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 FeZn derivative of purple acid phosphatase from porcine uterus (FeZnUf) and its phosphate complex (FeZnUf.PO4) have been characterized by X-ray absorption spectroscopy at both the iron and zinc K-edges, to gain insight into the nature of the FeZn active site as well as the phosphate binding mode. Pre-edge data show that both FeZnUf and FeZnUf.PO4 have a 6-coordinate iron site. The iron site has an average Fe-O/N bond length of 2.01-2.02 A, which can be resolved into subshells of 1.92 and 2.11 A for FeZnUf.PO4. On the other hand, the zinc site has a shell of scatterers at 2.02-2.03 A plus one scatterer at ca. 2.4 A. These metal-ligand bond lengths are consistent with the nature of the ligands deduced from spectroscopic studies or identified in the crystal structure of the related kidney bean purple acid phosphatase (KBPAP). The outer-sphere analysis indicates an Fe-Zn separation of approximately 3.3 A in both FeZnUf and FeZnUf.PO4, consistent with the presence of an M2(mu-OR)2 diamond core as found in the crystal structures of KBPAP, calcineurin, and protein phosphatase 1. The Fe-P and Zn-P bond distances in FeZnUf.PO4 are determined to be 3.23 and 3.18 A, respectively, indicating that phosphate binds to the dinuclear center in a bidentate mode; such a mode has been observed in oxoanion complexes of KBPAP, calcineurin, and alkaline phosphatase, as well as in a number of synthetic FeFe and FeZn complexes. The implications of these structural results on the mechanism of phosphatase action are discussed.
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PMID:X-ray absorption spectroscopic studies of the FeZn derivative of uteroferrin. 890 92

Differentiating chick limb-bud mesenchymal cells plated in micromass culture form a cartilage matrix that can be mineralized in the presence of 4 mM inorganic phosphate (Pi), and 1 mM calcium. Previous studies showed that when beta-glycerophosphate (beta GP) is used in place of Pi, the mineral crystals formed are larger and differ in distribution. The present study shows that the difference in distribution is not associated with alterations in cell proliferation, protein synthesis, or with collagen, proteoglycan core protein, or alkaline phosphatase gene expression. Cultures with 2.5, 5, and 10 mM beta GP did show different levels of alkaline phosphatase activity, and in the presence of low (0.3 mM) Ca had different Pi contents (4, 6 and 9 mM, respectively), indicating that the increase in CaxP product may in part be responsible for the altered pattern of mineralization. However, cultures with beta GP in which alkaline phosphatase activity was inhibited with levamisole still had an altered mineral distribution as revealed by Fourier transform-infrared (FT-IR) microspectroscopy. The presence of a casein kinase II-like activity in the mineralizing cultures, the ability of specific inhibitors of this enzyme to block mineralization, and the known ability of beta GP to block phosphoprotein phosphatase activity suggests that altered patterns of matrix protein phosphorylation may influence mineral deposition in these cultures.
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PMID:The mechanism of beta-glycerophosphate action in mineralizing chick limb-bud mesenchymal cell cultures. 891 77

Many of the hormone-regulated ion transport processes in distal nephron involve transcellular pathways which require a passive entry of ions at the apical membrane of the distal tubule cells. To investigate molecular mechanisms underlying the ionic permeability of the distal tubule apical membrane, a study was undertaken in which vesicles prepared from apical membranes from isolated rabbit distal tubules were fused onto a planar lipid bilayer. These experiments led to the identification of several ionic channels including a Cl(-)-permeable channel of 14 pS with a Na+ over Cl- permeability ratio, PNa/PCl < 0.09. The open channel probability (Po) showed a weak voltage dependency with Po increasing slightly at negative potential values (intracellular (trans) relative to extracellular (cis) for right-side-out vesicles). Channel activity was inhibited by NPPB at high concentrations (> 100 microM) and by DIDS (300 microM). A small inhibitory effect was also observed in the presence of DPC at concentrations ranging from 200 microM to 500 microM. The presence of SO4(2-) (32 mmol/l) in the trans solution caused a complete inhibition of channel activity, but no modification of channel behaviour was observed with the non-selective channel blocking agent gadolinium (Gd3+) at 100 microM. Finally, addition of the catalytic subunit of protein kinase A into the trans chamber (60 U/ml to 80 U/ml) led to an increase in channel activity characterized by a greater number of active channels coupled to an increase of the individual channel open probability. The action of the protein kinase A could be cancelled by the addition of a non specific protein phosphatase, such as alkaline phosphatase. Our results suggest that the apical membrane of the rabbit distal tubule contains a Cl- permeable channel of small conductance the activity of which may be modulated by hormones linked to the adenylate cyclase pathway.
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PMID:Evidence from incorporation experiments for an anionic channel of small conductance at the apical membrane of the rabbit distal tubule. 897 99

We examined the effect of okadaic acid, an inhibitor of protein phosphatase type 1 and 2A, on prostaglandin E1 (PGE1)-induced alkaline phosphatase (ALP) activity in osteoblast-like MC3T3-E1 cells. PGE1 increased ALP activity dose dependently in the range between 10 nM and 0.3 microM in these cells. The pretreatment with okadaic acid enhanced the PGE1-induced ALP activity in a dose-dependent manner in the range between 0.1 and 5 nM. On the other hand, 1-norokadaone, a less potent analogue of okadaic acid, had no effect on the PGE1-induced ALP activity. Tautomycin, an another inhibitor of protein phosphatase type 1 and 2A, also enhanced the PGE1-induced ALP activity. PGE1 stimulated cAMP accumulation dose dependently in the range between 10 nM and 0.3 microM. However, PGE1 had no effect on the formation of inositol phosphates. Okadaic acid did not affect the PGE1-induced cAMP accumulation. Okadaic acid dose dependently enhanced the dibutyryl cAMP-induced ALP activity. These results strongly suggest that protein phosphatase type 1 and/or 2A act as a regulator of ALP activity at a point downstream from protein kinase A in osteoblast-like cells.
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PMID:Okadaic acid enhances prostaglandin E1-induced alkaline phosphatase activity in osteoblast-like cells: regulation at a point downstream from protein kinase A. 898 33

Chronic immunosuppressive treatment with cyclosporin A (CsA) is associated with decreased bone density. However, in culture, CsA inhibits osteoclast differentiation and bone resorption. This raises the question as to whether CsA also affects osteoblast function. Immunophilin, one of the CsA-binding cyclophilins that is implicated in the immunosuppressive action of CsA via calcineurin, is a peptidyl prolyl cis-trans isomerase (PPl). CsA also binds a mitochondrial membrane PPl which is implicated in controlling permeability transition pores. Therefore, in the present study we tested the effect of CsA on cell mediated mineralization in parallel with mitochondrial rhodamine retention as an indicator of mitochondrial membrane potential Rat marrow stromal cells were grown in dexamethasone (DEX) medium to stimulate mineralization in culture, and CsA was added to various cultures using different treatment schedules. Low dose (0.1 microM) CsA inhibited mineralization, compared to controls, when present in the cultures during days 3-11 of DEX stimulation. Contrarily, high dose CsA (1.0 microM) resisted the inhibitory effect of the low dose. SDZ 220-384 (SDZ), a non-immunosuppressive derivative of CsA which is known, like CsA, to bind to mitochondrial cyclophilin but does not inhibit calcineurin, was also tested. Both high and low doses of SDZ decreased mineralization when present in the cultures from day 3 or from day 0. The similar effect of the low CsA dose and SDZ on mineralization is in accord with their ability to block permeability transition pores. The differential effect, on day 21 mineralization, between high CsA dose and SDZ took place in parallel to their opposing effects on mitochondrial membrane potential. On days 4-8, mitochondrial rhodamine retention was higher under CsA than under SDZ. Under these conditions there was no significant difference between the effects of these drugs on cell proliferation measured on day 11; there was a minor decrease in specific alkaline phosphatase activity by SDZ, too small to explain the extent of mineralization inhibition by SDZ. These results suggest that permeability transition pores might be involved in controlling mineralization. Unlike SDZ, CsA exhibits an additional effect on the mitochondrial membrane potential and on mineralization when applied at a high dose on day 3. Therefore identifying the additional activity of high dose CsA, which is missing in SDZ, may be beneficial. Such activity is expected to resist changes in rhodamine retention and decreased mineralization induced by SDZ, and yet enable preservation of immunosuppressive activity of CsA.
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PMID:Cyclosporin A and its non-immunosuppressive derivative exhibit a differential effect on cell-mediated mineralization in culture. 902 81

Western blotting studies revealed that connexin43 (Cx43), one of the major gap junction proteins in human vascular endothelial cells, is posttranslationally modified during mitosis. This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43(m). Cx43(m) was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all (32)P(i) from Cx43(m) by PP2A. Immunofluorescent confocal microscopy of mitotic cells revealed that Cx43 is intracellularly located, while in nonmitotic cells Cx43 is located at regions of cell-cell contact. Dye coupling studies revealed that mitotic endothelial cells were uncoupled from each other and from nonmitotic cells. After cytokinesis, sister cells resumed cell coupling independent of de novo protein synthesis. The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.
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PMID:A mitosis-specific phosphorylation of the gap junction protein connexin43 in human vascular cells: biochemical characterization and localization. 910 48

The Ah receptor binds aryl hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with high affinity. After binding aryl hydrocarbons, the receptor releases the 90-kDa heat shock protein and forms a dimer with the Arnt protein capable of binding at xenobiotic-responsive elements (XREs) and stimulating the transcription of genes involved in the metabolism of aryl hydrocarbons. The activity of the Ah receptor/ Arnt dimer can be decreased by treatments causing the down-regulation of protein kinase C and decreasing the nuclear accumulation of the receptor. Incubation with acid phosphatase or with alkaline phosphatase has been reported to block XRE binding. Thus the literature suggests that phosphorylation regulates Ah receptor activity by affecting DNA binding and/or nuclear transport. A reporter plasmid containing two XREs was used to investigate the effects of phosphatase inhibitors on TCDD-dependent transcription by the Hepa-1 mouse liver cell line. The inhibitors calyculin A and okadaic acid caused two- to threefold increases in TCDD-dependent transcription at concentrations capable of selectively inhibiting protein phosphatase 1 and protein phosphatase 2A. The inhibitor cyclosporin A doubled TCDD-dependent transcription at a concentration capable of selectively inhibiting protein phosphatase 2B. All three of the phosphatase inhibitors increased TCDD-dependent transcription without affecting transcription in the absence of TCDD. Nuclear extracts were prepared from cells treated with concentrations of okadaic acid or cyclosporin A which substantially stimulated TCDD-dependent transcription. Neither of the inhibitors significantly increased the level of TCDD-dependent XRE binding in the extracts. GAL4-Arnt fusion proteins were used to further investigate whether the phosphatase inhibitors affected a step other than DNA binding. Okadaic acid treatment specifically increased the ability of a GAL4 fusion protein containing the Arnt PAS and transactivation domains to stimulate transcription. These results suggest that serine/threonine-specific protein phosphatases can act at a level subsequent to XRE binding to inhibit the ability of the Ah receptor/Arnt dimer to stimulate transcription.
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PMID:Inhibitors of serine/threonine-specific protein phosphatases stimulate transcription by the Ah receptor/Arnt dimer by affecting a step subsequent to XRE binding. 912 79

We studied the role of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel as an HCO3- conductor during adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation in human airway epithelial cell lines. HCO3- or Cl- currents across the apical membrane were measured in the presence of an HCO3- or Cl- gradient under short-circuit conditions in intact and alpha-toxin-permeabilized monolayers, which allowed manipulation of the intracellular regulators cAMP and ATP. CFTR as the current carrier for HCO3- was identified by 1) stimulation by cAMP, 2) ATP dependence, 3) blocker sensitivity, 4) stimulation by genistein, and 5) lack of stimulation in CF epithelia bearing mutated delta F508 CFTR. In pulmonary alpha-toxin-permeabilized Calu-3 monolayers, cytosolic addition of 100 microM cAMP stimulated apical HCO3- currents from -9.4 +/- 1.6 to -31.1 +/- 3.9 microA/cm2 (n = 18), and apical Cl- currents increased from -54.1 +/- 7.1 to -203.2 +/- 15.4 microA/cm2 (n = 27). Average relative permselectivity for HCO3- vs. Cl- was approximately 15%. Absence of cytosolic ATP resulted in loss of cAMP stimulation of HCO3- and Cl- currents. Genistein (50 microM), which has been proposed to inhibit phosphatases controlling apical CFTR, as well as the alkaline phosphatase inhibitor (-)-p-bromotetramisole (1 mM) further activated cAMP-stimulated HCO3- and Cl- currents. Activated currents remained stimulated on removal of cAMP, suggesting inhibition of a protein phosphatase by genistein and bromotetramisole. The Cl- channel blockers glibenclamide (300 microM) and N-phenylanthranilic acid (5 mM), but not 4,4'-dinitro-2,2'-stilbenedisulfonic acid (100 microM), inhibited cAMP- and genistein-stimulated HCO3- and Cl- currents. Blocker effects were absent in human CF tracheal cells homozygous for the delta F508 mutation of CFTR (CFT1); Cl- and HCO3- currents were rescued in CFT1 cells recombinantly expressing wild-type CFTR. Thus CFTR functions as a HCO3- and Cl- conductor, and genistein and bromotetramisole maximize CFTR activity in airway epithelial cells.
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PMID:cAMP and genistein stimulate HCO3- conductance through CFTR in human airway epithelia. 914 51

The anabolic effect of 17beta-estradiol in osteoblastic MC3T3-E1 cells was investigated. The cells were cultured for 3 days in the medium containing either vehicle or 17beta-estradiol (10(-11)-10(-9) M). 17beta-Estradiol significantly increased alkaline phosphatase activity and protein concentration in the cells. The steroid (10(-9) M) also significantly elevated the cell numbers and the cellular DNA content. The anabolic effect by 17beta-estradiol was blocked by the presence of dipicolinate (10(-3) M), a chelator of zinc ion, suggesting a role of cellular zinc in osteoblastic cell function. The presence of zinc sulfate (10(-5) M) or beta-alanyl-L-histidinato zinc (AHZ) (10(-5) M) significantly enhanced the 17beta-estradiol (10(-10) or 10(-9) M)-induced increase of alkaline phosphatase activity and protein concentration in the cells; the effect of AHZ was greater than that of zinc sulfate. The enhancement by zinc compounds was not based on the augmentation of osteoblastic cell numbers. The co-addition of cycloheximide (10(-6) M), an inhibitor of protein synthesis, completely blocked the zinc compound (10(-5) M)-induced enhancement of 17beta-estradiol's (10(-9) M) effect to increase alkaline phosphatase activity and protein concentration in the cells. Moreover, the anabolic effect of 17beta-estradiol together with or without zinc compounds was abolished by the presence of staurosporine (10(-8) M), an inhibitor of protein kinase C, or of okadaic acid (10(-7) M), an inhibitor of protein phosphatase. The present study demonstrates that the anabolic effect of 17beta-estradiol is enhanced by zinc-chelating dipeptide in osteoblastic MC3T3-E1 cells, and that the enhancing effect may involve protein synthesis and protein kinase activity.
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PMID:Zinc enhancement of 17beta-estradiol's anabolic effect in osteoblastic MC3T3-E1 cells. 916 27

Orthovanadate is a known inhibitor of phosphotyrosyl protein phosphatase and is reported to stimulate osteogenic cell proliferation and differentiation when administered during the logarithmic growth phase and to potentiate the mitogenic effects of several growth factors. There is little information concerning the effects of orthovanadate on bone matrix deposition and mineralization, although there is some evidence that it increases collagen synthesis by osteogenic cells. To test the effects of orthovanadate on bone nodule formation and mineralization, osteogenic cells were exposed to 5-50 microM orthovanadate or 10(-7) M insulin-like growth factor-1 for 3, 7, and 21 days after plating. Exposure to orthovanadate produced differential effects on cellular proliferation and alkaline phosphatase activity following completion of the logarithmic growth phase, and on resultant bone nodule formation and mineralization by these populations. The effects of orthovanadate on osteogenic cultures were concentration dependent: 5 microM concentrations produced by a relatively large quantity of poorly mineralized matrix, while 30-50 microM concentrations produced a smaller quantity of heavily mineralized matrix. Thus, orthovanadate could possibly be used as a growth factor for bone, if administered at the critical dosage at the proper stage of the life cycle of the osteoblast.
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PMID:Enhancement by sodium orthovanadate of the formation and mineralization of bone nodules by chick osteoblasts in vitro. 922 45


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