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Cyclic Monophosphate (cyclic + monophosphate)
Selected AbstractsInhibition of carbachol-evoked oscillatory currents by the NO donor sodium nitroprusside in guinea-pig ileal myocytesEXPERIMENTAL PHYSIOLOGY, Issue 4 2005Seung-Soo Chung The effect of sodium nitroprusside (SNP) on carbachol (CCh)-evoked inward cationic current (Icat) oscillations in guinea-pig ileal longitudinal myocytes was investigated using the whole-cell patch-clamp technique and permeabilized longitudinal muscle strips. SNP (10 ,m) completely inhibited Icat oscillations evoked by 1 ,m CCh. 1H-(1,2,4) Oxadiazole [4,3-a] quinoxaline-1-one (ODQ; 1 ,m) almost completely prevented the inhibitory effect of SNP on Icat oscillations. 8-Bromo-guanosine 3,,5,-cyclic monophosphate (8-Br-cGMP; 30 ,m) in the pipette solution completely abolished Icat oscillations. However, a pipette solution containing Rp-8-Br-cGMP (30 ,m) almost completely abolished the inhibitory effect of SNP on Icat oscillations. When the intracellular calcium concentration ([Ca2+]i) was held at a resting level using BAPTA (10 mm) and Ca2+ (4.6 ,m) in the pipette solution, CCh (1 ,m) evoked only the sustained component of Icat without any oscillations and SNP did not affect the current. A high concentration of inositol 1,4,5-trisphosphate (IP3; 30 ,m) in the patch pipette solutions significantly reduced the inhibitory effect of SNP (10 ,m) on Icat oscillations. SNP significantly inhibited the Ca2+ release evoked by either CCh or IP3 but not by caffeine in permeabilized preparations of longitudinal muscle strips. These results suggest that the inhibitory effects of SNP on Icat oscillations are mediated, in part, by functional modulation of the IP3 receptor, and not by the inhibition of cationic channels themselves or by muscarinic receptors in the plasma membrane. This inhibition seems to be mediated by an increased cGMP concentration in a protein kinase G-dependent manner. [source] Functional Characterisation of the Volume-Sensitive Anion Channel in Rat Pancreatic ,-CellsEXPERIMENTAL PHYSIOLOGY, Issue 2 2001L. Best The whole-cell and perforated patch configurations of the patch-clamp technique were used to characterise the volume-sensitive anion channel in rat pancreatic ,-cells. The channel showed high permeability (P) relative to Cl, to extracellular monovalent organic anions (PSCN/PCll= 1.73, Pacetate/PCll= 0.39, Plactate/PCll= 0.38, Pacetoacetate/PCll= 0.32, Pglutamate/PCll= 0.28) but was less permeable to the divalent anion malate (Pmalate/PCll= 0.14). Channel activity was inhibited by a number of putative anion channel inhibitors, including extracellular ATP (10 mM), 1,9-dideoxyforskolin (100 ,M) and 4-OH tamoxifen (10 ,M). Inclusion of the catalytic subunit of protein kinase A in the pipette solution did not activate the volume-sensitive anion channel in non-swollen cells. Furthermore, addition of 8-bromoadenosine 3,,5,-cyclic monophosphate (8-BrcAMP) or forskolin failed to activate the channel in intact cells under perforated patch conditions. Addition of phorbol 12,13-dibutyrate (200 nM), either before or after cell swelling, also failed to affect channel activation. Our findings do not support the suggestion that the volume-sensitive anion channel in pancreatic ,-cells can be activated by protein kinase A. Furthermore, the ,-cell channel does not appear to be subject to regulation via protein kinase C. [source] Simulated conditions of microgravity increases progesterone production in I-10 cells of Leydig tumor cell lineINTERNATIONAL JOURNAL OF UROLOGY, Issue 3 2008Tomoyoshi Kaneko Background: The prospect of long-term residence in outer space is becoming a reality. Previously we found that testosterone secretion and spermatogenesis is reduced in model mice on which the shift of body fluids in space is simulated. The present study examines the effects of the microgravity (,G) environment on steroidogenesis of Leydig cells. Methods: I-10 cells were cultured for 24 h under a simulated ,G environment. Progesterone production was evaluated as steroidogenesis. Western blots of the enzyme, 3,hydroxy dehydrogenase (HSD) as well as of the transcription factors, Ad4bp/SF-1 and DAX-1, the amount of adenosine3,,5,-cyclic monophosphate (cAMP) and cell morphology were investigated. The ,G environment was simulated using clinostat rotation (CR). Rotational (Cont-1) and stationary (Cont-2) controls always received a regular gravity effect with and without rotatory motion, respectively. Results: Progesterone levels in cells under CR were significantly increased compared with those of both controls. Increased progesterone production was not related to the expression of either of the transcription factors or the enzyme. Although the cytoplasm expanded, the amount of cAMP produced under CR did not significantly differ from that produced under both control environments. Conclusions: Our findings showed that I-10 cells produced more progesterone under a ,G, than under control environments. Since the amounts of 3,HSD, Ad4bp/SF-1, DAX-1 and cAMP were not altered, the increase in steroid production under CR might be due to the expansion of cytoplasm, where steroidogenesis occurs. Steroidogenesis should be investigated in more detail to predict the effects of outer space on humans. [source] Effect of aging on corticosterone secretion in diestrous ratsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Ming-Jae Lo Abstract The roles of age and prolactin (PRL) in regulating glucocorticoid secretion in diestrous rats were investigated. Adrenal zona fasciculata-reticularis (ZFR) cells from young, adult, middle (mid)-aged, and old female rats were isolated. Estrous cycle stage was determined by light microscopy after vaginal smears. Blood samples were collected from right jugular vein at 0, 30, 60, and 120 min after challenge with adrenocorticotropin (ACTH). During the diestrous phase, plasma levels of estradiol and progesterone were lower in mid-aged and old rats than in either young or adult rats. Age-dependent increases of the basal levels of plasma PRL and corticosterone were observed. No difference of ACTH-increased plasma concentrations of corticosterone was observed among young, adult, mid-aged, and old rats. Aging increased the basal, ACTH-, PRL-, forskolin (an adenylate cyclase activator)-, and 3-isobutyl-l-methylxanthine (IBMX, a non-selective phosphodiesterase inhibitor)-stimulated release of corticosterone and production of adenosine 3,, 5,-cyclic monophosphate (cAMP) in ZFR cells. However, the 8-Br-cAMP (a membrane-permeable cAMP)-stimulated release of corticosterone was not affected by age. Taken together, these data indicated that aging increased corticosterone secretion in female rats during diestrous phase, which is in part due to an increase in cAMP accumulation. In conclusion, aging and PRL play a stimulatory role in the co-regulation of corticosterone secretion. J. Cell. Biochem. © 2005 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] Biological dosimetry of magnetic resonance imaging,JOURNAL OF MAGNETIC RESONANCE IMAGING, Issue 5 2002Concepción Guisasola MD Abstract Purpose To check the bioeffects of the components of magnetic resonance imaging (MRI). MRI is based on an assumedly harmless interaction between certain nuclei in the body when placed in a strong magnetic field and radio wave fields. There are three key factors actuating on the examining body: a powerful static magnetic field (SMF), magnetic gradient fields (MGFs), and pulsed radiofrequency (RF) radiation. Materials and Methods In vitro cells (L-132 cells) were used as biosensors, and different cellular compounds were used as biomarkers (heat shock proteins [HSPs] and their messenger ribonucleic acids [mRNAs], calcium, and adenosine-3,,5,-cyclic monophosphate [cAMP]). The biosensors were placed in the bore of a 1.5-T MRI machine and the different electromagnetic fields were operated. Results HSPs and their mRNAs and cAMP did not respond to SMF, MGFs, or RF radiation. RF radiation increased cytosolic calcium concentration (18%, P < 0.05). Conclusion Although MRI procedures do not induce any cellular stress response, it may cause an unfathomable calcium increase in vitro. Although the in vitro experimental conditions are not totally comparable to clinical situations, the usefulness of the in vivo biological dosimetry, circulating leukocytes as biosensors, and HSPs and/or calcium as biomarkers is suggested. J. Magn. Reson. Imaging 2002;15:584,590. © 2002 Wiley-Liss, Inc. [source] Nitric oxide inhibits mitochondrial movement in forebrain neurons associated with disruption of mitochondrial membrane potentialJOURNAL OF NEUROCHEMISTRY, Issue 3 2006Gordon L. Rintoul Abstract Nitric oxide (NO) has a number of physiological and pathophysiological effects in the nervous system. One target of NO is the mitochondrion, where it inhibits respiration and ATP synthesis, which may contribute to NO-mediated neuronal injury. Our recent studies suggested that impaired mitochondrial function impairs mitochondrial trafficking, which could also contribute to neuronal injury. Here, we studied the effects of NO on mitochondrial movement and morphology in primary cultures of forebrain neurons using a mitochondrially targeted enhanced yellow fluorescent protein. NO produced by two NO donors, papa non-oate and diethylamine/NO complex, caused a rapid cessation of mitochondrial movement but did not alter morphology. Movement recovered after removal of NO. The effects of NO on movement were associated with dissipation of the mitochondrial membrane potential. Increasing cGMP levels using 8-bromoguanosine 3,,5,-cyclic monophosphate, did not mimic the effects on mitochondrial movement. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of NO-induced activation of soluble guanylate cyclase, did not block the effects of NO. Thus, neither increasing nor decreasing cGMP levels had an effect on mitochondrial movement. Based on these data, we conclude that NO is a novel modulator of mitochondrial trafficking in neurons, which may act through the inhibition of mitochondrial function. [source] Modulation of perch connexin35 hemi-channels by cyclic AMP requires a protein kinase A phosphorylation siteJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2003Georgia Mitropoulou Abstract Retinal neurons are coupled via gap junctions, which function as electrical synapses that are gated by ambient light conditions. Gap junctions connecting either horizontal cells or AII amacrine cells are inhibited by the neurotransmitter dopamine, via the activation of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway. Fish connexin35 (Cx35) and its mouse ortholog, Cx36, are good candidates to undergo dopaminergic modulation, because they have been detected in the inner plexiform layer of the retina, where Type II amacrine cells establish synaptic contacts. We have taken advantage of the ability of certain connexins to form functional connexons (hemi-channels), when expressed in Xenopus oocytes, to investigate whether pharmacological elevation of cAMP modulates voltage-activated hemi-channel currents in single oocytes. Injection of perch Cx35 RNA into Xenopus oocytes induced outward voltage-dependent currents that were recorded at positive membrane potentials. Incubation of oocytes with 8-bromoadenosine 3,,5,-cyclic monophosphate (8-Br-cAMP), a membrane permeable cAMP analog, resulted in a dose-dependent and reversible inhibition of hemi-channel currents at the more positive voltage steps. In contrast, treatment with 8-Br-cAMP did not have any effect on hemi-channel currents induced by skate Cx35. Amino acid sequence comparison of the two fish connexins revealed, in the middle cytoplasmic loop of perch Cx35, the presence of a PKA consensus sequence that was absent in the skate connexin. The results obtained with two constructs in which the putative PKA phosphorylation site was either suppressed (perch Cx35R108Q) or introduced (skate Cx35Q108R) indicate that it is responsible for the inhibition of hemi-channel currents. These studies demonstrate that perch Cx35 is a target of the cAMP/PKA signaling pathway and identify a consensus PKA phosphorylation site that is required for channel gating. © 2003 Wiley-Liss, Inc. [source] Genetic Correlations Between Initial Sensitivity to Ethanol and Brain cAMP Signaling in Inbred and Selectively Bred MiceALCOHOLISM, Issue 6 2001Shelli L. Kirstein Background: Several lines of evidence have suggested a role for cAMP (adenosine 3,,5,-cyclic monophosphate) signaling in the acute and chronic effects of ethanol. This study investigated whether there is a genetic correlation between cAMP synthesis in the brain and the acute effects of ethanol [alcohol sensitivity or acute functional tolerance (AFT)]. Methods: By using nine inbred strains of mice, we measured initial sensitivity and AFT to ethanol with a test of balance on a dowel. Initial sensitivity was defined by the blood ethanol concentration (BEC0) at the loss of balance on a dowel after an ethanol injection [1.75 g/kg intraperitoneally (ip)]. When mice were able to regain balance on the dowel, BEC1 was determined, and a second ethanol injection was given (2 g/kg ip). Upon final regaining of balance, BEC2 was determined. AFT was defined by the difference between BEC1 and BEC2 (AFT =,BEC = BEC2, BEC1). Cyclic AMP synthesis was measured in whole-cell preparations in the cerebellum and other brain areas of mice of the nine inbred strains. Results: Significant differences in BEC0 and AFT were seen among the mice of the nine inbred strains. Cerebellar basal and forskolin- and isoproterenol-stimulated cAMP production differed significantly between the strains, and BEC0 was found to correlate significantly with forskolin- and isoproterenol-stimulated cAMP accumulation in the cerebellum (r= 0.70 and 0.94, respectively). When we measured cAMP production in mesencephalic and telencephalic tissue in three strains of mice that differed significantly in isoproterenol-stimulated cAMP accumulation in the cerebellum, significant differences between strains were found only in telencephalic tissue. The relative relationship between the rank order of the three strains for cAMP accumulation in the telencephalon and initial sensitivity to ethanol was identical to that seen with the cerebellum. However, AFT did not correlate with cAMP accumulation in the cerebellum or any other brain area tested. Conclusions: These results suggest that cAMP-generating systems of the cerebellum and possibly the brain areas contained in telencephalic tissues (e.g., basal ganglia) may have an important relationship to an animal's initial sensitivity to the incoordinating effects of ethanol. [source] Ethanol Uses cAMP-Independent Signal Transduction Mechanisms to Activate Proenkephalin Promoter Activity in Rat C6 Glioma CellsALCOHOLISM, Issue 7 2000Xiaoju Yang Background: Previous in vivo studies show that acute ethanol exposure sequentially increases protein kinase A (PKA) activity, the phosphorylation of the adenosine 3,:5,-cyclic monophosphate (cAMP) dependent transcription factor, CREB, and finally proenkephalin gene expression. The present study was conducted to determine if ethanol could activate directly the adenylyl cyclase pathway and thus enhance proenkephalin promoter activity. Methods: Cultured rat C6 glioma cells stably transfected with a segment of the five prime flanking region of rat proenkephalin promoter (nucleotide -2700 + 53) ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were employed to study the effects of ethanol on proenkephalin promoter activity. This region of proenkephalin promoter contains two cAMP response elements (CRE-1 and CRE-2) and one AP2 site located in the region upstream of the TATA box. Cultures were exposed to ethanol, isoproterenol, and phorbol-12, myristate 13-acetate (PMA) alone and in combination, in the presence and absence of PKA and protein kinase C (PKC) inhibitors. Results: Ethanol and isoproterenol increased proenkephalin promoter activity in a dose-dependent manner. Ethanol had an additive effect on maximal isoproterenol-stimulated proenkephalin promoter activity, which suggested that ethanol used a cAMP-independent signai transduction pathway to increase proenkephalin promoter activation. In contrast with isoproterenol, ethanol exposure did not increase cAMP accumulation, PKA activity, or the phosphorylated form of CREB. However, ethanol exposure modestly increased PKC activity. The PKA-specific inhibitor, Rp-cAMP, dampened isoproterenol-induced activation of CAT activity but did not alter ethanol's ability to increase CAT activity. However, the PKC inhibitors, chelerthyrine and G07874, abrogated ethanol's effect of CAT activity but did not alter isoproterenol's effects. Conclusions: Ethanol enhanced proenkephalin promoter activity and potentiated isoproterenol stimulated promoter activity through a cAMP-independent pathway. [source] KP4 fungal toxin inhibits growth in Ustilago maydis by blocking calcium uptakeMOLECULAR MICROBIOLOGY, Issue 4 2001Matthew J. Gage KP4 is a virally encoded fungal toxin secreted by the P4 killer strain of Ustilago maydis. From our previous structural studies , it seemed unlikely that KP4 acts by forming channels in the target cell membrane. Instead, KP4 was proposed to act by blocking fungal calcium channels, as KP4 was shown to inhibit voltage-gated calcium channels in rat neuronal cells, and its effects on fungal cells were abrogated by exogenously added calcium. Here, we extend these studies and demonstrate that KP4 acts in a reversible manner on the cell membrane and does not kill the cells, but rather inhibits cell division. This action is mimicked by EGTA and is abrogated specifically by low concentrations of calcium or non-specifically by high ionic strength buffers. We also demonstrate that KP4 affects 45Ca uptake in U. maydis. Finally, we show that cAMP and a cAMP analogue, N 6,2,-O-dibutyryladenosine 3,:5,-cyclic monophosphate, both abrogate KP4 effects. These results suggest that KP4 may inhibit cell growth and division by blocking calcium-regulated signal transduction pathways. [source] Endothelin A receptors mediate relaxation of guinea pig internal anal sphincter through cGMP pathwayNEUROGASTROENTEROLOGY & MOTILITY, Issue 9 2010S.-c. Huang Abstract Background, Endothelin (ET) modulates motility of the internal anal sphincter through unclear receptor subtypes. Methods, We measured relaxation of guinea pig internal anal sphincter strips caused by ET-related peptides and binding of 125I-ET-1 to cell membranes prepared from the internal anal sphincter muscle. Visualization of 125I-ET-1 binding sites in tissue was performed by autoradiography. Key Results , In the guinea pig internal anal sphincter, ET-1 caused a marked relaxation insensitive to tetrodotoxin, atropine, or ,-conotoxin GVIA. ET-2 was as potent as ET-1. ET-3 caused a mild relaxation. The relative potencies for ETs to cause relaxation were ET-1 = ET-2 > ET-3. The ET-1-induced relaxation was inhibited by BQ-123, an ETA antagonist, but not by BQ-788, an ETB antagonist. These indicate that ETA receptors mediate the relaxation. The relaxant response of ET-1 was attenuated by LY 83583, KT 5823, Rp-8CPT-cGMPS, tetraethyl ammonium, 4-aminopyridine and N(omega)-nitro-l-arginine, but not significantly affected by NG -nitro-l-arginine methyl ester, NG -methyl-l-arginine, charybdotoxin, apamin, KT 5720, and Rp-cAMPS. These suggest the involvement of cyclic guanosine 3,,5,-cyclic monophosphate (cGMP), and potassium channels. Autoradiography localized 125I-ET-1 binding to the internal anal sphincter. Binding of 125I-ET-1 to the cell membranes prepared from the internal anal sphincter revealed the presence of two subtypes of ET receptors, ETA and ETB receptors. Conclusions & Inferences, Taken together, these results demonstrate that ETA receptors mediate relaxation of guinea pig internal anal sphincter through the cGMP pathway. [source] Nitrergic,purinergic interactions in rat distal colon motilityNEUROGASTROENTEROLOGY & MOTILITY, Issue 1 2004K. Van Crombruggen Abstract, Responses of rat distal colon circular muscle strips to exogenous nitric oxide (NO) and adenosine 5,-triphosphate (ATP) and to electrical field stimulation (EFS) were assessed in the absence/presence of various agents that interfere with nitrergic,purinergic pathways. Exogenous NO (10,6 to 10,4 mol L,1) elicited concentration-dependent, tetrodotoxin (TTX)-insensitive relaxations. The soluble guanylyl-cyclase (sGC) inhibitor 1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced duration and amplitude; the small conductance Ca2+ -sensitive K+ (SK)-channel blocker apamin (APA) only shortened the relaxations. ODQ + APA showed a marked inhibitory effect on duration and amplitude. TTX, APA, the NO-synthase inhibitor N(omega)-nitro- l -arginine methyl ester (l -NAME) and the purinergic receptor P2Y antagonist Reactive Blue 2 (RB2) shortened the relaxations by exogenous ATP (10,3 mol L,1) but did not influence the amplitude. ODQ had no effect. TTX + l -NAME did not yield a more pronounced inhibitory effect than TTX alone. The effect of ATP- , -S was similar to that of ATP. Electrical field stimulation (EFS) (40 V, 0.05 ms, 0.5,4 Hz for 30 s) yielded TTX-sensitive relaxations that were not altered by l -NAME, ODQ or RB2. APA shortened the relaxations. l -NAME + APA nearly abolished these relaxations. ODQ + APA and RB2 +l -NAME reduced the duration. These results suggest that distinct sets of small conductance SK-channels are involved in the amplitude and the duration of the relaxations and that NO increases their sensitivity to NO and ATP via guanosine 3,,5,-cyclic monophosphate (cGMP). ATP elicits relaxations via P2Y receptors with subsequent activation of SK-channels and induces neuronal release of NO. Both nitrergic and purinergic pathways must be blocked to inhibit EFS-induced relaxations. [source] ,-Catenin expression in human neural cell lines following exposure to cytokines and growth factorsNEUROPATHOLOGY, Issue 2 2000Jun-ichi Satoh ,-Catenin acts as a key mediator of the Wnt/Wingless signaling pathway involved in cell proliferation, differentiation and survival. Recent studies have shown that an unstable interaction between ,-catenin and the mutant presenilin-1 induces neuronal apoptosis, and that ,-catenin levels are decreased in the brains of patients with Alzheimer's disease (AD). Since activated microglia and astrocytes play a role in the process of neuronal degeneration in AD, the cytokine/growth factor-regulated expression of ,-catenin in human neural cell lines, including NTera2 teratocarcinoma-derived differentiated neurons (NTera2-N), IMR-32 neuroblastoma, SKN-SH neuroblastoma and U-373MG astrocytoma, was studied quantitatively following exposure to epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), tumor necrosis factor-, (TNF-,), interleukin (IL)-1,, IL-6, interferon (IFN)-,, transforming growth factor (TGF)-,1, dibutyryl cyclic adenosine 3,,5,-cyclic monophosphate (cAMP) (dbcAMP) or phorbol 12-myristate 13-acetate (PMA). ,-Catenin mRNA expressed constitutively in all of these cell lines was unaffected by treatment with any factors examined. In contrast, ,-catenin protein levels were reduced markedly in NTera2-N cells by exposure to dbcAMP, EGF or bFGF, and in U-373MG cells by treatment with dbcAMP or PMA, but were unaffected in any cell lines by BDNF, TNF-,, IL-1,, IL-6, IFN-, or TGF-,1. These results indicate that ,-catenin is expressed constitutively in human neural cells and downregulated at a protein level by a set of growth factors in a cell type-specific manner. [source] A Phytochrome-like Protein AphC Triggers the cAMP Signaling Induced by Far-red Light in the Cyanobacterium Anabaena sp.PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2004Strain PCC7120 ABSTRACT In the filamentous, nitrogen-fixing cyanobacterium Anabaena sp. PCC7120, red light (630 nm) decreased, whereas far-red light (720 nm) increased cellular adenosine 3,,5,-cyclic monophosphate (cAMP) content. To find a red and far-red light photoreceptor that triggers the cAMP signal cascade, we disrupted 10 open reading frame having putative chromophore-binding GAF domains. The response of the cellluar cAMP concentration to red and far-red light in each open reading frame disruptant was determined. It was found that only the mutant of the gene all2699 failed to respond to far-red light. The open reading frame named as aphC encoded a protein with 920 amino acids including GAF domains similar to those involved in Cph2, a photoreceptor of Synechocystis sp. PCC6803. To determine which adenylate cyclase (AC) is responsible for far-red light signal, we disrupted all AC genes and found that CyaC was the candidate. The enzymatic activity of CyaC might be controlled by a far-red light photoreceptor through the phosphotransfer reaction. The site-specific mutant of the Asp59 residue of the receiver (R1) domain of CyaC lost its light-response capability. It was suggested that the far-red light signal was received by AphC and then transferred to the N-terminal response regulator domain of CyaC. Then its catalytic activity was stimulated, which increased the cellular cAMP concentration and drove the subsequent signal transduction cascade. [source] Effect of adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) hull extracts on testosterone release from rat Leydig cellsPHYTOTHERAPY RESEARCH, Issue 5 2009Shih-Min Hsia Abstract Adlay has been used as a traditional Chinese medicine for the treatment of many diseases. However, few studies have reported the effects of adlay seeds on the endocrine system. In the present study, the effects of methanol extracts of adlay hull (AHM) on testosterone synthesis were studied. Rat Leydig cells were incubated with different reagents including human chorionic gonadotropin, 8-bromo-adenosine-3,,5,-cyclic monophosphate, forskolin, A23187, progesterone and androstenedione in the presence or absence of AHM. The rat anterior pituitary (AP) gland was treated with gonadotropin-releasing hormone (GnRH) in vitro in the presence or absence of AHM, and the concentrations of luteinizing hormone (LH) in the media were measured. AHM decreased testosterone release via the inhibition of (1) the PKA and PKC signal transduction pathways, (2) 17, -HSD enzyme activity in rat Leydig cells, and (3) in vitro GnRH-induced LH secretion. Copyright © 2008 John Wiley & Sons, Ltd. [source] Systematic interpretation of cyclic nucleotide binding studies using KinetXBasePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2008Sonja Schweinsberg Abstract Functional proteomics aims to describe cellular protein networks in depth based on the quantification of molecular interactions. In order to study the interaction of adenosine-3,,5,-cyclic monophosphate (cAMP), a general second messenger involved in several intracellular signalling networks, with one of its respective target proteins, the regulatory (R) subunit of cAMP dependent protein kinase (PKA), a number of different methods was employed. These include fluorescence polarisation (FP), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), amplified luminescence proximity homogeneous assay (ALPHA-screen), radioligand binding or activity-based assays. Kinetic, thermodynamic and equilibrium binding data of a variety of cAMP derivatives to several cAMP binding domains were integrated in a single database system, we called KinetXBase, allowing for very distinct data formats. KinetXBase is a practical data handling system for molecular interaction data of any kind, providing a synopsis of data derived from different technologies. This supports ongoing efforts in the bioinformatics community to devise formal concepts for a unified representation of interaction data, in order to enable their exchange and easy comparison. KinetXBase was applied here to analyse complex cAMP binding data and highly site-specific cAMP analogues could be identified. The software package is free for download by academic users. [source] Involvement of H2O2 in superoxide-dismutase-induced enhancement of endothelium-dependent relaxation in rabbit mesenteric resistance arteryBRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2003Takeo Itoh The mechanism underlying the enhancement by superoxide dismutase (SOD) of endothelium-dependent relaxation was investigated in rabbit mesenteric resistance arteries. SOD (200 U ml,1) increased the production of H2O2 in smooth muscle cells (as indicated by the use of an H2O2 -sensitive fluorescent dye). Neither SOD nor catalase (400 U ml,1) modified either the resting membrane potential or the hyperpolarization induced by acetylcholine (ACh, 1 ,M) in smooth muscle cells. In arteries constricted with noradrenaline, the endothelium-dependent relaxation induced by ACh (0.01,1 ,M) was enhanced by SOD (200 U ml,1) (P<0.01). This action of SOD was inhibited by L - NG -nitroarginine (nitric oxide (NO)-synthase inhibitor) but not by either charybdotoxin+apamin (Ca2+ -activated-K+ -channel blockers) or diclofenac (cyclooxygenase inhibitor). Neither ascorbate (50 ,M) nor tiron (0.3 mM), superoxide scavengers, had any effect on the ACh-induced relaxation, but each attenuated the enhancing effect of SOD on the ACh-induced relaxation. Similarly, catalase (400 U ml,1) inhibited the effect of SOD without changing the ACh-induced relaxation. In endothelium-denuded strips constricted with noradrenaline, SOD enhanced the relaxation induced by the NO donor 1-hydroxy-2-oxo-3-(N -methyl-3-aminopropyl)-3-methyl-1-triazene (NOC-7) (P<0.05). Ascorbate and catalase each attenuated this effect of SOD. H2O2 (1 ,M) enhanced the relaxation on the noradrenaline contraction induced by NOC-7 and that induced by 8-bromo-cGMP, a membrane-permeable analogue of guanosine 3,,5, cyclic monophosphate (cGMP). SOD had no effect on cGMP production, whether measured in endothelium-intact strips following an application of ACh (0.1 ,M) or in endothelium-denuded strips following an application of NOC-7 (0.1 ,M). It is suggested that in rabbit mesenteric resistance arteries, SOD increases the ACh-induced, endothelium-dependent relaxation by enhancing the action of NO in the smooth muscle via its H2O2 -producing action (rather than via a superoxide-scavenging action). British Journal of Pharmacology (2003) 139, 444,456. doi:10.1038/sj.bjp.0705255 [source] | |||||||||||||