Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.16 (calcineurin)
 17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pK(a) approximately 8. In an initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU, all with pK(a) values lower than that of 4-MU, we found that one substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), was much improved for the detection of acid phosphatase activity. When measured at the preferred acid phosphatase reaction pH (5.0), DiFMUP yielded fluorescence signals that were more than 10-fold higher than those of 4-methylumbelliferyl phosphate (MUP). DiFMUP was also superior to MUP for the detection of protein phosphatase 1 activity at pH 7 and was just as sensitive as MUP for the detection of alkaline phosphatase activity at pH 10. A beta-galactosidase substrate was also prepared based on 6, 8-difluoro-4-methylumbelliferone. This substrate, 6, 8-difluoro-4-methylumbelliferyl beta-d-galactopyranoside (DiFMUG), was found to be considerably more sensitive than the commonly used substrate 4-methylumbelliferyl beta-d-galactopyranoside (MUG), for the detection of beta-galactosidase activity at pH 7. DiFMUP and DiFMUG should have great utility for the continuous assay of phosphatase and beta-galactosidase activity, respectively, at neutral and acid pH.
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PMID:Fluorogenic substrates based on fluorinated umbelliferones for continuous assays of phosphatases and beta-galactosidases. 1045 97

Fusions of the glycolytic genes TPI1, PGK1, ENO1, PYK1, PDC1, and ADH1 with the lacZ reporter gene of Escherichia coli and a lacZ fusion construct of a 390-bp fragment from the promoter of the HXT7 gene were assayed for beta-galactosidase activity. The glycolytic promoters were induced after addition of glucose to ethanol-grown cells, whereas the HXT7 promoter fragment showed a constitutive beta-galactosidase expression on both carbon sources. The genes coding for the seven enzymes of lower glycolysis Tdh, Pgk, Gpm, Eno, Pyk, Pdc, and Adh were simultaneously put under the control of the same strong promoter, a truncated HXT7 promoter that is constitutively active on ethanol as well as on glucose medium. Genomic expression of the glycolytic genes under the control of this promoter, resulted in an at least 2-fold overexpression. The gene MSG5 was isolated, coding for a protein phosphatase normally involved in cell cycle regulation, as a factor that possibly influences the expression of the HXT7 gene. However, overexpression of MSG5 had no effect on the expression of the HXT7/lacZ fusion, whereas a deletion of this gene resulted in a decreased expression of beta-galactosidase.
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PMID:Simultaneous genomic overexpression of seven glycolytic enzymes in the yeast Saccharomyces cerevisiae. 1086 74

The PPP1R3 gene encoding the G-subunit of protein phosphatase-1 has three polymorphisms in linkage disequilibrium in the Pima Indians: an mRNA-destabilizing element in the 3'-untranslated region (ARE1/ARE2 alleles), Arg883Ser, and Asp905Tyr substitutions. The ARE2 allele, Arg883, and Asp905 variants are associated with insulin resistance and higher prevalence of type 2 diabetes in the Pima Indians. The ARE2 allele is associated with lower PPP1R3 transcript and protein levels in muscle tissue. Here we determined the functional contribution of the amino acid substitutions independent of the ARE alleles to insulin-stimulated glycogen synthesis by adenoviral-mediated gene expression in L6 myotubes. Similar overexpression levels of the G-subunit variants increased glycogen synthase fractional activity in the presence ( approximately 1. 5-fold) of insulin compared to control myotubes transduced with adenovirus encoding beta-galactosidase. The glycogen synthesis rate of myotubes overexpressing the G-subunit variants also increased by approximately 1.7-fold over the control with and without insulin. However, these measures were not significantly different among the variants. This study does not support a role for Arg883 and Asp905 variants independent of the ARE2 allele in the impaired insulin-stimulated glycogen synthesis in the muscle of Pima Indians.
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PMID:Functional analyses of amino acid substitutions Arg883Ser and Asp905Tyr of protein phosphatase-1 G-subunit. 1087 97

Analysis of protein-protein interaction is one of the powerful methods for elucidating the new functions of functionally unknown proteins. Using this approach, we isolated two proteins interacting with PRIP-1, which was isolated as a new Ins(1,4,5) P3 binding protein from brain. One was protein phosphatase 1 catalytic subunit (PP1c) and the other was GABARAP (GABAA-receptor-associated protein). The region of PRIP-1 responsible for their interaction was the site preceding to the pleckstrin homology domain of PRIP-1 for PP1c and the EF-hand motifs of PRIP-1 for GABARAP, which were determined by beta-galactosidase assay of yeast two-hybrid system. The association between PRIP-1 and PP1c was confirmed in vitro by a pull-down assay, a far-western assay, an immunoprecipitation analysis and a surface plasmon resonance analysis. The interaction of PRIP-1 with PP1c resulted in inhibition of the catalytic activity of PP1c in a PRIP-1 concentration-dependent manner. The association between PRIP-1 and GABARAP was also confirmed by a pull-down assay, and we found that PRIP-1 competitively inhibited the binding of the gamma 2 subunit of the GABAA receptor to GABARAP in vitro. Our electrophysiological and behavioral analysis of PRIP-1 knockout mice revealed that PRIP-1 is essential for the function of GABAA receptors, especially in response to the agents acting on the gamma 2 subunit.
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PMID:[The analysis of protein-protein interaction with special reference to PRIP-1]. 1197 30

The Down syndrome critical region 1 (DSCR1) gene is present in the region of human chromosome 21 and the syntenic region of mouse chromosome 16, trisomy of which is associated with congenital heart defects observed in Down syndrome. DSCR1 encodes a regulatory protein in the calcineurin/NFAT signal transduction pathway. During valvuloseptal development in the heart, DSCR1 is expressed in the endocardium of the developing atrioventricular and semilunar valves, the muscular interventricular septum, and the ventricular myocardium. Human DSCR1 contains an NFAT-rich calcineurin-responsive element adjacent to exon 4. Transgenic mice generated with a homologous regulatory region of the mouse DSCR1 gene linked to lacZ (DSCR1(e4)/lacZ) show gene activation in the endocardium of the developing valves and aorticopulmonary septum of the heart, recapitulating a specific subdomain of endogenous DSCR1 cardiac expression. DSCR1(e4)/lacZ expression in the developing valve endocardium colocalizes with NFATc1 and, endocardial DSCR1(e4)/lacZ, is notably reduced or absent in NFATc1(-/-) embryos. Furthermore, expression of the endogenous DSCR1(e4) isoform is decreased in the outflow tract of NFATc1(-/-) hearts, and the DSCR1(e4) intragenic element is trans-activated by NFATc1 in cell culture. In trisomy 16 (Ts16) mice, expression of endogenous DSCR1 and DSCR1(e4)/lacZ colocalizes with anomalous valvuloseptal development, and transgenic Ts16 hearts have increased beta-galactosidase activity. DSCR1 and DSCR1(e4)/lacZ also are expressed in other organ systems affected by trisomy 16 in mice or trisomy 21 in humans including the brain, eye, ear, face, and limbs. Together, these results show that DSCR1(e4) expression in the developing valve endocardium is dependent on NFATc1 and support a role for DSCR1 in normal cardiac valvuloseptal formation as well as the abnormal development of several organ systems affected in individuals with Down syndrome.
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PMID:DSCR1 gene expression is dependent on NFATc1 during cardiac valve formation and colocalizes with anomalous organ development in trisomy 16 mice. 1473 82

Voluntary cage wheel exercise has been used extensively to determine the physiological adaptation of cardiac and skeletal muscle in mice. In this study, we tested the effect of different loading conditions on voluntary cage wheel performance and muscle adaptation. Male C57Bl/6 mice were exposed to a cage wheel with no-resistance (NR), low-resistance (LR), or high-resistance (HR) loads for 7 wk. Power output was elevated (3-fold) under increased loading (LR and HR) conditions compared with unloaded (NR) exercise training. Only unloaded (NR) exercise induced an increase in heart mass, whereas only loaded (LR and HR) exercise training induced an increase in skeletal (soleus) muscle mass. Moreover, unloaded and loaded exercise training had a differential impact on the cross-sectional area of muscle fibers, depending on the type of myosin heavy chain expressed by each fiber. The biochemical adaptation of the heart was characterized by a decrease in genes associated with pathological (but not physiological) cardiac hypertrophy and a decrease in calcineurin expression in all exercise groups. In addition, transcriptional activity of myocyte enhancer factor-2 (MEF-2) was significantly decreased in the hearts of the LR group as determined by a MEF-2-dependent transgene driving the expression of beta-galactosidase. Phosphorylation of glycogen synthase kinase-3beta, protein kinase B (Akt), and p70 S6 kinase was increased only in the hearts of the NR group, consistent with the significant increase in cardiac mass. In conclusion, unloaded and loaded cage wheel exercise have a differential impact on cage wheel performance and muscle (cardiac and skeletal) adaptation.
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PMID:Loaded wheel running and muscle adaptation in the mouse. 1573 90

The calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway has been shown to be of critical importance in regulating the growth response of cardiac myocytes. We have previously demonstrated that calcineurin A(beta) (CnA(beta)) mRNA and protein are increased in response to growth stimulation, although the precise regulatory mechanism underlying CnA(beta) upregulation is not clear. Here, we isolated the mouse CnA(beta) promoter and characterized its responsiveness to growth stimuli in vitro and in vivo. A 2.3-kb promoter fragment was strongly activated by phenylephrine and endothelin-1 stimulation and by cotransfection with constitutively active CnA, NFATc4, and GATA4. Using chromatin immunoprecipitation, sequence regions were identified within the 2.3-kb promoter that associated with NFAT and GATA4, as well as with acetylated histone H3, following agonist stimulation. Consistent with the chromatin immunoprecipitation experiments, deletion of the distal half of the CnA(beta) promoter severely reduced NFAT, GATA4, and hypertrophic agonist-mediated activation. To investigate in vivo activity, we generated beta-galactosidase (LacZ) containing transgenic mice under the control of the CnA(beta) 2.3-kb promoter. CnA(beta)-LacZ mice showed expression in the heart that was cyclosporine sensitive, as well as expression in the central nervous system and skeletal muscle from early embryonic stages through adulthood. CnA(beta)-LacZ mice were subjected to cardiac pressure overload stimulation and crossbreeding with mice containing cardiac-specific transgenes for activated calcineurin and NFATc4, which revealed inducible expression in the heart. These results indicate that the CnA(beta) 2.3-kb promoter is specifically activated by hypertrophic stimuli through a positive feedback mechanism involving NFAT and GATA4 transcription factors, suggesting transcriptional induction of CnA(beta) expression as an additional means of regulating calcineurin activity in the heart.
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PMID:Regulation of calcineurin through transcriptional induction of the calcineurin A beta promoter in vitro and in vivo. 1602

Proliferation of vascular smooth muscle cells (VSMC) is a primary cause of vascular disorders and is associated with major alterations in Ca2+ handling supported by loss of the sarco/endoplasmic reticulum calcium ATPase, SERCA2a. To determine the importance of SERCA2a in neointima formation, we have prevented loss of its expression by adenoviral gene transfer in a model of balloon injury of the rat carotid artery. Two weeks after injury, the intima/media ratio was significantly lower in SERCA2a-infected than in injured noninfected or injured beta-galactosidase-infected carotids (0.29+/-0.04 versus 0.89+/-0.19 and 0.72+/-0.14, respectively; P<0.05), and was comparable to that observed in control carotids (0.21+/-0.03). The pathways leading to proliferation were analyzed in serum-stimulated VSMC. Forced expression of SERCA2a arrested cell cycle at the G1 phase and prevented apoptosis. SERCA2a inhibits proliferation through inactivation of calcineurin (PP2B) and its target transcription factor NFAT (nuclear factor of activated T-cells) resulting in lowering of cyclin D1 and pRb levels. By using NFAT-competing peptide VIVIT, we showed that NFAT activity is strongly required to promote VSMC proliferation. In conclusion, we provide the first evidence that increasing SERCA2a activity inhibits VSMC proliferation and balloon injury-induced neointima formation.
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PMID:Sarco/endoplasmic reticulum Ca2+-ATPase gene transfer reduces vascular smooth muscle cell proliferation and neointima formation in the rat. 1608 70

Emerging new research suggests that the functions of the angiotensin (Ang) II type 1 (AT(1)) receptor are regulated in a complex manner. AT(1) receptor-associated protein (ATRAP) has been reported to reduce AT(1) receptor signaling with enhancement of AT(1) receptor internalization and to regulate the calcineurin/nuclear factor of activated T cells (NFAT) pathway. We examined the possibility that ATRAP could attenuate AT(1) receptor-mediated vascular senescence via inactivation with the calcineurin/NFAT pathway. Ang II stimulation significantly increased senescence-associated beta-galactosidase (SA-beta-gal)-stained cells, oxidative stress, and expression of p53 and p21 in wild-type (WT) vascular smooth muscle cells (VSMC). Moreover, in WT VSMC, Ang II stimulation enhanced NFAT transcriptional activity, which was prevented by CAML-siRNA treatment. NFAT-siRNA treatment attenuated Ang-II-increased SA-beta-gal activity and p53 and p21 expression. Treatment with a calcineurin activity inhibitor, cyclosporin A, reduced Ang-II-induced NFAT transcriptional activity and senescent VSMC. In contrast, VSMC prepared from ATRAP transgenic (ATRAP-Tg) mice exhibited attenuation of Ang-II-induced SA-beta-gal activity, oxidative stress, NFAT transcriptional activity, and expression of p53 and p21. Moreover, ATRAP-Tg VSMC showed a more reduction of Ang-II-induced NFAT transcriptional activity by CAML-siRNA treatment than WT VSMC. Furthermore, we demonstrated that in ATRAP-Tg VSMC, NFAT activity and senescent cells induced by ultraviolet irradiation were decreased compared with those in WT VSMC. Treatment with an AT(1) receptor blocker, valsartan, blocked these senescent cells but did not change NFAT activity in both cells. These results suggest that ATRAP negatively regulates VSMC senescence by reducing AT(1) receptor signaling, and that ATRAP-mediated inactivation of the calcineurin/NFAT pathway could be at least partly involved in prevention of VSMC senescence, irrespective of AT(1) receptor blockade in some conditions.
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PMID:Angiotensin II type 1 receptor-associated protein prevents vascular smooth muscle cell senescence via inactivation of calcineurin/nuclear factor of activated T cells pathway. 1976 83


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