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Okadaic Acid (okadaic + acid)
Kinds of Okadaic Acid Selected AbstractsSerine/Threonine Phosphatase Inhibitors Decrease Adrenergic Arylalkylamine N -Acetyltransferase Induction in the Rat Pineal GlandJOURNAL OF NEUROENDOCRINOLOGY, Issue 7 2001R. Spessert Abstract Adrenergic regulation of the pineal enzyme serotonin N -acetyltransferase [arylalkylamine N -acetyltransferase (AA-NAT); EC 2.3.1.87] accounts for the circadian rhythm in melatonin formation. In the present study, the role of protein phosphatases in the adrenergic regulation of rat pineal AA-NAT was investigated using specific inhibitors. In cultured pineals, the serine/threonine phosphatase type 1 and type 2A inhibitors okadaic acid and calyculin A significantly decreased adrenergically or cAMP-induced AA-NAT activity, whereas the serine/threonine phosphatase type 2B inhibitor cypermethrin and tyrosine phosphatase inhibitor dephostatin were ineffective. Reverse transcriptase-polymerase chain reaction (RT-PCR) data indicate that okadaic acid exerts its effect on cAMP-dependent AA-NAT induction by downregulating the amount of AA-NAT transcript. The ,third' messengers, inducible cAMP early repressor (ICER) and Fos-related antigene-2 (Fra-2), are believed to play a negative role in pineal AA-NAT transcription. Okadaic acid increased the cAMP responsiveness of neither ICER mRNA nor Fra-2 mRNA. Therefore, the regulatory role of pineal serine/threonine phosphatases in adrenergically stimulated AA-NAT expression probably does not depend on ICER or Fra-2. [source] Mitochondrial clustering at the vertebrate neuromuscular junction during presynaptic differentiationDEVELOPMENTAL NEUROBIOLOGY, Issue 6 2006Chi Wai Lee Abstract During vertebrate neuromuscular junction (NMJ) development, presynaptic motor axons differentiate into nerve termini enriched in synaptic vesicles (SVs). At the nerve terminal, mitochondria are also concentrated, but how mitochondria become localized at these specialized domains is poorly understood. This process was studied in cultured Xenopus spinal neurons with mitochondrion-specific probe MitoTracker and SV markers. In nerve-muscle cocultures, mitochondria were concentrated stably at sites where neurites and muscle cells formed NMJs, and mitochondria coclustered with SVs where neurites were focally stimulated by beads coated with growth factors. Labeling with a mitochondrial membrane potential-dependent probe JC-1 revealed that these synaptic mitochondria were with higher membrane potential than the extrasynaptic ones. At early stages of bead-stimulation, actin-based protrusions and microtubule fragmentation were observed in neurites at bead contact sites, suggesting the involvement of cytoskeletal dynamics and rearrangement during presynaptic differentiation. Treating the cultures with an actin polymerization blocker, latrunculin A (Ltn A), almost completely abolished the formation of actin-based protrusions and partially inhibited bead-induced mitochondrial and SV clustering, whereas the microtubule disrupting agent nocodazole was ineffective in inhibiting the clustering of mitochondria and SVs. Lastly, in contrast to Ltn A, which blocked bead-induced clustering of both mitochondria and SVs, the ser/thr phosphatase inhibitor okadaic acid inhibited SV clustering but not mitochondrial clustering. These results suggest that at developing NMJs, synaptogenic stimuli induce the clustering of mitochondria together with SVs at presynaptic terminals in an actin cytoskeleton-dependent manner and involving different intracellular signaling molecules. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Morphological and biochemical changes associated with apoptosis induced by okadaic acid in human amniotic FL cellsENVIRONMENTAL TOXICOLOGY, Issue 5 2009Ming-luan Xing Abstract The marine toxin okadaic acid (OA) is an apoptosis inducer and a tumor promoter. During recent years, extensive studies have demonstrated that OA can induce apoptosis in a wide variety of cell types. In contrast to the relatively longer incubation time or higher treatment concentrations of OA in apoptosis shown previously, relatively lower concentrations (,100 nM) and shorter time (4 h) were designed in the current study to observe the toxic effects of OA in human amniotic cells (FL cells). The present study was undertaken to determine the morphological and biochemical changes of FL cells induced by OA. Results indicated that externalization of phosphatidylserine, cytoskeletal disruption, DNA strand breaks and decrease of Bcl-2 protein expression levels as well as increase of PP2A-A subunit protein were all involved in the apoptosis of FL cells induced by OA. This work not only provided further evidence of apoptosis induced by OA but also suggested that PP2A might play a pivotal role in apoptosis induced by protein phosphatases inhibitors. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009. [source] Characteristics of okadaic acid,induced cytotoxic effects in CHO K1 cellsENVIRONMENTAL TOXICOLOGY, Issue 6 2003C. Huynh-Delerme Abstract This article reports the results of investigations into the process of cell death induced in the Chinese hamster ovary cell K1 subclone (CHO K1) by okadaic acid (OA), a hydrophobic polyether produced by marine dinoflagellates. The IC50 was about 13 nM OA after 24 h of treatment, as determined using neutral red. With the MTT assay, the IC50 was 25 nM, although in this case 25% of the initial staining was still observed at 100 nM. Hoechst staining showed that mitotic figures accumulated at 12 nM OA after a 24- or 48-h treatment. In experiments limited to a 3-day treatment without changing the medium, CHO K1 cells were engaged in the death process at 50 nM OA after about 20 h and at 10 nM OA after 48 h. In many cells nuclear fragmentation that resulted in the apparent appearance of vesicles correlated with increasing cellular volume. But additional cell fragmentation was not observed with any treatment, and the chromatin material seemed to progressively disappear inside the cells. DNA fragmentation was analyzed by electrophoresis and with the TUNEL technique. With both techniques, the DNA was fragmented by 48 h in both 25 and 50 nM OA. Electrophoresis showed that both adherent and nonadherent cells were affected. Annexin-positive/ propidium iodide (PI),negative cells were rarely observed after OA treatment. Some were seen under the scanning cytometer after 20 h at 50 nM OA or after 48 h at 10 nM OA, but they were never detected by flow cytometry. Most of the time scanning cytometry showed either unstained cells or PI-positive (annexin-positive or -negative) cells (48 h, 50 nM, or 72 h, 10 nM). Flow cytometry cytograms showed two cell subpopulations: one composed of a majority of smaller cells, the other of larger cells. The larger cells markedly decreased with time and OA treatment (50 and 100 nM). Stained-cell counting showed that all cells that stained were both annexin- and PI positive and that most PI-positive cells were smaller. Ki67 antigen labeling showed the proliferative activity of CHO K1 cultures but also demonstrated the loss of this activity in smaller cells treated with 50 nM OA for 48 h. We concluded that in our culture conditions the main OA target within CHO K1 cultures was dividing cells. Our results suggest that cells with disturbed metaphase,anaphase enter apoptosis, leading to necrotic daughter cells. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 383,394, 2003 [source] Metaplasticity of the late-phase of long-term potentiation: a critical role for protein kinase A in synaptic taggingEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2006Jennie Z. Young Abstract The late-phase of long-term potentiation (L-LTP) in hippocampal area CA1 requires gene expression and de novo protein synthesis but it is expressed in an input-specific manner. The ,synaptic tag' theory proposes that gene products can only be captured and utilized at synapses that have been ,tagged' by previous activity. The mechanisms underlying synaptic tagging, and its activity dependence, are largely undefined. Previously, we reported that low-frequency stimulation (LFS) decreases the stability of L-LTP in a cell-wide manner by impairing synaptic tagging. We show here that a phosphatase inhibitor, okadaic acid, blocked homosynaptic and heterosynaptic inhibition of L-LTP by prior LFS. In addition, prior LFS homosynaptically and heterosynaptically impaired chemically induced synaptic facilitation elicited by forskolin/3-isobutyl-1-methylxanthine, suggesting that there is a cell-wide dampening of cAMP/protein kinase A (PKA) signaling concurrent with phosphatase activation. We propose that prior LFS impairs expression of L-LTP by inhibiting synaptic tagging through its actions on the cAMP/PKA pathway. In support of this notion, we show that hippocampal slices from transgenic mice that have genetically reduced hippocampal PKA activity display impaired synaptic capture of L-LTP. An inhibitor of PKA, KT-5720, also blocked synaptic capture of L-LTP. Moreover, pharmacological activation of the cAMP/PKA pathway can produce a synaptic tag to capture L-LTP expression, resulting in persistent synaptic facilitation. Collectively, our results show that PKA is critical for synaptic tagging and for input-specific L-LTP. PKA-mediated signaling can be constrained by prior episodes of synaptic activity to regulate subsequent L-LTP expression and perhaps control the integration of multiple synaptic events over time. [source] Proteasome-driven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells, a serotonin producing mast cell lineFEBS JOURNAL, Issue 19 2002Yoshiko Iida We previously demonstrated in mast cell lines RBL2H3 and FMA3 that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by 26S-proteasomes [Kojima, M., Oguro, K., Sawabe, K., Iida, Y., Ikeda, R., Yamashita, A., Nakanishi, N. & Hasegawa, H. (2000) J. Biochem (Tokyo) 2000, 127, 121,127]. In the present study, we have examined an involvement of TPH phosphorylation in the rapid turnover, using non-neural TPH. The proteasome-driven degradation of TPH in living cells was accelerated by okadaic acid, a protein phosphatase inhibitor. Incorporation of 32P into a 53-kDa protein, which was judged to be TPH based on autoradiography and Western blot analysis using anti-TPH serum and purified TPH as the size marker, was observed in FMA3 cells only in the presence of both okadaic acid and MG132, inhibitors of protein phosphatase and proteasome, respectively. In a cell-free proteasome system constituted mainly of RBL2H3 cell extracts, degradation of exogenous TPH isolated from mastocytoma P-815 cells was inhibited by protein kinase inhibitors KN-62 and K252a but not by H89. Consistent with the inhibitor specificity, the same TPH was phosphorylated by exogenous Ca2+/calmodulin-dependent protein kinase II in the presence of Ca2+ and calmodulin but not by protein kinase A (catalytic subunit). TPH protein thus phosphorylated by Ca2+/calmodulin-dependent protein kinase II was digested more rapidly in the cell-free proteasome system than was the nonphosphorylated enzyme. These results indicated that the phosphorylation of TPH was a prerequisite for proteasome-driven TPH degradation. [source] PPAR, and PP2A are involved in the proapoptotic effect of conjugated linoleic acid on human hepatoma cell line SK-HEP-1INTERNATIONAL JOURNAL OF CANCER, Issue 11 2007Giuliana Muzio Abstract Conjugated linoleic acid (CLA), found in dairy products, in beef and lamb has been demonstrated to possess anticancer properties protecting several tissues from developing cancer. Moreover, it has been shown to modulate apoptosis in several cancer cell lines. The aim of this study was to investigate which signaling transduction pathways were modulated in CLA-induced apoptosis in human hepatoma SK-HEP-1 cells. The cells exposed to CLA were evaluated for PPAR,, PP2A, pro-apoptotic proteins Bak, Bad and caspases, and anti-apoptotic proteins Bcl-2 and Bcl-XL. Cells were also treated with okadaic acid, a PP2A inhibitor, or with Wy-14643, a specific PPAR, agonist. The CLA-induced apoptosis was concomitant to the increase of percentage of cells in the S phase, PPAR,, PP2A and pro-apoptotic proteins; simultaneously, antiapoptotic proteins decreased. Inhibition of PP2A prevented apoptosis, and PPAR, agonist showed similar effect as CLA. The increased PP2A could be responsible for the dephosphorylation of Bcl-2 and Bad, permitting apoptotic activity of Bax and Bad. The increase of caspase 8 and 9 suggested that both the intrinsic and extrinsic apoptotic pathways were induced. PP2A was probably increased by PPAR,, since putative PPRE sequences were found in genes encoding its subunits. In conclusion, CLA induces apoptosis in human hepatoma SK-HEP-1 cells, by increasing PPAR,, PP2A and pro-apoptotic proteins. © 2007 Wiley-Liss, Inc. [source] The nonconserved N-terminus of protein phosphatase 2B confers its properties to protein phosphatase 1IUBMB LIFE, Issue 2 2009Xiu-Jie Xie Abstract The protein phosphatase 1 catalytic subunit (PP1c) and the protein phosphatase 2B (PP2B or calcineurin) catalytic subunit (CNA) contain nonconserved N-terminal regions followed by conserved phosphatase cores. To examine the role of the N-termini of these two phosphatases, we substituted the residues 1,8 of PP1c with residues 1,42 of CNA, which is designated CNA(1-42)-PP1(9-330). The activities of CNA(1-42)-PP1(9-330) were similar to those of PP2B and different from those of PP1. The chimera was at least fourfold less sensitive to inhibition by okadaic acid, but was stimulated by nickel ions and chlorogenic acid, characteristics of PP2B not of PP1. These observations suggest that the N-terminus of CNA shifts the properties of PP1 toward those of PP2B. Our findings provide evidence that the nonconserved N-terminus of PP2B not only functions as important regulatory domain but also confers itself particular characteristics. This region may be targeted for regulation of PP2B activities in vivo. © 2008 IUBMB IUBMB Life, 61(2): 178,183, 2009 [source] Overexpression of RGPR-p117 enhances regucalcin gene promoter activity in cloned normal rat kidney proximal tubular epithelial cells: Involvement of TTGGC motifJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Natsumi Sawada Abstract A novel protein RGPR-p117 was discovered as regucalcin gene promoter region-related protein that binds to the TTGGC motif using a yeast one-hybrid system. RGPR-p117 is localized in the nucleus of kidney cells, and overexpression of RGPR-p117 can modulate regucalcin protein and its mRNA expression in the cloned normal rat kidney proximal tubular epithelial NRK52E cells. This study was undertaken to determine whether overexpression of RGPR-p117 enhances the regucalcin promoter activity using the ,710/+18 LUC construct (wild-type) or ,710/+18 LUC construct (mutant) with deletion of ,523/,435 including TTGGC motif. NRK52E cells (wild-type) or stable HA-RGPR-p117/phCMV2-transfected cells (transfectant) were cultured in Dulbecco's minimum essential medium (DMEM) containing 5% bovine serum (BS). Wild-type cells or transfectants were transfected with the ,710/+18 LUC construct vector or the ,710/+18 LUC construct with deletion of ,523/,435. Wild-type cells or transfectants with subconfluency were cultured for 48 h in a DMEM medium containing either vehicle, BS (5%), or parathyroid hormone (1,34) (PTH; 10,7 M). Luciferase activity in wild-type cells was significantly increased with culture of BS or PTH. This increase was significantly blocked in the presence of various protein kinase inhibitors (staurosporine and PD 98059). Luciferase activity in transfectants was significantly increased as compared with that of wild-type cells in the absence of BS or PTH. The increase in luciferase activity in transfectants was completely decreased in mutant with deletion of ,523/,435 sequence of regucalcin promoter. This was also seen using the ,710/+18 LUC construct with deletion of ,523/,503 sequence containing TTGGC motif. The increase in luciferase activity in transfectants was not significantly enhanced with culture of BS (5%), PTH (10,7 M), Bay K 8644 (10,6 M), phorbol 12-myristate 13-acetate (PMA; 10,6 M), or N6, 2,-dibutyryl cyclic adenosine 3,, 5,-monophosphate (DcAMP; 10,4 M). The increase in luciferase activity in transfectants was completely inhibited with culture of dibucaine (10,6 M), staurosporine (10,9 M), PD 98059 (10,8 M), wortmannin (10,8 M), genistein (10,6 M), vanadate (10,6 M), or okadaic acid (10,6 M) which are inhibitors of various kinases and protein phosphatases. This study demonstrates that RGPR-p117 can enhance the regucalcin promoter activity which is related to the NF-1 consensus sequences including TTGGC motif, and that its enhancing effect is partly mediated through phosphorylation and dephosphorylation in NRK52E cells. J. Cell. Biochem. 99: 589,597, 2006. © 2006 Wiley-Liss, Inc. [source] Mass spectrometric analysis of the marine lipophilic biotoxins pectenotoxin-2 and okadaic acid by four different types of mass spectrometersJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 8 2008Arjen Gerssen Abstract The performances of four different mass spectrometers [triple-quadrupole (TQ), time-of-flight (ToF), quadrupole ToF (Q-ToF) and ion trap (IT)] for the detection of the marine lipophilic toxins pectenotoxin-2 (PTX2) and okadaic acid (OA) were investigated. The spectral data obtained with the different mass spectrometric analyzers were used to propose fragmentation schemes for PTX2 in the positive electrospray mode and for OA in the negative electrospray mode. TQ data were used to obtain product ions, while ToF and Q-ToF-MS produced accurate mass data of the precursor ion and product ions, respectively. IT data provided a better understanding of the fragmentation pathways using MSn experiments. With respect to analytical performance, all four mass analyzers showed a good linearity (R2 > 0.97) and repeatability (CV < 20%). Detection limits (LoDs) (S/N = 3) were the lowest on triple-quad MS: 12.2 and 2.9 pg on-column for PTX2 and OA, respectively. Copyright © 2008 John Wiley & Sons, Ltd. [source] PACAP and C2-ceramide generate different AP-1 complexes through a MAP-kinase-dependent pathway: involvement of c-Fos in PACAP-induced Bcl-2 expressionJOURNAL OF NEUROCHEMISTRY, Issue 4 2006Nicolas Aubert Abstract The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) inhibits C2-ceramide-induced cell death through blockade of the mitochondrial apoptotic pathway in rat cerebellar granule neurones. However, the gene induction processes and transcription factors involved in the anti-apoptotic effect of PACAP remain unknown. Here, we show that PACAP and C2-ceramide activate activator protein-1 (AP-1) DNA binding in a dose- and time-dependent manner, but generate different AP-1 dimers. Thus, PACAP increased the proportion of c-Fos and Jun D while C2-ceramide increased c-Jun and reduced c-Fos in AP-1 complexes. In addition, PACAP strongly activated c-Fos gene expression while C2-ceramide markedly increased c-Jun phosphorylation. The effect of PACAP on c-Fos expression was blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitor, U0126, while phosphorylation of c-Jun induced by C2-ceramide was abrogated by the protein phosphatase 2A (PP2A) inhibitor, okadaic acid. Transfection of immature granule cells with c-Fos siRNA, which strongly reduced basal and PACAP-stimulated levels of the protein, totally prevented the stimulatory effect of PACAP on Bcl-2 expression. The present study demonstrates that AP-1 complexes containing c-Fos mediate the effect of PACAP on Bcl-2 gene expression in cerebellar granule neurones. Our data also indicate that different AP-1 dimers are associated with the pro-apoptotic effect of C2-ceramide and the anti-apoptotic effect of PACAP. [source] Apoptosis in cerebellar granule neurons is associated with reduced interaction between CREB-binding protein and NF-,BJOURNAL OF NEUROCHEMISTRY, Issue 2 2003Asligul Yalcin Abstract Cerebellar granule neurons undergo apoptosis when switched from medium containing depolarizing levels of potassium (high K+ medium, HK) to medium containing low K+ (LK). NF-,B, a ubiquitously expressed transcription factor, is involved in the survival-promoting effects of HK. However, neither the expression nor the intracellular localization of the five NF-,B proteins, or of I,B-, and I,B-,, are altered in neurons primed to undergo apoptosis by LK, suggesting that uncommon mechanisms regulate NF-,B activity in granule neurons. In this study, we show that p65 interacts with the transcriptional co-activator, CREB-binding protein (CBP), in healthy neurons. The decrease in NF-,B transcriptional activity caused by LK treatment is accompanied by a reduction in the interaction between p65 and CBP, an alteration that is accompanied by hyperphosporylation of CBP. LK-induced CBP hyperphosphorylation can be mimicked by inhibitors of protein phosphatase (PP) 2A and PP2A-like phosphatases such as okadaic acid and cantharidin, which also causes a reduction in p65,CBP association. In addition, treatment with these inhibitors induces cell death. Treatment with high concentrations of the broad-spectrum kinase inhibitor staurosporine prevents LK-mediated CBP hyperphosphorylation and inhibits cell death. In vitro kinase assays using glutathione- S -transferase (GST)-CBP fusion proteins map the LK-regulated site of phosphorylation to a region spanning residues 1662,1840 of CBP. Our results are consistent with possibility that LK-induced apoptosis is triggered by CBP hyperphosphorylation, an alteration that causes the dissociation of CBP and NF-,B. [source] Constitutive Phosphorylation of the Vesicular Inhibitory Amino Acid Transporter in Rat Central Nervous SystemJOURNAL OF NEUROCHEMISTRY, Issue 4 2000Cécile Bedet Abstract:,-Aminobutyric acid (GABA) and glycine are stored into synaptic vesicles by a recently identified vesicular inhibitory amino acid transporter [VIAAT, also called vesicular GABA transporter (VGAT)]. Immunoblotting analysis revealed that rat brain VIAAT migrated as a doublet during sodium dodecyl sulfate,polyacrylamide gel electrophoresis, with a predominant slower band in all areas examined except olfactory bulb and retina. The slower band corresponded to a phosphorylated form of VIAAT as it was converted to the faster one by treating brain homogenates with alkaline phosphatase or with an endogenous phosphatase identified as type 2A protein,serine/threonine phosphatase using okadaic acid. In contrast, the recombinant protein expressed in COS-7 or PC12 cells co-migrated with the faster band of the brain doublet and was insensitive to alkaline phosphatase. To investigate the influence of VIAAT phosphorylation on vesicular neurotransmitter loading, purified synaptic vesicles were treated with alkaline phosphatase and assayed for amino acid uptake. However, neither GABA nor glycine uptake was affected by VIAAT phosphorylation. These results indicate that VIAAT is constitutively phosphorylated on cytosolic serine or threonine residues in most, but not all, regions of the rat brain. This phosphorylation does not regulate the vesicular loading of GABA or glycine, suggesting that it is involved at other stages of the synaptic vesicle life cycle. [source] Regulation of Tyrosine Hydroxylase Activity and Phosphorylation at Ser19 and Ser40 via Activation of Glutamate NMDA Receptors in Rat StriatumJOURNAL OF NEUROCHEMISTRY, Issue 6 2000Niklas Lindgren Abstract: The activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by phosphorylation. In this study, we examined the effects of activation of NMDA receptors on the state of phosphorylation and activity of tyrosine hydroxylase in rat striatal slices. NMDA produced a time-and concentration-dependent increase in the levels of phospho-Ser19 -tyrosine hydroxylase in nigrostriatal nerve terminals. This increase was not associated with any changes in the basal activity of tyrosine hydroxylase, measured as DOPA accumulation. Forskolin, an activator of adenylyl cyclase, stimulated tyrosine hydroxylase phosphorylation at Ser40 and caused a significant increase in DOPA accumulation. NMDA reduced forskolin-mediated increases in both Ser40 phosphorylation and DOPA accumulation. In addition, NMDA reduced the increase in phospho-Ser40 -tyrosine hydroxylase produced by okadaic acid, an inhibitor of protein phosphatase 1 and 2A, but not by a cyclic AMP analogue, 8-bromo-cyclic AMP. These results indicate that, in the striatum, glutamate decreases tyrosine hydroxylase phosphorylation at Ser40 via activation of NMDA receptors by reducing cyclic AMP production. They also provide a mechanism for the demonstrated ability of NMDA to decrease tyrosine hydroxylase activity and dopamine synthesis. [source] A Gonadotropin-Releasing Hormone Insensitive, Thapsigargin-Sensitive Ca2+ Store Reduces Basal Gonadotropin Exocytosis and Gene Expression: Comparison with Agonist-Sensitive Ca2+ StoresJOURNAL OF NEUROENDOCRINOLOGY, Issue 2 2003J. D. Johnson Abstract We examined whether distinct Ca2+ stores differentially control basal and gonadotropin (GTH-II)-releasing hormone (GnRH)-evoked GTH-II release, long-term GTH-II secretion and contents, and GTH-II- , mRNA expression in goldfish. Thapsigargin (Tg)-sensitive Ca2+ stores mediated neither caffeine-evoked GTH-II release, nor salmon (s)GnRH- and chicken (c)GnRH-II-stimulated secretion; the latter responses were previously shown to involve ryanodine (Ry)-sensitive Ca2+ stores. Surprisingly, Tg decreased basal GTH-II release. This response was attenuated by prior exposure to sGnRH and caffeine, but was insensitive to the phosphatase inhibitor okadaic acid, the inhibitor of constitutive release brefeldin A and cGnRH-II. GTH-II- , mRNA expression was decreased at 24 h by 2 µm Tg, and by inhibiting (10 µm Ry) and stimulating (1 nm Ry) Ry receptors. Transient increases in GTH-II- , mRNA were observed at 2 h and 12 h following 10 µm and 1 nm Ry treatment, respectively. Effects of Tg, Ry and GnRH on long-term GTH-II secretion, contents and apparent production differed from one another, and these changes were not well correlated with changes in GTH-II- , mRNA expression. Our data show that GTH-II secretion, storage and transcription can be independently controlled by distinct Ca2+ stores. [source] Serine/Threonine Phosphatase Inhibitors Decrease Adrenergic Arylalkylamine N -Acetyltransferase Induction in the Rat Pineal GlandJOURNAL OF NEUROENDOCRINOLOGY, Issue 7 2001R. Spessert Abstract Adrenergic regulation of the pineal enzyme serotonin N -acetyltransferase [arylalkylamine N -acetyltransferase (AA-NAT); EC 2.3.1.87] accounts for the circadian rhythm in melatonin formation. In the present study, the role of protein phosphatases in the adrenergic regulation of rat pineal AA-NAT was investigated using specific inhibitors. In cultured pineals, the serine/threonine phosphatase type 1 and type 2A inhibitors okadaic acid and calyculin A significantly decreased adrenergically or cAMP-induced AA-NAT activity, whereas the serine/threonine phosphatase type 2B inhibitor cypermethrin and tyrosine phosphatase inhibitor dephostatin were ineffective. Reverse transcriptase-polymerase chain reaction (RT-PCR) data indicate that okadaic acid exerts its effect on cAMP-dependent AA-NAT induction by downregulating the amount of AA-NAT transcript. The ,third' messengers, inducible cAMP early repressor (ICER) and Fos-related antigene-2 (Fra-2), are believed to play a negative role in pineal AA-NAT transcription. Okadaic acid increased the cAMP responsiveness of neither ICER mRNA nor Fra-2 mRNA. Therefore, the regulatory role of pineal serine/threonine phosphatases in adrenergically stimulated AA-NAT expression probably does not depend on ICER or Fra-2. [source] Protein phosphatase 2A,negative regulation of the protective signaling pathway of Ca2+/CaM-dependent ERK activation in cerebral ischemiaJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2008Jie Zhao Abstract Extracellular-signal-regulated kinase (ERK) undergoes rapid inactivation following the intense activation evoked by cerebral ischemia and reperfusion. However, the precise mechanism of this inactivation has not been elucidated. To investigate how phosphatases regulate the ERK cascade following ischemia, the PP2A inhibitors cantharidin and okadaic acid were administrated to the CA1 subregion of the rat hippocampus. The resulting sustained ERK activity implies that PP2A is a major phosphatase contributing to the rapid inactivation, but not activation, of ERK following cerebral ischemia. The increase in PP2A activity induced by ceramide has a weak effect on the activation of Raf via dephosphorylation of Ser259 in response to ischemia. In contrast, ketamine (Keta) and cyclosporine A (CsA), two chemicals that block calcium signal in ischemia, decrease ERK activity by blocking Raf dephosphorylation of Ser259. We also observed that activation of an upstream protein, Ras-GRF, leads to calcium/calmodulin-dependent activation of the ERK signaling cascade in response to ischemic stimuli. In addition, the activity of cyclic AMP response element-binding protein (CREB) and estrogen receptor , (ER,), target proteins of ERK and protective elements against ischemic lesion, parallels the activity of ERK. These data indicate that PP2A plays a significant role in blocking the protective effect induced by the ERK kinase pathway and that fast inactivation of ERK is the result of cross talk between calcium/calmodulin-dependent, positively regulated signal cascades and a ceramide-dependent negative signaling pathway. © 2008 Wiley-Liss, Inc. [source] Alteration of argyrophilic nucleolar organizer region associated (Ag-NOR) proteins in apoptosis-induced human salivary gland cells and human oral squamous carcinoma cellsJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 4 2001Yasuhiro Morimoto Abstract: The level of argyrophilic nucleolar organizer regions (AgNORs) and AgNOR-associated proteins (Ag-NOR proteins) varies with cell activity, including ribosomal biogenesis occurring in proliferating cells. Proteins associated with some AgNORs are detected by a specific silver staining. To investigate a possible relationship between apoptosis and the AgNORs or Ag-NOR proteins, we examined the changes of AgNORs and Ag-NOR proteins during apoptosis in a human salivary gland cell line, HSG cells, and a human oral squamous carcinoma cell line, SCC-25 cells. Apoptosis was induced by treatment of HSG and SCC-25 cells with okadaic acid. Proteins prepared from HSG and SCC-25 cells treated with varying concentrations of okadaic acid (OA) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to transfer membranes and staining for Ag-NOR proteins by modified Western blot analysis. Four major bands (110 kDa, 43 kDa, 39kDa, and 37 kDa) were detected in the proteins obtained from the control cells. The level of the 110-kDa protein decreased in the proteins prepared from OA-induced apoptotic cells; however, the reaction intensity of the other three bands was changed in apoptotic cells. An additional band of an 80-kDa Ag-NOR protein appeared and increased in the apoptotic cells. Cellular fractionation of HSG cells and SCC-25 cells was done with or without apoptotic induction. An 80-kDa Ag-NOR protein was detected in the nuclear fraction prepared from the apoptotic cells, while the 110-kDa protein decreased in the nuclear fraction of these cells. The 110-kDa Ag-NOR protein may be nucleolin (C23) as deduced from its AgNOR staining features, including molecular weight. The 80-kDa protein may be the cleavage product of the 110-kDa protein. In the cell-free apoptotic system, in which intact nuclei of HSG cells were incubated with the cytosol fraction of apoptotic HSG and SCC-25 cells, the 80-kDa Ag-NOR protein was detected in nuclei incubated with the cytosol fraction of apoptotic cells, while the level of the 110-kDa protein decreased. The changes of Ag-NOR proteins in nuclei prepared from SCC-25 cells incubated with cytosol fractions prepared from HSG and SCC-25 cells were identical to those of the HSG cells. The alternation of AgNORs in apoptosis-induced HSG cells was also examined using double staining with Hoechst 33342 and silver nitrate. Hoechst staining revealed typical apoptotic nuclei, which exhibited highly fluorescent condensed chromatin in OA-treated HSG cells. Silver grains representing AgNORs were not detected in the cells undergoing apoptosis. The dual-imposition view confirmed that AgNORs, which are visible as dots in nucleoli in the control cells, disappeared from the apoptotic nuclei of HSG cells. Our results indicate that the 110-kDa nucleolar Ag-NOR protein is associated with apoptosis and is cleaved during apoptosis. [source] Oxidative metabolism by Thalassiosira weissflogii (Bacillariophyceae) of a diol-ester of okadaic acid, the diarrhetic shellfish poisoningJOURNAL OF PHYCOLOGY, Issue 2 2000Anthony J. Windust Previous investigations into the comparative toxicity of the diarrhetic shellfish poisoning (DSP) toxins to Thalassiosira weissflogii (Grun.) Fryxell et Hasle found that this diatom oxidatively metabolized okadaic acid diol-ester (OA diol-ester) to a more water-soluble product. This oxidative transformation of OA diol-ester by the diatom is significant for two reasons. First, it is known that dinophysistoxin-4 (DTX-4), the primary DSP toxin produced by the dinoflagellate Exuviaella lima (Ehr.) Butschli, will be hydrolyzed to the diol-ester following cell rupture (e.g. ingestion by a predator). Second, it implies that the ester, an uncharged, lipophilic intermediate, can easily enter cells and therefore may play an important role in the uptake and transfer of DSP toxins through the food web. It has been suggested that the water soluble DTX-4 may also be the form in which DSP toxins are excreted from the producing cell. Therefore, the stability of DTX-4 was examined when incubated either in fresh seawater medium into which washed cells of E. lima were introduced or in seawater medium conditioned by E. lima cells. Rapid hydrolysis of DTX-4 to the diol-ester took place in both cases. Thus, regardless of the route by which DTX-4 is liberated from the cell, either by cell disruption or excretion, the diol-ester will be the dominant form of the toxin to challenge associated organisms. To examine the metabolism of OA diol-ester by T. weissflogii in more detail, serial cultures of the diatom were challenged with OA diol-ester at a concentration of 2.0 ,g·mL,1. The metabolism and fate of the diol-ester in both cellular and medium fractions were monitored over 3 days using liquid chromatography with either ultraviolet (LC-UV) or mass spectrometric (LC-MS) detection. During the course of the experiment, all of the diol-ester was metabolized. LC-MS analysis revealed the presence of multiple oxidative products of OA diol-ester in the medium fraction, including a carboxylic acid derivative. The major metabolites were isolated in sufficient quantity to permit structural elucidation by NMR and MS. All the metabolites identified resulted from oxidation of the diol-ester side chain with the primary sites of attack at the terminal, subterminal, and unsaturated carbons. OA was found in both cellular and medium fractions, and its production was directly correlated with the metabolism of the diol-ester. The relative partitioning of both OA diol-ester and its oxidation products between cells and medium supports the contention that OA diol-ester can readily enter cells, be metabolized, and then excreted in more water-soluble forms. [source] Increased recombination frequency showing evidence of loss of interference is associated with abnormal testicular histopathologyMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2003Susannah Varmuza Abstract Nondisjunction leading to aneuploid gametes has been linked genetically to both increases and decreases in recombination frequency on the aneuploid chromosome. In the present study, we present physical evidence of increased frequency of recombination nodules as measured by Mut-S-like homologue-1 (MLH1) foci on pachytene chromosomes from sterile male mice homozygous for a mutation in the protein phosphatase 1c, (PP1c,) gene. The pattern of elevated recombination frequency in PP1c, mutant spermatocytes is consistent with a loss of interference. Previous studies demonstrated: (1) spermiogenesis is impaired starting at step 8 with a severe reduction in elongating and condensed spermatids; (2) spermatids and sperm exhibit elevated rates of DNA fragmentation; and (3) haploid gametes exhibit elevated levels of aneuploidy. Morphometric analysis of developing testes revealed that the first wave of meiosis proceeds at a normal rate in mutant testes, a surprising result given that the PP1 inhibitor okadaic acid has been shown to accelerate progression of spermatocytes from pachytene to the first meiotic division (MI). Evidence of abnormal testicular histopathology is apparent at 3 weeks, before the appearance of haploid gametes, eliminating the possibility that the mutant phenotype is caused by the presence of abnormal spermatids, but coincident with the appearance of the first set of mid to late pachytene spermatocytes. These observations lead us to conclude that the PP1c, mutation causes a complex phenotype, including subtle adverse effects on meiosis, possibly mediated by defective signaling between germ cells and Sertoli cells. Mol. Reprod. Dev. 64: 499,506, 2003. © 2003 Wiley-Liss, Inc. [source] A protein phosphatase 2A from Fagus sylvatica is regulated by GA3 and okadaic acid in seeds and related to the transition from dormancy to germinationPHYSIOLOGIA PLANTARUM, Issue 1 2006Mary Paz González-García Several gibberellic acid (GA3)-induced cDNA fragments encoding putative serine/threonine protein phosphatase (PP) 2A catalytic subunits were obtained by means of differential reverse transcriptase-PCR approach. The full-length clone, named FsPP2A1, isolated from a beechnut cDNA library, exhibited all the features of and homology to members of the PP2A family. By transient expression of FsPP2A1 in tobacco and Arabidopsis cells as a green fluorescent fusion protein, we have obtained evidence supporting the subcellular localization of this protein in both the cytosol and the nucleus. Analysis of FsPP2A1 expression during seed stratification shows that these transcripts increase in the presence of GA3, a treatment proved to be efficient in breaking the dormancy of Fagus sylvatica seeds, but they are almost undetectable in dormant seeds or when dormancy is maintained after treatment with either abscisic acid or the gibberellin biosynthesis inhibitor paclobutrazol. The PP inhibitor okadaic acid (OKA) has a clear effect in decreasing both seed germination and FsPP2A1 expression. Furthermore, FsPP2A1 is specifically expressed in seed tissues, not being detected in other vegetative tissues examined. These results show the regulation of this PP by GA3 and OKA in these seeds. Its relationship with the processes taking place during the transition from dormancy to germination is also discussed. [source] The phosphoproteome of Fusarium graminearum at the onset of nitrogen starvationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2010Christof Rampitsch Abstract Fusarium graminearum grown under stress, such as nutrient deprivation, activates, among others, the trichothecene pathway that produces the mycotoxin deoxynivalenol and its derivatives. The kinase inhibitor staurosporine reduced the production of trichothecenes by 39% compared with control in vitro. On the other hand, phosphatase inhibitor okadaic acid increased the amount by 72% compared with the control in vitro. This suggests that phosphorylation events are involved in the signalling pathway, leading to the activation of the trichothecene pathway. Three approaches were used to study the phosphoproteome of F. graminearum under nitrogen-limiting conditions: 2-DE (2-DE: IEF×SDS-PAGE) in combination with MS protein identification; SDS-PAGE in combination with off-line IMAC and TiO2 enrichment and gel electrophoresis LC-MS analysis; and a gel-free approach using strong anion exchange chromatography, IMAC and LC-MS. A total of 348 phosphorylation sites localized in 301 peptides from 241 proteins were identified. By 2-DE, 20 phosphoproteins were identified, nine of which underwent changes during the time course examined. Using gel electrophoresis LC-MS 231 phosphopeptides were identified from three samples (ten gel slices each) at time points of nitrogen starvation t=0, 6, and 12,h. The gel-free analysis added 70 peptides from 65 proteins to the total. Proteins of unknown function and enzymes of known function comprised the largest groups overall. Ten protein kinases and seven transcription factors were identified. This is the first reported phosphoproteome of F. graminearum. [source] Phosphorylation at S384 regulates the activity of the TaALMT1 malate transporter that underlies aluminum resistance in wheatTHE PLANT JOURNAL, Issue 3 2009Ayalew Ligaba Summary In this study we examined the role of protein phosphorylation/dephosphorylation in the transport properties of the wheat (Triticum aestivum) root malate efflux transporter underlying Al resistance, TaALMT1. Pre-incubation of Xenopus laevis oocytes expressing TaALMT1 with protein kinase inhibitors (K252a and staurosporine) strongly inhibited both basal and Al3+ -enhanced TaALMT1-mediated inward currents (malate efflux). Pre-incubation with phosphatase inhibitors (okadaic acid and cyclosporine A) resulted in a modest inhibition of the TaALMT1-mediated currents. Exposure to the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), enhanced TaALMT1-mediated inward currents. Since these observations suggest that TaALMT1 transport activity is regulated by PKC-mediated phosphorylation, we proceeded to modify candidate amino acids in the TaALMT1 protein in an effort to identify structural motifs underlying the process regulating phosphorylation. The transport properties of eight single point mutations (S56A, S183A, S324A, S337A, S351-352A, S384A, T323A and Y184F) generated in amino acid residues predicted to be phosphorylation sites and examined electrophysiologically. The basic transport properties of mutants S56A, S183A, S324A, S337A, S351-352A, T323A and Y184F were not altered relative to the wild-type TaALMT1. Likewise the sensitivity of these mutants to staurosporine resembled that observed for the wild-type transporter. However, the mutation S384A was noticeable, as in oocytes expressing this mutant protein TaALMT1-mediated basal and Al-enhanced currents were significantly inhibited, and the currents were insensitive to staurosporine or PMA. These findings indicate that S384 is an essential residue regulating TaALMT1 activity via direct protein phosphorylation, which precedes Al3+ enhancement of transport activity. [source] V-ATPase deactivation in blowfly salivary glands is mediated by protein phosphatase 2CARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2009Martin Voss Abstract The activity of vacuolar H+ -ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5-HT). 5-HT induces, via protein kinase A, the phosphorylation of V-ATPase subunit C and the assembly of V-ATPase holoenzymes. The protein phosphatase responsible for the dephosphorylation of subunit C and V-ATPase inactivation is not as yet known. We show here that inhibitors of protein phosphatases PP1 and PP2A (tautomycin, ocadaic acid) and PP2B (cyclosporin A, FK-506) do not prevent V-ATPase deactivation and dephosphorylation of subunit C. A decrease in the intracellular Mg2+ level caused by loading secretory cells with EDTA-AM leads to the activation of proton pumping in the absence of 5-HT, prolongs the 5-HT-induced response in proton pumping, and inhibits the dephosphorylation of subunit C. Thus, the deactivation of V-ATPase is most probably mediated by a protein phosphatase that is insensitive to okadaic acid and that requires Mg2+, namely, a member of the PP2C protein family. By molecular biological techniques, we demonstrate the expression of at least two PP2C protein family members in blowfly salivary glands. © 2009 Wiley Periodicals, Inc. [source] Regulation by FK506 and rapamycin of Ca2+ release from the sarcoplasmic reticulum in vascular smooth muscle: the role of FK506 binding proteins and mTORBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2009D MacMillan Background and purpose:, The sarcoplasmic reticulum (SR), regulates the cytoplasmic Ca2+ concentration ([Ca2+]cyto) in vascular smooth muscle. Release from the SR is controlled by two intracellular receptor/channel complexes, the ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (IP3R). These receptors may be regulated by the accessory FK506-binding protein (FKBP) either directly, by binding to the channel, or indirectly via FKBP modulation of two targets, the phosphatase, calcineurin or the kinase, mammalian target of rapamycin (mTOR). Experimental approach:, Single portal vein myocytes were voltage-clamped in whole cell configuration and [Ca2+]cyto measured using fluo-3. IP3Rs were activated by photolysis of caged IP3 and RyRs activated by hydrostatic application of caffeine. Key results:, FK506 which displaces FKBP from each receptor (to inhibit calcineurin) increased the [Ca2+]cyto rise evoked by activation of either RyR or IP3R. Rapamycin which displaces FKBP (to inhibit mTOR) also increased the amplitude of the caffeine-evoked, but reduced the IP3 -evoked [Ca2+]cyto rise. None of the phosphatase inhibitors, cypermethrin, okadaic acid or calcineurin inhibitory peptide, altered either caffeine- or IP3 -evoked [Ca2+]cyto release; calcineurin did not contribute to FK506-mediated potentiation of RyR- or IP3R-mediated Ca2+ release. The mTOR inhibitor LY294002, like rapamycin, decreased IP3 -evoked Ca2+ release. Conclusions and implications:, Ca2+ release in portal vein myocytes, via RyR, was modulated directly by FKBP binding to the channel; neither calcineurin nor mTOR contributed to this regulation. However, IP3R-mediated Ca2+ release, while also modulated directly by FKBP may be additionally regulated by mTOR. Rapamycin inhibition of IP3 -mediated Ca2+ release may be explained by mTOR inhibition. [source] | |||||||||||||||||||||||||||||||