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)

The in vivo state of phosphorylation and the modification of two Cys residues of neuromodulin/ GAP-43 (Nm) were analyzed by electrospray ionization-mass spectrometry (ES-MS). The protein was purified from rat brain with homogenization buffer containing 1% Nonidet P-40, protease inhibitors, protein phosphatase inhibitors, and sulfhydryl reagent, 4-vinylpyridine. Nm was purified by HPLC and ion-exchange chromatography, and the various fractions were identified by ES-MS as unphosphorylated and mono-, di-, tri-, and tetraphosphorylated species. All of these Nm species contained 2 mol of added 4-vinylpyridine per mol of Nm, suggesting that the two Cys residues are in the reduced form in the brain. In vivo, the majority of Nm is in the phosphorylated form (approximately 80%), of which the levels of the mono- and diphospho forms are higher than those of the tri- and tetraphospho species. Four in vivo phosphorylation sites, Ser41, Thr95, Ser142, and Thr172, were identified by amino acid sequencing and tandem ES-MS of the peptides derived from Lys-C endoproteinase digestion. Among these sites, only Ser41 is a known target of PKC, whereas the kinases responsible for the phosphorylation of the other three novel sites are unknown. Hypoxia/ischemia caused a preferential dephosphorylation of Ser41 and Thr172, whereas Thr95 is the least susceptible to dephosphorylation.
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PMID:Hypoxia/ischemia induces dephosphorylation of rat brain neuromodulin/GAP-43 in vivo. 1003 3

Astrocytes, the most abundant glial cell type in the brain, are considered to have physiological and pathological roles in neuronal activities. We found that reperfusion of cultured astrocytes after Ca2+ depletion causes delayed cell death and that the Na(+)-Ca2+ exchanger in the reverse mode is responsible for this Ca(2+)-mediated cell injury (Ca2+ paradox injury). The Ca2+ paradox injury of cultured astrocytes is considered to be an in vitro model of ischemia/reperfusion injury, since a similar paradoxical change in extracellular Ca2+ concentration is reported in ischemic brain tissue. Furthermore, we demonstrated that heat shock proteins, glutathione and calcineurin inhibitors protected astrocytes against Ca2+ paradox-induced cell toxicity. We also observed DNA fragmentation, a typical apoptotic ladder, 2-3 days after hydrogen peroxide exposure. In addition, laser microscopic observation showed that reperfusion after the exposure to hydrogen peroxide caused nuclear condensation of astrocytes. Hydrogen peroxide-induced cell injury and DNA fragmentation were attenuated by the NF-kappa B inhibitor ammonium pyrrolidinedithiocarbamate, 1,10-phenanthroline and a caspase 3 inhibitor. These findings suggest that astrocytes are one of the targets for ROS and the oxidative stress-induced delayed death of astrocytes is at least due to apoptosis.
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PMID:[Apoptosis of astroglial cells]. 1019 Jan 27

Small heat shock proteins (hsp) have been implicated in mediation of classic preconditioning in the rabbit, Hsp27 is a terminal substrate of the p38 MAPK cascade. One and 2D gel electrophoresis and immunoblotting of cell fractions was used to determine p38 MAPK and hsp27 phosphorylation levels, respectively, during in vitro ischemia in control, calyculin A (Cal A)-treated (protein phosphatase inhibitor), SB203580-treated (p38MAPK inhibitor) and preconditioned (IPC) isolated adult rabbit cardiomyocytes. The dual phosphorylation of p38 MAPK was increased by early ischemia (30-60 min), after which there was a loss of total cytosolic p38 MAPK. The ischemic increase of p38 MAPK dual phosphorylation was enhanced by IPC. Cal A strongly activated dual phosphorylation of p38 MAPK in oxygenated cells and this was maintained into early ischemia, SB203580 inhibited the dual phosphorylation of p38 MAPK and attenuated the loss of total cytosolic p38 MAPK. In each protocol, ischemia translocated hsp27 from the cytosolic fraction to the cytoskeletal fraction at similar rates and extents, Hsp27 phosphorylation was quantitated as the fraction of diphosphorylated hsp27, based on IEF mobility shifts of hsp27 phosphorylation isoforms. In oxygenated control cells, cytosolic and cytoskeletal hsp27 was highly phosphorylated. After 90 min ischemia, cytoskeletal hsp27 was markedly dephosphorylated. Cal A slightly increased control cytoskeletal hsp27 phosphorylation. During ischemic incubation, Cal A blocked ischemic dephosphorylation, SB203580 accelerated ischemic hsp27 dephosphorylation and injury, IPC insignificantly decreased the initial rate of ischemic dephosphorylation of hsp27, but not the extent of dephosphorylation in later ischemia. Phosphorylation is regulated by both kinase and phosphatase activities. IPC protection was not correlated with a significant increase in cytosolic or cytoskeletal hsp27 phosphorylation levels during prolonged (> 60-90 min) ischemia.
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PMID:Phosphorylation state of hsp27 and p38 MAPK during preconditioning and protein phosphatase inhibitor protection of rabbit cardiomyocytes. 1019 87

We investigated the changes in the enzyme activity and immunoreactivity of calcineurin in the rat hippocampus after transient forebrain ischemia. Immediately after 20-min transient forebrain ischemia, calcineurin activity decreased to about 40% of the control in the CA1 region and to about 55% in other regions. Protein phosphatase 2A activity showed no remarkable changes. By 12 h after ischemia, calcineurin activity recovered, more in the CA1 region than in other regions. At 24 h it decreased again, but only in the CA1 region. Immunohistochemical- and immunoblot analyses showed no remarkable change in calcineurin in any region of the hippocampus within 12 h after ischemia. Thus, the activity of calcineurin is dissociated from its immunoreactivity and quantity. Several studies have suggested that unknown inhibitory factor(s) and/or reversible changes in calcineurin act to modify enzyme activity after ischemia. In contrast, phosphatase 2A activity underwent no obvious changes during the post-ischemia period we examined. This unique time course of calcineurin activity may contribute to the mechanism of ischemic neuronal injury.
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PMID:Activities of calcineurin and phosphatase 2A in the hippocampus after transient forebrain ischemia. 1032 Jul 33

We have previously shown that the calcium-calmodulin-regulated phosphatase calcineurin (PP2B) is sufficient to induce cardiac hypertrophy that transitions to heart failure in transgenic mice. Given the rapid onset of heart failure in these mice, we hypothesized that calcineurin signaling would stimulate myocardial cell apoptosis. However, utilizing multiple approaches, we determined that calcineurin-mediated hypertrophy protected cardiac myocytes from apoptosis, suggesting a model of heart failure that is independent of apoptosis. Adenovirally mediated gene transfer of a constitutively active calcineurin cDNA (AdCnA) was performed in cultured neonatal rat cardiomyocytes to elucidate the mechanism whereby calcineurin affected myocardial cell viability. AdCnA infection, which induced myocyte hypertrophy and atrial natriuretic factor expression, protected against apoptosis induced by 2-deoxyglucose or staurosporine, as assessed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) labeling, caspase-3 activation, DNA laddering, and cellular morphology. The level of protection conferred by AdCnA was similar to that of adenoviral Bcl-x(L) gene transfer or hypertrophy induced by phenylephrine. In vivo, failing hearts from calcineurin-transgenic mice did not demonstrate increased TUNEL labeling and, in fact, demonstrated a resistance to ischemia/reperfusion-induced apoptosis. We determined that the mechanism whereby calcineurin afforded protection from apoptosis was partially mediated by nuclear factor of activated T cells (NFAT3) signaling and partially by Akt/protein kinase B (PKB) signaling. Although calcineurin activation protected myocytes from apoptosis, inhibition of calcineurin with cyclosporine was not sufficient to induce TUNEL labeling in Gqalpha-transgenic mice or in cultured cardiomyocytes. Collectively, these data identify a calcineurin-dependent mouse model of dilated heart failure that is independent of apoptosis.
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PMID:Calcineurin-mediated hypertrophy protects cardiomyocytes from apoptosis in vitro and in vivo: An apoptosis-independent model of dilated heart failure. 1067 75

Protein phosphorylation and dephosphorylation mediated by protein kinases and protein phosphatases, respectively, represent essential steps in a variety of vital neuronal processes that could affect susceptibility to ischemic stroke. In this study, the role of the neuron-specific gamma isoform of protein kinase C (gammaPKC) in reversible focal ischemia was examined using mutant mice in which the gene for gammaPKC was knocked-out (gammaPKC-KO). A period of 150 minutes of unilateral middle cerebral artery and common carotid artery (MCA/CCA) occlusion followed by 21.5 hours of reperfusion resulted in significantly larger (P < 0.005) infarct volumes (n = 10; 31.1+/-4.2 mm3) in gammaPKC-KO than in wild-type (WT) animals (n = 12; 22.6+/-7.4 mm3). To control for possible differences related to genetic background, the authors analyzed Balb/cJ, C57BL/6J, and 129SVJ WT in the MCA/CCA model of focal ischemia. No significant differences in stroke volume were detected between these WT strains. Impaired substrate phosphorylation as a consequence of gammaPKC-KO might be corrected by inhibition of protein dephosphorylation. To test this possibility, gammaPKC-KO mice were treated with the protein phosphatase 2B (calcineurin) inhibitor, FK-506, before ischemia. FK-506 reduced (P < 0.008) the infarct volume in gammaPKC-KO mice (n = 7; 24.6+/-4.6 mm3), but at this dose in this model, had no effect on the infarct volume in WT mice (n = 7; 20.5+/-10.7 mm3). These results indicate that gammaPKC plays some neuroprotective role in reversible focal ischemia.
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PMID:Interplay between the gamma isoform of PKC and calcineurin in regulation of vulnerability to focal cerebral ischemia. 1069 72

The development of immunosuppressive agents reflects the progress in understanding the cellular and molecular mechanisms which mediate allograft rejection. Six paradigms represent the evolution of immunosuppressive strategies for organ transplantation. The proliferation paradigm advances agents which interrupt lymphocyte cell division (azathioprine, cyclophosphamide, mycophenolic acid). The depletion paradigm conscripts drugs that bind to lymphocyte cell surface markers, thereby producing cell lysis and/or inactivation (polyclonal ATGAM and thymoglobulin, and monoclonal OKT3 antilymphocyte antibodies). The cytokine paradigm uses agents that interrupt lymphocyte maturational events; eg, synthesis (calcineurin inhibitors: cyclosporine/tacrolimus), binding to surface receptors (anti-CD25 mAbs), or signal transduction phases of cytokine stimulation (sirolimus). The introduction of calcineurin inhibitors markedly reduces the rate of acute rejection episodes and increases short-term graft survival rates; nephrotoxicity and chronic allograft attrition remain as unanswered challenges. The cyclosporine A (CsA) sparing property of sirolimus permits the use of lower exposure to calcineurin agents, allows for early withdrawal of steroid therapy, and may delay allograft senescence. Furthermore, the combination of SRL with anti-IL-2R mAbs proffers an induction approach which allows prolonged periods of holiday from calcineurin inhibitors. To address the tissue nonselectivity of the calcineurin and mTOR inhibitors, which presumably causes the drug toxicities, new agents are being developed to selectively inhibit the T cell target Janus Kinase 3. In the costimulation paradigm, the accessory signals generated by antigen-presenting cells are interrupted by distinct agents: the receptor conjugate CTLA4-immunoglobulin and anti-B7 or anti-CD40 ligand mAbs. Another set of drugs (selectin blocking agents, anti-ICAM-1 antisense deoxy oligonucleotides, and the lymphocyte homing inhibitor FTY720) seeks to modulate the ischemia-reperfusion injury, which exacerbates cytokine-mediated events in the donor and the subsequent procurement injury and may also accelerate the progression of transplant senescence. Finally, the transplantation tolerance paradigm is based on the development of strategies which distort alloimmune recognition by antigen reactive cells (MHC peptides or proteins), produce anergy (costimulation blockers), functional inactivation, or deletion of antigen-reactive cells (donor bone marrow infusions and gene therapy). Presently, the optimal immunosuppressive strategy uses combinations of agents that act in synergistic fashion to provide the potency, freedom from toxic reactions, convenience of administration, and cost appropriate for the individual patient.
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PMID:Immunosuppressive agents in organ transplantation: past, present, and future. 1074 55

Alpha B Crystallin (alpha BC) is a putative effector protein of ischemic preconditioning (IPC), that is phosphorylated on Ser 45 by ERK1/2 and Ser 59 by the p38 MAPK substrate, MAPKAPK-2. Translocation and phosphorylation of alpha BC was determined in cytosolic and cytoskeletal fractions by 1D SDS-PAGE and IEF, or using Ser 45 and Ser 59 phospho-specific antibodies in: (1) control rabbit cardiomyocytes; (2) cells preconditioned by 10 min in vitro ischemia; or after pre-treatment with specific inhibitors of (3) Ser/Thr protein phosphatase 1/2A (calyculin A); (4) p38 MAPK (SB203580); or (5) ERK 1/2 (PD98059); all prior to 180 min ischemia. Ischemia induced a cytosolic to cytoskeletal translocation of alpha BC, which was similar in all the groups. Highly phosphorylated isoforms (D1/2) of alpha BC were present in cytosolic but not cytoskeletal fractions at 0 min ischemia. By 60-90 min ischemia, D1/2 isoforms had translocated to the cytoskeletal fraction. Calyculin A maintained D1/2 levels throughout prolonged ischemia. SB203580 decreased alpha BC phosphorylation. Neither PD98059 nor IPC altered alpha BC phosphorylation during prolonged ischemia. It is concluded that alpha BC phosphorylation during ischemia is regulated by p38 MAPK but not by ERK 1/2. The inability to detect a correlation between IPC protection and either alpha BC translocation or phosphorylation suggests that the proteins in the highly phosphorylated isoform bands of alpha BC quantitated in this study are not protective end effectors of classical IPC.
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PMID:Differential translocation or phosphorylation of alpha B crystallin cannot be detected in ischemically preconditioned rabbit cardiomyocytes. 1086 Jul 71

Heat stress proteins (HSPs), in particular HSP72, seem to play a major role in cell protection against lethal stresses such as hyperthermia or ischemia. HSP synthesis is negatively regulated by protein phosphatases, which are implicated in dephosphorylation processes. In the present study, we have investigated the effect of okadaic acid (OA, a protein phosphatase inhibitor) on heat stress-induced HSP72 synthesis and thermotolerance in smooth muscle cells (SMC). SMC were heat stressed (42 degrees C for 20 minutes) in the presence of 250 nM OA (HS+OA cells) or its vehicle (HS+V cells). Control (OA or V) cells were not heat stressed. HSP72 mRNA expression was determined 1, 1.5, 3, and 6 hours after heat stress by RT-PCR, and HSP72 synthesis was determined 6, 12, 24, 48, and 72 hours after heat stress by Western blotting. SMC survival of lethal hyperthermia (47 degrees C for 90 minutes) was assessed 6, 24, and 48 hours after heat stress by a tetrazolium assay. The maximal expression of HSP72 mRNA was markedly prolonged in HS+OA cells (until 6 hours after heat stress) compared to HS+V cells (1 hour after heat stress). The kinetics of HSP72 synthesis and thermotolerance of SMC were not different between HS+OA and HS+V cells. Baseline HSP72 mRNA and protein expression were similar in control V and OA cells. In conclusion, okadaic acid treatment of SMC potentiated HSP72 mRNA expression without affecting heat stress-induced HSP72 synthesis and thermotolerance.
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PMID:Effect of okadaic acid, a protein phosphatase inhibitor, on heat stress-induced HSP72 synthesis and thermotolerance. 1099 52

Brain ischemia and reperfusion engage multiple independently-fatal terminal pathways involving loss of membrane integrity in partitioning ions, progressive proteolysis, and inability to check these processes because of loss of general translation competence and reduced survival signal-transduction. Ischemia results in rapid loss of high-energy phosphate compounds and generalized depolarization, which induces release of glutamate and, in selectively vulnerable neurons (SVNs), opening of both voltage-dependent and glutamate-regulated calcium channels. This allows a large increase in cytosolic Ca(2+) associated with activation of mu-calpain, calcineurin, and phospholipases with consequent proteolysis of calpain substrates (including spectrin and eIF4G), activation of NOS and potentially of Bad, and accumulation of free arachidonic acid, which can induce depletion of Ca(2+) from the ER lumen. A kinase that shuts off translation initiation by phosphorylating the alpha-subunit of eukaryotic initiation factor-2 (eIF2alpha) is activated either by adenosine degradation products or depletion of ER lumenal Ca(2+). Early during reperfusion, oxidative metabolism of arachidonate causes a burst of excess oxygen radicals, iron is released from storage proteins by superoxide-mediated reduction, and NO is generated. These events result in peroxynitrite generation, inappropriate protein nitrosylation, and lipid peroxidation, which ultrastructurally appears to principally damage the plasmalemma of SVNs. The initial recovery of ATP supports very rapid eIF2alpha phosphorylation that in SVNs is prolonged and associated with a major reduction in protein synthesis. High catecholamine levels induced by the ischemic episode itself and/or drug administration down-regulate insulin secretion and induce inhibition of growth-factor receptor tyrosine kinase activity, effects associated with down-regulation of survival signal-transduction through the Ras pathway. Caspase activation occurs during the early hours of reperfusion following mitochondrial release of caspase 9 and cytochrome c. The SVNs find themselves with substantial membrane damage, calpain-mediated proteolytic degradation of eIF4G and cytoskeletal proteins, altered translation initiation mechanisms that substantially reduce total protein synthesis and impose major alterations in message selection, down-regulated survival signal-transduction, and caspase activation. This picture argues powerfully that, for therapy of brain ischemia and reperfusion, the concept of single drug intervention (which has characterized the approaches of basic research, the pharmaceutical industry, and clinical trials) cannot be effective. Although rigorous study of multi-drug protocols is very demanding, effective therapy is likely to require (1) peptide growth factors for early activation of survival-signaling pathways and recovery of translation competence, (2) inhibition of lipid peroxidation, (3) inhibition of calpain, and (4) caspase inhibition. Examination of such protocols will require not only characterization of functional and histopathologic outcome, but also study of biochemical markers of the injury processes to establish the role of each drug.
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PMID:Brain ischemia and reperfusion: molecular mechanisms of neuronal injury. 1105 82


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