Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using 32P-labeled phosphocasein or phosphohistones as exogenous substrates it was possible to detect a phosphoprotein phosphatase activity on the outer surface of intact normal and transformed 3T3 fibroblasts. Incubation of monolayers of intact cells in buffered salt solution with the radioactively labeled substrate resulted in the release of alkali-labile 32P counts into the surrounding medium. The reaction was: (a) linear with time (at least up to 20 min); (b) proportional to the cell density; (c) dependent on the temperature and pH of the incubation medium; (d) stimulated by K+; and (e) inhibited by sodium fluoride, inorganic pyrophosphate, zinc chloride and relatively impermeant sulfhydryl reagents. Less than 2% of the externally located phosphoprotein phosphatase activity was detectable in pooled cell-free washings of the intact cell monolayer. Phosphocasein did not cause any detectable leakage of intracellular lactate dehydrogenase or soluble phosphoprotein phosphatase activity into the external medium; incubation of the cells with phosphohistones, on the other hand, resulted in appreciable leakage of both these cytoplasmic activities. Neoplastic transformation was associated with a nearly two-fold decrease in the activity of the surface phosphoprotein phosphatase. Addition of serum to either non-transformed 3T3 or spontaneously transformed 3T6 cells resulted in a rapid and remarkeable drop in the cell surface dephosphorylating activity. Acrylamide gel electrophoresis of the dephosphorylated casein or histone substrate revealed no proteolytic degradation or change in electrophoretic mobility. The intact cells showed no damage upon microscopic examination as a result of exposure to phosphocasein or phosphohistones.
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PMID:Phosphoprotein phosphatase activity at the outer surface of intact normal and transformed 3T3 fibroblasts. 22 67

Okadaic acid (OA), a potent inhibitor of protein phosphatase type 1 and protein phosphatase type 2A was studied for its effect on bone resorption in neonatal mouse calvaria. OA (0.01 to 1000 ng/ml) had no effect on the basal bone resorption rate, except at 1000 ng/ml, were a small inhibitory effect was observed. Resorption stimulated by parathyroid hormone (10(-8) M) was abolished in the presence of OA, half maximal inhibition being observed at 1 ng/ml. However, at 50 ng/ml or higher, OA significantly increased lactate dehydrogenase activity in the medium, indicating a cytotoxic effect at these concentrations. Similar inhibitory effects were observed when bone resorption was stimulated by 1,25-dihydroxycholecalciferol (10(-8) M) or prostaglandin E2 (10(-6) M). From this it is concluded that protein dephosphorylation may represent an important regulatory mechanism in the bone resorption process.
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PMID:Inhibitory effect of okadaic acid on bone resorption in neonatal mouse calvaria in vitro. Protein dephosphorylation as an important regulatory mechanism in the bone resorption process. 165 Jan 97

To learn whether autophagy might be dependent on any of the major cytoskeletal elements, the effect of various cytoskeleton inhibitors on autophagy and cytoskeletal organization was studied in isolated rat hepatocytes. Autophagy, measured as the sequestration of endogenous lactate dehydrogenase, was completely inhibited in isolated rat hepatocytes by the protein phosphatase inhibitor okadaic acid (30 nM). Only small effects were seen with vinblastine (10 microM) or cytochalasin D (10 microM). Indirect immunofluorescence microscopy with antibody to a 55-kDa cytokeratin, corresponding to human cytokeratin 8 (CK8), revealed that whereas control cells contained a well-organized network of cytokeratin intermediate filaments, okadaic acid disrupted this network into small spherical aggregates. Treatment with cytochalasin D or vinblastine, which disrupt microfilaments and microtubules, respectively, had no detectable effect on the cytokeratin filament distribution. Neither the microtubule network (detected by indirect immunofluorescence with antibodies against alpha- and beta-tubulin) nor the actin microfilament network (detected by rhodamine-palloidin) was disrupted by okadaic acid. Naringin (100 microM), a putative protein kinase-inhibitory flavonoid, offered complete protection against the autophagy-inhibitory and cytokeratin-disruptive effects of okadaic acid. Two other flavonoids, genistein (100 microM) and prunin (100 microM), as well as KN-62 (10 microM), a specific inhibitor of Ca2+/calmodulin-dependent kinase II), likewise displayed a good ability to protect against the effect of okadaic acid upon cytokeratin organization, while no such protection was seen with H-89 (20 microM), an inhibitor of the cyclic nucleotide-dependent protein kinases, or with H-7 (100 microM), which in addition inhibits protein kinase C. The results suggest that the cytokeratin cytoskeleton of hepatocytes is subject to rapid control by phosphorylation and dephosphorylation and that cytokeratin filaments may somehow be involved in the autophagic process.
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PMID:Disruption of the cytokeratin cytoskeleton and inhibition of hepatocytic autophagy by okadaic acid. 754 Sep 86

Six monoclonal antibodies (MAbs) to microcystin-LR (MCLR), a cyclic heptapeptide hepatotoxin isolated from the cyanobacterium Microcystis aeruginosa, were produced. They showed the protective effects on hepatotoxicity of MCLR in vitro and in vivo, and on the inhibition of protein phosphatase by MCLR. Competitive enzyme-linked immunosorbent assays with various microcystins revealed that the six MAbs recognized a part of the molecule, in particular, a tertial structure around Adda, 3-amino-9-methoxy-2,6,8,-trimethyl-10-phenyldeca-4,6-dienoic acid. The specificity of these MAbs varied slightly. In primary rat hepatocyte cultures, all MAbs showed protective effects against the MCLR-induced cell damages, assessed by morphological changes, lactate dehydrogenase release into the medium, and a calorimetric assay to measure the cell viability using a tetrazolium dye. The M8H5 MAb showing the highest affinity for MCLR blocked the lethal effects and hepatocellular damage to mice. In addition, M8H5 MAb recovered protein phosphatase 2A inhibition by MCLR in a dose-dependent manner, while phosphatase inhibition by okadaic acid was not affected. Thus, the MAbs specifically reacted with the microcystins and prevented their biological activities. This is the first report on the protective effects of specific monoclonal antibodies on MCLR-induced toxicity.
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PMID:Novel monoclonal antibodies against microcystin and their protective activity for hepatotoxicity. 761 39

Autophagy, measured as the sequestration of electroinjected [3H]raffinose or endogenous lactate dehydrogenase, was inhibited in isolated rat hepatocytes by the protein phosphatase inhibitors okadaic acid, calyculin A and microcystin-LR. Okadaic acid, the most potent inhibitor, suppressed autophagy almost completely at 15 nM, suggesting inhibition of a protein phosphatase of type 2A. Okadaic acid had no effect on ATP levels, protein synthesis or cellular viability at this concentration, but caused a disruption of the hepatocytic cytoskeleton and a consequent reduction in organelle sedimentability, potentially interfering with the autophagy assay unless the necessary precautions are taken. Lysosomal (propylamine-sensitive) degradation of endogenous protein was inhibited by okadaic acid, whereas non-lysosomal (propylamine-resistant) degradation was unaffected. The autophagy-inhibitory effect of okadaic acid was not affected by inhibitors of cAMP-dependent protein kinase or protein kinase C (H-7, H-89, calphostin C) but eliminated by the non-specific inhibitor K-252a and its analogues (KT-5720, KT-5823, KT-5926) and by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II. Protein phosphorylation by this kinase would thus seem to play a role in regulation of the autophagic-lysosomal degradation pathway.
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PMID:Inhibition of hepatocytic autophagy by okadaic acid and other protein phosphatase inhibitors. 839 87

The present work was undertaken to test whether cytoskeletal components are involved in the control of rat-liver carnitine palmitoyltransferase I (CPT-I) activity by cellular effectors. The microtubule stabilizer taxol abolished the changes in CPT-I activity induced by the effectors tested. Taxol also prevented OA-induced shrinkage of hepatocytes as well as the enhanced release of lactate dehydrogenase from digitonin-permeabilized hepatocytes. On the basis of its relative sensitivity to tautomycin and OA, the modulation of CPT-I activity seemed to involve mostly protein phosphatase 1. These data suggest that the short-term control of hepatic CPT-I by cellular effectors may involve modulation of interactions between CPT-I and cytoskeletal components.
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PMID:Are cytoskeletal components involved in the control of hepatic carnitine palmitoyltransferase I activity? 871 18

In a model of anoxia/reoxygenation (A/R) of cultured rat cardiomyocytes, protection of cellular damage, activation of protein kinase C (PKC) and PKC-mediated protein phosphorylation by anoxic preconditioning (APC) can be demonstrated as shown by the increase of cell viability, attenuation of formation of lipid peroxides (MDA) and lowering of the leakage of lactate dehydrogenase (LDH) and protein from cells. The results also show that transient anoxia mimicked the outcomes of activation of PKC as shown by increased incorporation of 32P, especially in the 66 kD and 31 kD protein fractions. Preincubation of cardiomyocytes with H7 (an inhibitor of PKC) completely abolished these protective effects of transient anoxia. Protein phosphatase inhibitor OA mimicked the protective effects of A/R, while protein phosphatase activator BDM induced a complete abolishment. In short, we conclude that the protective effect of APC is medicated by activation of PKC.
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PMID:[Effect of anoxic preconditioning on protein kinase C activity in neonatal rat cardiomyocytes]. 981 75

Activation of the transcription factor nuclear factor of activated T cells by the calcium-sensitive serine/threonine phosphatase calcineurin has been proposed as one of the molecular mechanisms by which motor nerve activity establishes the slow muscle phenotype. To investigate whether the calcineurin pathway can regulate the large spectrum of slow muscle characteristics in vivo, we treated rats for three weeks with cyclosporin A (an inhibitor of calcineurin). In soleus (slow muscle), but not in plantaris (fast muscle), the proportion of slow myosin heavy chain (MHC-1) and slow sarcoplasmic reticulum ATPase (SERCA2a) was decreased, whereas that of fast MHC (MHC-2A) and fast SERCA1 increased, indicating a slow to fast contractile phenotype transition. Cytosolic isoforms of creatine kinase and lactate dehydrogenase (most abundant in fast fibers), as well as mitochondrial creatine kinase and citrate synthase activities (elevated in fast/oxidative fibers) were dose dependently increased by cyclosporin A treatment in soleus muscle, with no change in plantaris. Calcineurin catalytic subunit was more abundant in soleus muscle fibers compared with plantaris. Taken together these results suggest that the calcineurin pathway co-regulates a set of multigenic protein families involved in the transition between slow oxidative (type I) to fast oxidative (type IIa) phenotype in soleus muscle.
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PMID:Calcineurin Co-regulates contractile and metabolic components of slow muscle phenotype. 1077 82

L-Deprenyl, an irreversible MAO-B (monoamine oxidase B, EC 1.4.3.4) inhibitor, is used for the treatment of Parkinson's disease and to delay the progression of Alzheimer's disease. L-Deprenyl also exhibits protective effects against neuronal apoptosis which are independent of its ability to inhibit MAO-B. The purpose of this study was to compare the antiapoptotic efficacy of L-deprenyl against different types of apoptotic inducers in three neuronal cell culture models. The level of apoptosis was quantified by measuring the activation of caspase-3 enzyme, which is the main apoptotic executioner in neuronal cells. MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] and LDH (lactate dehydrogenase, EC 1. 1.1.27) assays were used to demonstrate the cytotoxic response of apoptotic treatments. Our results showed that okadaic acid, an inhibitor of protein phosphatase 1 and 2A, induced a prominent increase in caspase-3 activity both in cultured hippocampal and cerebellar granule neurons as well as in Neuro-2a neuroblastoma cells. Interestingly, L-deprenyl offered a significant protection against the apoptotic response induced by okadaic acid in all three neuronal models. The best protection appeared at the concentration level of 10(-9) M. L-Deprenyl also provided a protection against apoptosis after AraC (cytosine beta-D-arabinoside) treatment in hippocampal neurons and Neuro-2a cells and after etoposide treatment in Neuro-2a cells. However, L-deprenyl did not offer any protection against apoptosis caused by serum withdrawal or potassium deprivation. Okadaic acid treatment in vivo is known to induce an Alzheimer's type of hyperphosphorylation of tau protein, formation of beta-amyloid plaques, and a severe memory impairment. Our results show that the okadaic acid model provides a promising tool to study the molecular basis of Alzheimer's disease and to screen the neuroprotective capacity of L-deprenyl derivatives.
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PMID:Protective effect of L-deprenyl against apoptosis induced by okadaic acid in cultured neuronal cells. 1079 57

The effects of marine substances with various cytotoxic mechanisms on the integrity of the human intestinal Caco-2 cell monolayer were examined by measuring the transepithelial electrical resistance (TEER). TEER was rapidly decreased by apical exposure of the monolayers to discodermin A, a membrane pore-forming substance. The decrease in TEER occurred in an earlier stage of incubation than the release of intracellular lactate dehydrogenase (LDH) which is commonly used as a parameter of cell damage or death. Mycalolide B (an actin-depolymerizing substance), calyculin A and okadaic acid (protein phosphatase inhibitors) also rapidly decreased the TEER value, although no cell membrane damage or resultant LDH release by these toxicants were detected. The TEER decrease caused by the toxicants was associated with the increased transepithelial permeability of the cell monolayer. Treatment with these toxicants, except calyculin A, caused morphological changes in the intracellular actin filament, suggesting that these toxicants altered the cytoskeletal structure, by which the tight junction was opened. Calyculin A was likely to loosen the cellular junctions rapidly and induce cell detachment from the monolayer. Although onnamide A, a protein synthesis inhibitor, did not cause any decrease in TEER, at least during a 90-min incubation, TEER sensitively reflects the cytotoxic effects of various types of toxicants with acute toxicity.
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PMID:Assessment of the marine toxins by monitoring the integrity of human intestinal Caco-2 cell monolayers. 1080 72


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