR1, NR2A, 2B, estrogen receptor beta (ER,), and aromatase cytochrome P450 (P450arom) protein expressions of hippocampus in male rat offspring during postnatal development. Western-blotting analyses showed that perinatal exposure to BPA significantly affected the expression of NMDAR subunits. At the lower doses of 0.05 to 50 mg/kg/d, BPA concentration dependently inhibited the expression of NMDAR subunits. However, at the higher dose (200 mg/kg/d), the effects of BPA on these subunits were different, with a stronger inhibition of NR1 expression and a slighter inhibition of NR2A, 2B expression when compared with those at the lower dosage of BPA. In addition, perinatal exposure to BPA inhibited the expression of ER, protein, but increased P450arom protein expression in a concentration-dependent manner, especially during the early postnatal period (the first 1,3 postnatal weeks). No significant influence of BPA on P450arom was observed at postnatal week 8. These data suggest that environmental BPA exposure may affect the development of the brain, enhancing the local biosynthesis of estrogen in the brain, inhibiting ER, and NMDAR expressions. Environ. Toxicol. Chem. 2010;29:176,181. © 2009 SETAC [source] Cold liver ischemia-reperfusion injury critically depends on liver T cells and is improved by donor pretreatment with interleukin 10 in miceHEPATOLOGY, Issue 6 2000Olivier Le Moine M.D. Kupffer cells are thought to mediate most of the deleterious effects of liver ischemia-reperfusion injury. The role of liver T cells and the impact of resident cell deactivation by interleukin 10 (IL-10) have never been addressed. Using a model of ex vivo liver cold ischemia and reperfusion, we assessed liver injury, tumor necrosis factor (TNF) and interferon gamma (IFN-,) release from livers of balb/c mice, nude mice, nude mice reconstituted with T cells, and gadolinium balb/c pretreated mice. The anti-inflammatory cytokine IL-10 was then used to define the best strategy of administration potentially able to modulate ischemia-reperfusion injury. For this purpose IL-10 was administered to the donor before liver harvesting, in the preservation medium during cold ischemia or during reperfusion. TNF and IFN-, were released time dependently and paralleled liver injury after reperfusion of cold preserved livers. Reperfused livers from nude or gadolinium pretreated mice disclosed a dramatic decrease in TNF and IFN-, release. Tissue injury was reduced by 51% in the absence of T cells and by 88% when Kupffer cells were deactivated. This effect was reverted by T-cell transfer to nude mice. Only donor pretreatment with IL-10 or IL-10 infusion during reperfusion led to a significant decrease in liver injury, TNF, and IFN-, release (,66% or ,41%, ,95% or ,94%, and ,70% or ,70%, respectively). In conclusion, liver resident T cells are critically involved in cold ischemia-reperfusion injury and pretreatment of the donor with IL-10 decreases liver injury and the release of T-cell, and macrophage-dependent cytokines. [source] Dose-dependent long-term effects of Tat in the rat hippocampal formation: A design-based stereological studyHIPPOCAMPUS, Issue 4 2010Sylvia Fitting Abstract The human immunodeficiency virus type 1 (HIV-1) protein transactivator of transcription (Tat) is believed to play a critical role in mediating central nervous system (CNS) pathology in pediatric HIV-1 infection. Long-term neurotoxicity was investigated in a design-based stereology study following intrahippocampal injection of Tat on postnatal day (P)10, a time period that approximates the peak in the rats' rate of brain growth and mimics clinical HIV-1 CNS infection at labor/delivery. The goal was to examine the impact of P10 intrahippocampal Tat injection on the anatomy of the adult hippocampus (5 month) to gain a better understanding about how timing of infection influences the rate of progression of pediatric HIV-1 infection [cf. Fitting et al. (2008a) Hippocampus 18:135,147]. Male P10 Sprague-Dawley rats were bilaterally injected with vehicle or one of three different doses of Tat (5, 25, or 50 ,g). Unbiased stereological estimates were used to quantify total neuron number (Nissl stain) in five major subregions of the rat hippocampus: granular layer (GL), hilus of the dentate gyrus (DGH), cornu ammonis fields (CA)2/3, CA1, and subiculum (SUB). Glial cells (astrocytes and oligodendrocytes) were quantified in the DGH and SUB. No significant reduction of neuron number was noted for any of the five hippocampal subregions, in contrast to the very prominent reductions reported when Tat was administered on P1 [Fitting et al. (2008a) Hippocampus 18:135,147]. However, for glial cells, the number of astrocytes in the DGH and SUB as well as the number of oligodendrocytes in the DGH were linear dose dependently increased as a function of dose of Tat. In conjunction with previous stereological research [Fitting et al., (2008a) Hippocampus 18:135,147], the present data suggest that variability in the progression of pediatric HIV/acquired immunodeficiency syndrome (AIDS) may be better understood with the knowledge of the factor of timing of HIV-1 CNS infection. © 2009 Wiley-Liss, Inc. [source] Gliotoxin, an inhibitor of nuclear factor-kappa B, attenuates peptidoglycan-polysaccharide-induced colitis in ratsINFLAMMATORY BOWEL DISEASES, Issue 3 2002Dr. Leo R. Fitzpatrick Abstract Gliotoxin is a fungal metabolite that has immunosuppressive properties. First, we determined if gliotoxin could inhibit bacterial peptidoglycan,polysaccharide-stimulated tumor necrosis factor-, production, as well as nuclear factor-kappa B (NF-,B), in a rat macrophage (NR8383) cell line. Next, the apoptosis-inducing potential of gliotoxin was also evaluated in this cell line. Finally, we evaluated whether gliotoxin could reduce peptidoglycan,polysaccharide-induced colitis in rats. Gliotoxin (2 mg/kg/day) was dosed from day 14 after the initial intramural colonic injection of peptidoglycan,polysaccharide until day 21. A gross colonic injury score, myeloperoxidase activity, and cytokine levels were all evaluated on day 21. Gliotoxin dose dependently inhibited cytokine production, as well as NF-,B, and also induced apoptosis in the NR8383 cell line. On day 21, gliotoxin significantly reduced gross colonic injury (adhesions, nodules, mucosal lesions) in rats. Gliotoxin-treated rats also had partially normalized biochemical indices of colitis, such as colonic cytokine levels. The colonic level of NF-,B was also partially normalized in gliotoxin treated rats. Gliotoxin also exhibited an antiarthritis effect in peptidoglycan,polysaccharide-treated rats. In summary, gliotoxin effectively attenuated the chronic reactivation phase of peptidoglycan,polysaccharide-induced colitis. This anticolitis effect may be related to the inhibition of NF-,B in Lewis rats. [source] Cigarette smoke extract affects functional activity of MRP1 in bronchial epithelial cellsJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 5 2007Margaretha van der Deen Abstract Cigarette smoke is the principal risk factor for development of chronic obstructive pulmonary disease (COPD). Multidrug resistance-associated protein 1 (MRP1) is a member of the ATP-binding cassette (ABC) superfamily of transporters, which transport physiologic and toxic substrates across cell membranes. MRP1 is highly expressed in lung epithelium. This study aims to analyze the effect of cigarette smoke extract (CSE) on MRP1 activity. In the human bronchial epithelial cell line 16HBE14o,, MRP1 function was studied flow cytometrically by cellular retention of carboxyfluorescein (CF) after CSE incubation and MRP1 downregulation by RNA interference (siRNA). Cell survival was measured by the MTT assay. Immunocytochemically, it was shown that 16HBE14o, expressed MRP1 and breast cancer resistance protein. Coincubation of CSE IC50 (1.53% ± 0.22%) with MK571 further decreased cell survival 31% (p, = 0.018). CSE increased cellular CF retention dose dependently from 1.7-fold at 5% CSE to 10.3-fold at 40% CSE (both p < 0.05). siRNA reduced MRP1 RNA expression with 49% and increased CF accumulation 67% versus control transfected cells. CSE exposure further increased CF retention 24% (p = 0.031). A linear positive relation between MRP1 function and CSE-modulating effects (r = 0.99, p =0.089) was shown in untransfected, control transfected, and MRP1 downregulated 16HBE14o, cells analogous to blocking effects with MRP1 inhibitor MK571 (r = 0.99, p = 0.034). In conclusion, cigarette smoke extract affects MRP1 activity probably competitively in bronchial epithelial cells. Inhibition of MRP1 in turn results in higher CSE toxicity. We propose that MRP1 may be a protective protein for COPD development. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:243,251, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20187 [source] Dietary vitamin E reduces labile iron in rat tissuesJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 5 2005Wissam Ibrahim Abstract Previous studies have shown that dietary vitamin E reduced generation and/or levels of superoxide. As superoxide has potential to release iron from its transport and storage proteins, and labile or available form of iron is capable of catalyzing the formation of reactive hydroxyl radicals, the effect of dietary vitamin E on labile iron pool was studied in rats. One-month-old Sprague-Dawley male and female rats were fed a basal vitamin E-deficient diet supplemented with 0, 20, 200, or 2,000 IU vitamin E/kg diet for 90 days. The levels of labile iron were measured in the liver, kidney, spleen, heart and skeletal muscle. Additionally, the levels of lipid peroxidation products were measured. The results showed that, except for labile iron in the heart of male rats, dietary vitamin E dose dependently reduced the levels of labile iron and lipid peroxidation products in all tissues of male and female rats. The findings suggest that dietary vitamin E may protect against oxidative tissue damage by reducing the generation and/or level of superoxide, which in turn attenuates the release of iron from its protein complexes. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:298,303, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20094 [source] Intermittently Administered Human Parathyroid Hormone(1,34) Treatment Increases Intracortical Bone Turnover and Porosity Without Reducing Bone Strength in the Humerus of Ovariectomized Cynomolgus MonkeysJOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2001David B. Burr Abstract Cortical porosity in patients with hyperparathyroidism has raised the concern that intermittent parathyroid hormone (PTH) given to treat osteoporotic patients may weaken cortical bone by increasing its porosity. We hypothesized that treatment of ovariectomized (OVX) cynomolgus monkeys for up to 18 months with recombinant human PTH(1,34) [hPTH(1,34)] LY333334 would significantly increase porosity in the midshaft of the humerus but would not have a significant effect on the strength or stiffness of the humerus. We also hypothesized that withdrawal of PTH for 6 months after a 12-month treatment period would return porosity to control OVX values. OVX female cynomolgus monkeys were given once daily subcutaneous (sc) injections of recombinant hPTH(1,34) LY333334 at 1.0 ,g/kg (PTH1), 5.0 ,g/kg (PTH5), or 0.1 ml/kg per day of phosphate-buffered saline (OVX). Sham OVX animals (sham) were also given vehicle. After 12 months, PTH treatment was withdrawn from half of the monkeys in each treatment group (PTH1-W and PTH5-W), and they were treated for the remaining 6 months with vehicle. Double calcein labels were given before death at 18 months. After death, static and dynamic histomorphometric measurements were made intracortically and on periosteal and endocortical surfaces of sections from the middiaphysis of the left humerus. Bone mechanical properties were measured in the right humeral middiaphysis. PTH dose dependently increased intracortical porosity. However, the increased porosity did not have a significant detrimental effect on the mechanical properties of the bone. Most porosity was concentrated near the endocortical surface where its mechanical effect is small. In PTH5 monkeys, cortical area (Ct.Ar) and cortical thickness (Ct.Th) increased because of a significantly increased endocortical mineralizing surface. After withdrawal of treatment, porosity in PTH1-W animals declined to sham values, but porosity in PTH5-W animals remained significantly elevated compared with OVX and sham. We conclude that intermittently administered PTH(1,34) increases intracortical porosity in a dose-dependent manner but does not reduce the strength or stiffness of cortical bone. [source] Basic Fibroblast Growth Factor Stimulates Vascular Endothelial Growth Factor Release in Osteoblasts: Divergent Regulation by p42/p44 Mitogen-Activated Protein Kinase and p38 Mitogen-Activated Protein KinaseJOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2000Haruhiko Tokuda Abstract We previously showed that basic fibroblast growth factor (bFGF) activates p38 mitogen-activated protein (MAP) kinase via Ca2+ mobilization, resulting in interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of bFGF on the release of vascular endothelial growth factor (VEGF) in these cells. bFGF stimulated VEGF release dose dependently in the range between 10 and 100 ng/ml. SB203580, an inhibitor of p38 MAP kinase, markedly enhanced the bFGF-induced VEGF release. bFGF induced the phosphorylation of both p42/p44 MAP kinase and p38 MAP kinase. PD98059, an inhibitor of upstream kinase of p42/p44 MAP kinase, reduced the VEGF release. SB203580 enhanced the phosphorylation of p42/p44 MAP kinase induced by bFGF. The enhancement by SB203580 of the bFGF-stimulated VEGF release was suppressed by PD98059. The depletion of extracellular Ca2+ by [ethylenebis-(oxyethylenenitrilo)]tetracetic acid (EGTA) or 1,2-bis-(O -aminophinoxy)-ethane- N,N,N,N -tetracetic acid tetracetoxymethyl ester (BAPTA/AM), a chelator of intracellular Ca2+, suppressed the bFGF-induced VEGF release. A23187, a Ca ionophore, or thapsigargin, known to induce Ca2+ release from intracellular Ca2+ store, stimulated the release of VEGF by itself. A23187 induced the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase. PD98059 suppressed the VEGF release induced by A23187. SB203580 had little effect on either A23187-induced VEGF release or the phosphorylation of p42/p44 MAP kinase by A23187. These results strongly suggest that bFGF stimulates VEGF release through p42/p44 MAP kinase in osteoblasts and that the VEGF release is negatively regulated by bFGF-activated p38 MAP kinase. [source] Endothelin-1 Modulates the Arrhythmogenic Activity of Pulmonary VeinsJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 3 2008AMEYA R. UDYAVAR M.D. Objective: Endothelin-1 has important cardiovascular effects and is activated during atrial fibrillation. Pulmonary veins (PVs) play a critical role in the pathophysiology of atrial fibrillation. The aim of this study was to evaluate whether endothelin-1 affects PV arrhythmogenic activity. Methods: Conventional microelectrodes were used to record the action potentials (APs) and contractility in isolated rabbit PV tissue specimens before and after the administration of endothelin-1 (0.1, 1, 10 nM). The ionic currents of isolated PV cardiomyocytes were investigated before and after the administration of endothelin-1 (10 nM) through whole-cell patch clamps. Results: In the tissue preparation, endothelin-1 (1, 10 nM) concentration dependently shortened the AP duration and decreased the PV firing rates. Endothelin-1 (10 nM) decreased the resting membrane potential. Endothelin-1 (0.1, 1, 10 nM) decreased the contractility and increased the resting diastolic tension. In single PV cardiomyocytes, endothelin-1 (10 nM) decreased the PV firing rates from 2.7 ± 1.0 Hz to 0.8 ± 0.5 Hz (n = 16). BQ-485 (100 ,M, endothelin-1 type A receptor blocker) reversed and prevented the chrono-inhibitory effects of endothelin-1 (10 nM). Endothelin-1 (10 nM) reduced the L-type calcium currents, transient outward currents, delayed rectifier currents, transient inward currents, and sodium,calcium exchanger currents in the PV cardiomyocytes with and without pacemaker activity. Endothelin-1 (10 nM) increased the inward rectifier potassium current, hyperpolarization-induced pacemaker current, and the sustained outward potassium current in PV cardiomyocytes with and without pacemaker activity. Conclusion: Endothelin-1 may have an antiarrhythmic potential through its direct electrophysiological effects on the PV cardiomyocytes and its action on multiple ionic currents. [source] Platelet-derived growth factor-BB phosphorylates heat shock protein 27 in cardiac myocytesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2004Motoki Takenaka Abstract It is recognized that heat shock protein 27 (HSP27) is highly expressed in heart. In the present study, we investigated whether platelet-derived growth factor (PDGF) phosphorylates HSP27 in mouse myocytes, and the mechanism underlying the HSP27 phosphorylation. Administration of PDGF-BB induced the phosphorylation of HSP27 at Ser-15 and -85 in mouse cardiac muscle in vivo. In primary cultured myocytes, PDGF-BB time dependently phosphorylated HSP27 at Ser-15 and -85. PDGF-BB stimulated the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily. SB203580, a specific inhibitor of p38 MAP kinase, reduced the PDGF-BB-stimulated phosphorylation of HSP27 at both Ser-15 and -85, and phosphorylation of p38 MAP kinase. However, PD98059, a specific inhibitor of MEK, or SP600125, a specific inhibitor of SAPK/JNK, failed to affect the HSP27 phosphorylation. These results strongly suggest that PDGF-BB phosphorylates HSP27 at Ser-15 and -85 via p38 MAP kinase in cardiac myocytes. © 2003 Wiley-Liss, Inc. [source] Sonic hedgehog is involved in osteoblast differentiation by cooperating with BMP-2JOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2002Takahito Yuasa The roles of Sonic hedgehog (Shh) and Bone morphogenetic protein-2 (Bmp-2) in osteoblast differentiation were investigated using in vitro cell systems. Recombinant amino-terminal portion of SHH (rSHH-N) dose dependently stimulated ALP activity in C3H10T1/2 and MC3T3-E1 cells. rSHH-N induced expression of Osteocalcin mRNA in C3H10T1/2 cells. A soluble form of the receptor for type IA BMP receptor antagonized rSHH-N-induced ALP activity in C3H10T1/2 and MC3T3-E1 cells, indicating that BMPs are involved in SHH-induced osteoblast differentiation. Simultaneous supplement with rSHH-N and BMP-2 synergistically induced ALP activity and expression of Osteocalcin mRNA in C3H10T1/2 cells. Pretreatment with rSHH-N for 6 h enhanced the response to BMP-2 by increasing ALP activity in C3H10T1/2 and MC3T3-E1 cells. Stimulatory effects of rSHH-N and additive effects with rSHH-N and BMP-2 on ALP activity were also observed in mouse primary osteoblastic cells. Transplantation of BMP-2 (1 ,g) into muscle of mice induced formation of ectopic bone, whereas transplantation of r-SHH-N (1,5 ,g) failed to generate it. These results indicate that Shh plays important roles in osteoblast differentiation by cooperating with BMP. © 2002 Wiley-Liss, Inc. [source] Inhibition of plasminogen activator inhibitor-1 expression by siRNA in rat hepatic stellate cellsJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 12 2008Ping-Fang Hu Abstract Background and Aim:, The plasminogen activator/plasmin system is known to regulate the extracellular matrix turnover. The aim of this study was to detect the role of plasminogen activator inhibitor-1 (PAI-1) during liver fibrogenesis and investigate the functional effects of PAI-1 gene silencing in rat hepatic stellate cells (HSCs) using small interfering RNA (siRNA). Methods:, Hepatic fibrosis in rats was induced through serial subcutaneously injections of CCl4 and the expression of PAI-1 was detected by immunohistochemistry and reverse transcription,polymerase chain reaction (PCR). PAI-1 siRNA molecules were constructed and transiently transfected into HSC-T6 using the cell suspension transfection method. The pSUPER RNA interfering system was used to establish the HSC stable cell line pSUPER-shPAI. Expression of alpha-smooth muscle actin, transforming growth factor-beta, tissue inhibitor of metalloproteinases-1, and collagen types I and III were evaluated by real-time PCR. Cell proliferation and the cell cycle were determined by the methyl thiazolyl tetrazolium (MTT) method and flow cytometry. Collagen content in HSCs supernatant was evaluated by enzyme-linked immunosorbent assay. Results:, The results showed that PAI-1 was upregulated during liver fibrosis, and its expression was closely correlated with the deposition of collagens. SiRNA molecules were successfully transfected into HSCs and induced inhibition of PAI-1 expression time dependently. Moreover, PAI-1 siRNA treatment downregulated alpha-smooth muscle actin, transforming growth factor-beta, tissue inhibitor of metalloproteinases-1 expression, and inhibited collagen types I and III synthesis both at the mRNA and protein level in transiently and stably transfected HSCs. Conclusions:, This study suggests a significant functional role for PAI-1 in the development of liver fibrosis and that downregulating PAI-1 expression might present as a potential strategy to treat liver fibrosis. [source] Hypocretin-1 Dose-Dependently Modulates Maternal Behaviour in MiceJOURNAL OF NEUROENDOCRINOLOGY, Issue 8 2006K. L. D'Anna Increases in neuronal activity of hypocretin (HCRT), a peptide involved in arousal, and in HCRT-1 receptor mRNA expression have recently been identified in association with lactation. HCRT is released within brain regions regulating maternal behaviour and it is possible that increased HCRT neurotransmission during lactation supports maternal care. The present study examined for the first time the behavioural effects of HCRT on lactating mice. At intermediate doses, intracerebroventricular (i.c.v.) injections of HCRT-1 (0.06 and 0.1 µg) elevated levels of licking and grooming of pups (but not self-grooming) and number of nursing bouts without affecting other behaviours. At the highest dose, HCRT-1 (0.3 µg, i.c.v) delayed latency to nurse, decreased nursing, increased time off nest, and decreased maternal aggression. Intraperitoneal injections of the HCRT-1 receptor antagonist, SB-334867, exhibited a general trend towards increasing time spent low-arched back nursing (P = 0.053) and decreasing licking and grooming of pups while high-arched back nursing (P = 0.052). This suggests that the endogenous release of HCRT, working independently or dependently with other neuromodulators, may be necessary for full maternal behaviour expression. Possible sites of HCRT action in enhancing and impairing maternal care were identified via examinations of c,Fos immunoreactivity in association with i.c.v. HCRT injections. Together, these finding support the idea of HCRT modulating maternal behaviour, with intermediate levels (0.06 and 0.1 µg) supporting (even augmenting) some behaviours, but with levels that are too high (0.3 µg HCRT, i.c.v.), maternal behaviour and aggression are suppressed. [source] Focal adhesion kinase mediates human leukocyte histocompatibility antigen class II-induced signaling in gingival fibroblastsJOURNAL OF PERIODONTAL RESEARCH, Issue 6 2007S. Yoshizawa Background and Objective:, The role of human leukocyte antigen class II molecules on nonantigen-presenting cells has been a matter of controversy. We previously reported that human leukocyte antigen class II molecules on human gingival fibroblasts do not present antigens, but transduce signals into the cells by making a complex with antigenic peptide T-cell receptor or by stimulating cell surface human leukocyte antigen-DR molecules with human leukocyte antigen-DR antibody (L243), which mimics the formation of the human leukocyte antigen class II,antigenic peptide T-cell receptor complex, resulting in the expression of several cytokines. The aim of this study was to detect human leukocyte antigen class II-associated molecules mediating human leukocyte antigen class II-induced signals into the cells. Material and Methods:, Antibody-based protein-microarray analysis was performed to detect activated signaling molecules in gingival fibroblasts stimulated via human leukocyte antigen class II molecules. Then, we examined if these molecules structurally associate with human leukocyte antigen class II and actually transduce signals into the cells. Results:, Stimulation of human leukocyte antigen class II on gingival fibroblasts by L243 resulted in enhanced phosphorylation of focal adhesion kinase. Focal adhesion kinase was co-immunoprecipitated with human leukocyte antigen-DR by L243. Stimulation of gingival fibroblasts with L243 induced phosphorylation of focal adhesion kinase. Luteolin, a putative focal adhesion kinase inhibitor, suppressed phosphorylation of focal adhesion kinase and dose dependently inhibited human leukocyte antigen class II-induced cytokine production. Conclusion:, Focal adhesion kinase is structurally associated with human leukocyte antigen-DR and mediates human leukocyte antigen class II-induced signals in gingival fibroblasts. [source] Milk basic protein increases alveolar bone formation in rat experimental periodontitisJOURNAL OF PERIODONTAL RESEARCH, Issue 1 2007H. Seto Background and Objective:, It is conceivable that the active components extracted from milk whey protein (i.e. milk basic protein, MBP) stimulate bone formation and suppress bone resorption. Periodontitis is characterized by excessive alveolar bone resorption. We examined whether milk basic protein could recover alveolar bone loss in rat experimental periodontitis. Material and Methods:, A nylon ligature was placed around the cervix of molars in 8-wk-old male Fischer rats for 20 d. Then, the ligature was removed and a powder diet containing 0.2 or 1.0% milk basic protein was provided daily for another 45,90 d. On days 45 and 90, the maxillae were extracted and analyzed using microcomputerized tomography (micro-CT), followed by histological analysis. Results:, Micro-CT images showed that alveolar bone resorption was severely induced around the molar by the 20-d ligature procedure. Treatment with high-dose milk basic protein (1.0%) clearly recovered ligature-induced alveolar bone resorption on days 45 and 90, whereas low-dose milk basic protein (0.2%) did not show such a clear effect. Histological examination clarified that the osteoid thickness of alveolar bone was dose dependently increased by milk basic protein treatment for 90 d. Conclusion:, These findings suggest that a systemic administration of milk basic protein may be effective for the recovery of alveolar bone loss in periodontitis. [source] Anti-angiogenic, antinociceptive and anti-inflammatory activities of Lonicera japonica extractJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 6 2008Hye-Jung Yoo This study aimed to elucidate some novel pharmacological activities of Lonicera japonica (Caprifoliaceae), which is widely used in Oriental folk medicine. The ethanolic extract of L. japonica (LJ) dose dependently inhibited chick chorioallantoic membrane angiogenesis. The antinociceptive activity of LJ was assessed using the acetic acid-induced constriction model in mice. LJ showed anti-inflammatory activity in two in-vivo models: the vascular permeability and air pouch models. LJ suppressed the production of nitric oxide via down-regulation of inducible nitric oxide synthase in lipopolysaccharide-stimulated RAW264.7 macrophage cells. However, LJ was unable to suppress induction of cyclooxygenase-2 in the stimulated macrophage cells. LJ decreased the reactive oxygen species level in the stimulated macrophage cells. In brief, the flowers of L. japonica possess potent anti-angiogenic and antinociceptive activities, in addition to anti-inflammatory activity, which partly supports its therapeutic efficacy. [source] Inhibitory effect of apigenin on benzo(a)pyrene-mediated genotoxicity in Swiss albino miceJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 12 2006Tajdar Husain Khan Apigenin, a bioflavonoid, is abundantly present in fruits and vegetables and possesses potential chemopreventive properties against a wide variety of chronic diseases. In this study we investigated the anti-genotoxic effects of apigenin against a known genotoxicant, benzo(a)pyrene (B(a)P) (125 mg kg,1 orally) toxicity in Swiss albino mice. B(a)P administration led to induction of cytochrome P-450 (CYP), aryl hydrocarbon hydroxylase (AHH) and DNA strand breaks (P < 0.001), which was suppressed by apigenin (2.5 and 5 mg kg,1 orally) dose dependently (P < 0.001). B(a)P-induced depletion in the level of reduced glutathione (GSH), quinone reductase (QR) and glutathione-S-transferase (GST) was also shown to be restored by apigenin pre-treatment (P < 0.001). A simultaneous significant and dose-dependent reduction was noted in DNA strand breaks and in-vivo DNA damage (P < 0.001), which gives some insight into restoration of DNA integrity in modulator groups. These results strongly support the protective nature of apigenin against B(a)P-induced toxicity. [source] Effects of recombinant human activated protein C on the coagulation system: a study with rotational thromboelastometryACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 9 2008C. U. NILSSON Background: Recombinant human activated protein C (rhAPC) is an anticoagulant that can be used for treatment of patients with severe sepsis. The use of rhAPC is accompanied by an increased risk of severe bleeding. Rotational thromboelastometry is a method for measuring the status of the coagulation. The aim of the study was to investigate whether rotational thromboelastometry could be used for monitoring the effects of rhAPC on the coagulation. Methods: Whole blood was mixed in vitro with concentrations of rhAPC ranging from 0 to 75 ng/ml and analysed with rotational thromboelastometry. Results: The parameter Coagulation Time was significantly prolonged by increasing the concentrations of rhAPC (P=0.002). Other parameters were not significantly affected. Conclusion: rhAPC dose dependently affects the early humoral parts of the coagulation, while platelet function and fibrinogen to fibrin conversion seem virtually unaffected. [source] Role of Dopamine D1 Receptors and Extracellular Signal Regulated Kinase in the Motivational Properties of Acetaldehyde as Assessed by Place Preference ConditioningALCOHOLISM, Issue 4 2010Liliana Spina Background:, The role of dopamine D1 receptors and Extracellular signal Regulated Kinase (ERK) in the motivational properties of drugs can be studied by place-conditioning. Recent advances have shown that the motivational properties of ethanol, determined by place-conditioning, are mediated by its metabolic conversion into acetaldehyde. To date, the role of D1 receptors and ERK activation in acetaldehyde-elicited place preference has not been determined. The aim of this study was to assess the role of D1 receptors blockade and MEK inhibition in the acquisition of acetaldehyde-elicited conditioned place preference. Methods:, Male Sprague,Dawley rats were subjected to repeated pairings with 1 compartment of the conditioning apparatus immediately following acetaldehyde (20 mg/kg i.g.) or ethanol (1 g/kg i.g.) administration. The D1 receptor antagonist, SCH 39166 (50 ,g/kg s.c.), was administered 10 minutes before acetaldehyde or ethanol administration. In order to study the role of activated ERK in the acetaldehyde-elicited place preference, rats were administered the MEK inhibitor, PD98059 (1, 30, and 90 ,g i.c.v.), 10 or 30 minutes before acetaldehyde. To verify the specificity of these effects, we also studied whether PD98059 pretreatment could affect morphine (1 mg/kg s.c.)-elicited place preference. Results:, Both acetaldehyde and ethanol elicited significant place preferences and these were prevented by pretreatment with SCH 39166. In addition, pretreatment with PD98059, dose (30 and 90 but not 1 ,g i.c.v.) and time (10 but not 30 minutes before) dependently, prevented the acquisition of acetaldehyde- and significantly reduced the acquisition of morphine-elicited conditioned place preference. Conclusions:, These results confirm that acetaldehyde and ethanol elicit conditioned place preference and demonstrate that D1 receptors are critically involved in these effects. Furthermore, the finding that PD98059 prevents the acquisition of acetaldehyde-elicited conditioned place preference highlights the importance of the D1 receptor,ERK pathway in its motivational effects. [source] Signaling mechanisms of melatonin in antiproliferation of hormone-refractory 22Rv1 human prostate cancer cells: implications for prostate cancer chemopreventionJOURNAL OF PINEAL RESEARCH, Issue 2 2007Chun W. Tam Abstract:, There is an unmet clinical demand for safe and effective pharmaceuticals/nutraceuticals for prostate cancer prevention and hormone-refractory prostate cancer treatment. Previous laboratory and human studies of our laboratory demonstrated an association between the antiproliferative action of melatonin and melatonin MT1 receptor expression in prostate cancer. The aim of this study was to determine, using a pharmacological approach, the signaling mechanisms of melatonin in hormone-refractory 22Rv1 human prostate cancer cell antiproliferation. Both immunoreactive MT1 and MT2 subtypes of G protein-coupled melatonin receptor were expressed in 22Rv1 cells. Melatonin inhibited, concentration dependently, cell proliferation, upregulated p27Kip1 gene transcription and protein expression, and downregulated activated androgen signaling in 22Rv1 cells. While the effects of melatonin were mimicked by 2-iodomelatonin, a high-affinity nonselective MT1 and MT2 receptor agonist, melatonin effects were blocked by luzindole, a nonselective MT1 and MT2 receptor antagonist, but were unaffected by 4-phenyl-2-propionamidotetraline, a selective MT2 receptor antagonist. Importantly, we discovered that the antiproliferative effect of melatonin exerted via MT1 receptor on p27Kip1 gene and protein upregulation is mediated by a novel signaling mechanism involving co-activation of protein kinase C (PKC) and PKA in parallel. Moreover, we also showed that a melatonin/MT1/PKC mechanism is involved in melatonin-induced downregulation of activated androgen signal transduction in 22Rv1 cells. Taken together with the known molecular mechanisms of prostate cancer progression and transition to androgen independence, our data provide strong support for melatonin to be a promising small-molecule useful for prostate cancer primary prevention and secondary prevention of the development and progression of hormone refractoriness. [source] Stimulatory and entraining effect of melatonin on tuberoinfundibular dopaminergic neuron activity and inhibition on prolactin secretionJOURNAL OF PINEAL RESEARCH, Issue 4 2000Yeh-Shiu Chu The aims of the present study were to determine if melatonin exerts an effect on prolactin (PRL) secretion via the tuberoinfundibular dopaminergic (TIDA) neurons and if endogenous or exogenous melatonin has an entraining effect on the rhythmic changes of TIDA neuronal activity and PRL secretion. Melatonin given in the morning (10:00 h), dose- (0.01,1 mg/kg, ip) and time- (at 15 and 60 min, but not at 30 min) dependently stimulated TIDA neuronal activity in ovariectomized (OVX), estrogen-treated rats as determined by 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the median eminence (ME). Serum PRL was concurrently inhibited by the injection. Melatonin administered in the afternoon (15:00 h) was even more effective in stimulating the lowered TIDA neuronal activity and inhibiting the increased PRL level than that given in the morning (10:00 h). S-20098, a melatonin agonist was also effective in stimulating the TIDA neurons. In contrast, S-20928, a putative melatonin antagonist, while it had no effect by itself, blocked the effect of S-20098. Although S-20928 failed to prevent melatonin's effect on ME DOPAC levels, six interspaced injections of S-20928, from 18:00 to 01:30 h, significantly blocked the increase of ME DOPAC levels at 03:00 h, indicating that the endogenous melatonin may play a role. We further used rats that received daily injection of melatonin (1 mg/kg, ip) at 18:00 h for 10 days and found that the injection augmented basal TIDA neuronal activity at 11:00 h and blunted the afternoon PRL surge. In all, melatonin can have an inhibitory effect on PRL secretion by stimulating the TIDA neurons, and it may help to entrain the circadian rhythms of both TIDA neuronal activity and PRL secretion. [source] Growth and Hematological Changes of Rockfish, Sebastes schlegeli (Hilgendorf) Exposed to Dietary Cu and CdJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 2 2005Ju-Chan Kang Cd and Cu toxicological effect on growth and hematological parameters was investigated in juvenile rockfish Sebasres schlegeli after sub-chronic dietary Cd (0, 0.5, 5, 25, and 125 mg/kg) and Cu exposure (0, 50, 125, 250, and 500 mg/kg) for 60 d. In the experiment of dietary Cd exposure, weight and length SGR of the rockfish were significantly different from control, and a significant inverse relationship was observed between weight gain and the exposure concentration of dietary Cd at 25, 125 mg/kg (P > 0.05). Hematwrit and hemoglobin decreased significantly and were dose dependently in all Cd exposure. Glucose in serum was also increased significantly (P < 0.05). The concentration of total protein in serum was significantly lower than control at 5, 25, and 125 mgkg. No differences were observed in serum calcium concentration. Magnesium concentration in serum was increased signillcantly with dietary Cd concentration. In the experiment of dietary Cu exposure, Cu was inhibited weight gain and growth rate. No differences were observed in hematocrit, hemoglobin and RBCs compared to control. Contents of total protein, glucose, and Ca in serum remained stable. Mg concentration in serum was increased significantly at 500 mg/kg. [source] Two subpopulations of thrombin-activated platelets differ in their binding of the components of the intrinsic factor X-activating complexJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 11 2005M. A. PANTELEEV Summary., Binding of fluorescein-labeled coagulation factors IXa, VIII, X, and allophycocyanin-labeled annexin V to thrombin-activated platelets was studied using flow cytometry. Upon activation, two platelet subpopulations were detected, which differed by 1,2 orders of magnitude in the binding of the coagulation factors and by 2,3 orders of magnitude in the binding of annexin V. The percentage of the high-binding platelets increased dose dependently of thrombin concentration. At 100 nm of thrombin, platelets with elevated binding capability constituted ,4% of total platelets and were responsible for the binding of ,50% of the total bound factor. Binding of factors to the high-binding subpopulation was calcium-dependent and specific as evidenced by experiments in the presence of excess unlabeled factor. The percentage of the high-binding platelets was not affected by echistatin, a potent aggregation inhibitor, confirming that the high-binding platelets were not platelet aggregates. Despite the difference in the coagulation factors binding, the subpopulations were indistinguishable by the expression of general platelet marker CD42b and activation markers PAC1 (an epitope of glycoprotein IIb/IIIa) and CD62P (P-selectin). Dual-labeling binding studies involving coagulation factors (IXa, VIII, or X) and annexin V demonstrated that the high-binding platelet subpopulation was identical for all coagulation factors and for annexin V. The high-binding subpopulation had lower mean forward and side scatters compared with the low-binding subpopulation (,80% and ,60%, respectively). In its turn, the high-binding subpopulation was not homogeneous and included two subpopulations with different scatter values. We conclude that activation by thrombin induces the formation of two distinct subpopulations of platelets different in their binding of the components of the intrinsic fX-activating complex, which may have certain physiological or pathological significance. [source] Interstitial Cystitis and the Therapeutic Effect of Suplatast TosilateLUTS, Issue 2009Yukio HAYASHI Painful bladder syndrome (PBS)/interstitial cystitis (IC) can be a chronic and debilitating disease characterized by urinary urgency, frequency, and bladder pain, which are often very difficult to treat, regardless of currently-proposed treatments. Suplatast tosilate (IPD-1151T) is an immunoregulator that suppresses Th2 cytokine production, immunoglobulin E (IgE) synthesis, chemical mediator release from mast cells, and eosinophilic recruitment. In a preliminary, open-label clinical study of IPD-1151T in 14 women with IC, treatment with IPD-1151T significantly increased bladder capacity and decreased urinary urgency, urinary frequency, and lower abdominal pain, as measured by the IC symptom index, in patients with non-ulcerative IC. A concomitant reduction in immunological parameters (eosinophils, IgE, and urine T cells) was observed. Also, in basic experimental studies using hydrochloric acid-induced chronic cystitis rats, the oral administration of IPD-1151T (0.1,100 mg/kg/day) for 7 days after the induction of cystitis dose dependently increased the intercontraction intervals and micturition volume. In addition, the infiltration of mast cells and eosinophils into the bladder was suppressed by IPD-1151T. These findings suggest that IPD-1151T could be a new medicine for treating debilitating symptoms, such as bladder pain and urinary frequency in PBS/IC. [source] The roles of prostaglandin E receptor subtypes in the cytoprotective action of prostaglandin E2 in rat stomachALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 2000H. Araki Summary Aim: To investigate the EP receptor subtype involved in the gastroprotective action of prostaglandin (PG) E2 using various EP receptor agonists in rats, and using knockout mice lacking EP1 or EP3 receptors. Methods: Male SD rats and C57BL/6 mice were used after an 18-h fast. Gastric lesions were induced by oral administration of HCl/ethanol (150 m m HCl in 60% ethanol). Rats were given various EP agonists i.v. 10 min before HCl/ethanol: PGE2, sulprostone (EP1/EP3 agonist), butaprost (EP2 agonist), 17-phenyl-,-trinorPGE2 (17-phenylPGE2: EP1 agonist), ONO-NT012 (EP3 agonist) and 11-deoxyPGE1 (EP3/EP4 agonist). In a separate study, the effect of PGE2 on HCl/ethanol lesions was examined in EP1 - and EP3 -receptor knockout mice. Results: Gastric lesions induced by HCl/ethanol were dose dependently prevented by PGE2; this effect was mimicked by sulprostone and 17-phenylPGE2 and was significantly antagonized by ONO-AE-829, an EP1 antagonist. Neither butaprost, ONO-NT012 nor 11-deoxyPGE1 exhibited any protective activity against HCl/ethanol-induced gastric lesions. PGE2 caused an inhibition of gastric motility as well as an increase of mucosal blood flow and mucus secretion, the effects being mimicked by prostanoids activating EP1 receptors, EP2/EP3/EP4 receptors and EP4 receptors, respectively. On the other hand, although HCl/ethanol caused similar damage in both wild-type mice and knockout mice lacking EP1 or EP3 receptors, the cytoprotective action of PGE2 observed in wild-type and EP3 -receptor knockout mice totally disappeared in mice lacking EP1 receptors. Conclusion: The gastric cytoprotective action of PGE2 is mediated by activation of EP1 receptors. This effect may be functionally associated with inhibition of gastric motility but not with increased mucosal blood flow or mucus secretion. [source] The Receptors and Role of Angiotensin II in Knee Joint Blood Flow Regulation and Role of Nitric Oxide in Modulation of Their FunctionMICROCIRCULATION, Issue 5 2003H. NAJAFIPOUR ABSTRACT Objectives: Angiotensin-converting enzyme (ACE) upregulation in the stroma cells of arthritis rheumatoid joints may produce a higher tissue concentration of angiotensin II (angII), which is a vasoconstrictor and mitogen factor that causes local hypoxia and synovial proliferation. No study in the literature has examined the role of angII in joint blood flow (JBF) regulation and the potential effect of ACE inhibitors on JBF. Methods: The study was performed on 20 Dutch white rabbits to examine the JBF response to angII, angII receptor subtypes, and the role of nitric oxide (NO) in angII effects in knee joint blood vessels. Drugs were administered locally through retrograde saphenous artery cannulation. Joint vascular resistance (JVR) was calculated by dividing the arterial blood pressure by the JBF. Results: AngII increased JVR dose dependently. The angII type 1 (AT1) receptor antagonist losartan did not change the basal JVR but completely blocked the effect of angII on JVR. N, -nitro-L-arginin methyl ester (L-NAME) increased JVR by a mean (±SEM) of 25.8 ± 8.7% (p < 0.05) but did not affect the joint vessel response to angII and losartan. Conclusions: AngII receptors are from the AT1 subtype in normal joint blood vessels, but angII plays no significant role in JBF regulation. The basal release of NO plays a role in resting JBF regulation, but NO does not affect the AT1 receptor-mediated vasoconstriction of joint blood vessels. [source] The Ustilago maydis Cys2His2 -type zinc finger transcription factor Mzr1 regulates fungal gene expression during the biotrophic growth stageMOLECULAR MICROBIOLOGY, Issue 6 2008Yan Zheng§ Summary The smut fungus Ustilago maydis establishes a biotrophic relationship with its host plant maize to progress through sexual development. Here, we report the identification and characterization of the Cys2His2 -type zinc finger protein Mzr1 that functions as a transcriptional activator during host colonization. Expression of the U. maydis mig2 cluster genes is tightly linked to this phase. Upon conditional overexpression, Mzr1 confers induction of a subset of mig2 genes during vegetative growth and this requires the same promoter elements that confer inducible expression in planta. Furthermore, expression of the mig2-4 and mig2-5 genes during biotrophic growth is strongly reduced in cells deleted in mzr1. DNA-array analysis led to the identification of additional Mzr1-induced genes. Some of these genes show a mig2 -like plant-specific expression pattern and Mzr1 is responsible for their high-level expression during pathogenesis. Mzr1 function requires the b -dependently regulated Cys2His2 -type cell cycle regulator Biz1, indicating that two stage-specific regulators mediate gene expression during host colonization. In spite of a role as transcriptional activator during biotrophic growth, mzr1 is not essential for pathogenesis; however, conditional overexpression interfered with proliferation during vegetative growth and mating ability, caused a cell separation defect, and triggered filamentous growth. We discuss the implications of these findings. [source] amfR, an essential gene for aerial mycelium formation, is a member of the AdpA regulon in the A-factor regulatory cascade in Streptomyces griseusMOLECULAR MICROBIOLOGY, Issue 4 2003Haruka Yamazaki Summary In Streptomyces griseus, A-factor (2-isocapryloyl-3R -hydroxymethyl-,-butyrolactone) acts as a chemical signalling molecule that triggers morphological differentiation and secondary metabolism. A transcriptional activator, AdpA, in the A-factor regulatory cascade switches on a number of genes required for both processes, thus forming an AdpA regulon. amfR encoding a regulatory protein similar to response regulators of bacterial two-component regulatory systems and essential for aerial mycelium formation was found to be a member of the AdpA regulon. AdpA bound two sites at nucleotide positions approximately ,200 (site 1) and ,60 (site 2), with respect to the major transcriptional start point of amfR, and accelerated the transcription of amfR by assisting RNA polymerase in forming an open complex at an appropriate region including the transcriptional start point. Site 2 contributed more to the transcriptional activation of amfR by AdpA than site 1, although AdpA showed a much lower affinity to site 2 than to site 1. The amfR transcription enhanced by AdpA subsequently ceased at day 2 when aerial hyphae began to be formed in the wild-type strain, whereas in an adsA null mutant amfR was continuously transcribed even until day 3. This implied that amfR was repressed growth dependently by a gene product under the control of ,-AdsA. Transcription of the promoter upstream of amfT depended on amfR, which is consistent with the idea that AmfR serves as an activator for amfTSBA in the amf operon. The observations that the amfR gene contains a TTA codon, a potential target for bldA -mediated regulation, and a conserved Asp-54 residue, which might be phosphorylated by a sensor kinase, suggest that the amf operon is under transcriptional, translational and post-translational control systems. [source] | |||||||||||||||||||||||||||||||