Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of regucalcin, a regulatory protein of Ca2+ signaling, on guanosine-5'-triphosphatase (GTPase) activity in isolated rat liver plasma membranes was investigated. GTPase activity was significantly increased by the addition of Ca2+ (25-100 microM) in the enzyme reaction mixture. Such an increase was not seen by other metals (Mg, Co, Zn, Cu, Ni and Mn) with 50 microM. The activatory effect of calcium (50 microM) was significantly decreased by calmodulin (2.5 and 5 microg/ml), indicating that it does not depend on calmodulin. The presence of regucalcin (0.1-0.5 microM) in the enzyme reaction mixture caused a significant increase in GTPase activity. This increase was not significantly enhanced by calcium (50 microM). GTPase activity was significantly increased by dithiothreitol (DTT; 5 mM), a protecting reagent of thiol (SH)-groups, while it was decreased by N-ethylmaleimide (NEM; 5 mM), a modifying reagent of SH-groups. The effect of calcium or regucalcin in increasing GTPase activity was not seen in the presence of NEM. Also, the activatory effect of calcium or regucalcin on GTPase was not seen in the presence of vanadate, an inhibitor of protein phosphorylation, which could inhibit GTPase activity. Moreover, the effect of regucalcin was not seen in the presence of digitonin (0.01%), a solubilizing reagent of membranous lipids, while the effect of calcium was not inhibited by digitonin. The present study demonstrates that regucalcin has an activatory effect on GTPase activity independently of Ca2+ in rat liver plasma membranes.
Mol Cell Biochem 2001 Aug
PMID:Activatory effect of regucalcin on GTPase activity in rat liver plasma membranes. 1169 88

The aim of this study was to produce the Bet v 1-related major hazelnut allergen Cor a 1.0401 and variants thereof as recombinant allergens, and to compare their immuno-reactivity with the major hazel pollen allergen using sera of patients whose hazelnut allergy recently was confirmed by double-blind placebo-controlled food challenges (DBPCFC) in a multicenter study. Total RNA was isolated from immature hazelnuts and transcribed into cDNA. Full length coding DNA obtained by PCR-strategy was subcloned into pTYB11 vector and expressed in E. coli ER2566 cells. Native non-fusion target proteins were purified by DTT-induced self-cleavage of the intein-tagged N-terminal fusion proteins. IgE reactivity of the recombinant allergens was tested by enzyme allergosorbent test (EAST), EAST-inhibition, immunoblot-inhibition and histamine release assays. Four recombinant allergens were produced showing deduced amino acid sequence identities among each other of 97-99%, and were considered as variants Cor a 1.0401 (GenBank Accession no.: AF136945), Cor a 1.0402 (AF323973), Cor a 1.0403 (AF323974) and Cor a 1.0404 (AF323975). Cor a 1.0402 and 03 only differed in a C4S exchange. Cor a 1.0404 had a unique proline residue in position 99. Surprisingly, only 63% identity was revealed with hazel pollen Cor a 1. EAST with 43 sera of patients with positive DBPCFC to hazelnut indicated IgE reactivity to Cor a 1.0401 in 95% of the sera, to Cor a 1.0402 in 93%, to Cor a 1.0403 in 91%, and in only 74% of the sera to the proline variant Cor a 1.0404. The allergenic activity of the four variants was confirmed by histamine release assays in 15 hazelnut-allergic patients stimulated with the four variants and controls. Eleven sera were positive with extract from native hazelnut, 13 with rCor a 1.0401, 12 with rCor a 1.0402, 11 with rCor a 1.0403, and only two with rCor a 1.0404 containing the proline exchange. The high IgE binding variant Cor a 1.0401 showed only partial IgE cross-reactivity with pollen Cor a 1. IgE-binding and histamine release capacity led to a concordant ranking of the allergenic activity of the recombinant variants: Cor a 1.0401>Cor a 1.0402 and 03>Cor a 1.0404 (the proline variant). Similar results for Cor a 1.0402 and 03 suggest a minor influence in IgE binding of cysteine in position 4, whereas proline in position 99 appears to be responsible for the decrease in IgE reactivity in Cor a 1.0404. It appears that the epitopes of hazelnut Cor a 1.04 are less related to pollen Cor a 1 than to Bet v 1 from birch pollen. Low IgE binding variants or mutants of Cor a 1.04 are candidate compounds for developing a novel and safe approach of specific immunotherapy of hazelnut allergy.
Mol Immunol 2002 Jan
PMID:Comparison of four variants of a major allergen in hazelnut (Corylus avellana) Cor a 1.04 with the major hazel pollen allergen Cor a 1.01. 1175 Jun 53

Here we report the cloning and characterization of a gene, cypA, from Aspergillus niger that encodes a peptidyl prolyl cis-trans isomerase (PPIase) belonging to the cyclophilin family. Sequencing of both genomic and cDNA clones revealed two ORFs in cypA, one encoding a 19-kDa protein of 174 amino acid residues and the other a 24-kDa protein of 219 amino acid residues, with overall identities of 27-77% to the homologous cyclophilins from prokaryotic and eukaryotic organisms. Expression of the 19-kDa CYPA-(His)(6) in E. coli shows that the purified protein has PPIase activity which is inhibited by cyclosporin A. Northern analysis shows two specific cypA transcripts, the smaller transcript encodes the cytosolic 19-kDa CYPA protein, the larger transcript encodes the putative mitochondrial 24 kDa CYPA protein. The transcript for the cytosolic CYPA is expressed at a higher basal level than that for the mitochondrial protein. The presence of tunicamycin, DTT or cyclosporin A in the medium does not affect the expression level of cypA. Its expression is however slightly induced by heat shock. Growing A. niger mycelium in the presence of cyclosporin A leads to an increase in hyphal branching prior to growth arrest. Overexpression of cypA under the control of its own promoter in A. niger results in increased sensitivity to cyclosporin A, suggesting that cypA encodes the cellular target for cyclosporin A in A. niger.
Mol Genet Genomics 2001 Dec
PMID:The Aspergillus niger cypA gene encodes a cyclophilin that mediates sensitivity to the immunosuppressant cyclosporin A. 1181 Feb 23

Here we report the isolation and characterization of the cypB gene from Aspergillus niger. The cypB gene encodes a protein with a predicted molecular weight of 20.7 kDa, which shows a high degree of identity to the cyclophilin family of peptidyl prolyl cis-trans isomerases (PPIases) from other eukaryotes. The 5' untranslated region of cypB includes three sequences resembling UPREs (unfolded protein response elements). The expression of cypB is upregulated by tunicamycin and DTT, suggesting that at least one UPRE is functional. The CYPB protein also has a 23-amino acid sequence which serves to target the protein to the endoplasmic reticulum (ER), and the ER retention sequence HEEL. CYPB-(His)(6) was expressed in Escherichia coli; the purified protein is capable of isomerizing a substrate peptide in vitro. This is also the first report to show that C-terminal addition of the sequence HEEL is sufficient to ensure retention of the green fluorescent protein (GFP) within the ER.
Mol Genet Genomics 2001 Dec
PMID:The foldase CYPB is a component of the secretory pathway of Aspergillus niger and contains the endoplasmic reticulum retention signal HEEL. 1181 Feb 24

The function of a gene closely linked to nitrate assimilation loci from Chlamydomonas reinhardtii has been investigated. Gene expression analysis shows that its mRNA accumulation is modulated by light, carbon source and adaptation to light/dark cyclic conditions of growth. A full-length cDNA was isolated for the light-regulated transcript, and sequence characterization indicates that it encodes the NADP-malate dehydrogenase from C. reinhardtii (NADP-MDH;Cr). The primary structure of NADP-MDH;Cr is closely related to plant, mossfern and algal NADP-malate dehydrogenases, and shares structural determinants for chloroplast targeting, cofactor binding and catalysis. Sequence conservation extends to the carboxy end of the protein, where plant and mossfern enzymes have two cysteines and an acidic C-terminus with a critical role for regulation of NADP-MDH activity by the thioredoxin/ferredoxin system. Accordingly, incubation with DTT activates NADP-MDH enzyme in cell-free extracts from C. reinhardtii. Like NADP-malate dehydrogenases from two other green algae, the N-terminal extension of NADP-MDH;Cr lacks two thiol residues whose reduction constitutes the rate-limiting step in the activation reaction of plant enzymes. Homology-based 3D modelling of NADP-MDH;Cr, the first structure predicted for NADP-malate dehydrogenase from a lower eukaryote, evidences close positioning of two new cysteines in an accessible region of the protein surface. These results suggest that the algal enzyme has a different arrangement of regulatory disulfide bridges, which might involve the existence of new mechanisms that control functioning of the malate valve, the main system to export reducing power from the chloroplast of plant cells.
Plant Mol Biol 2002 Feb 01
PMID:NADP-malate dehydrogenase from Chlamydomonas: prediction of new structural determinants for redox regulation by homology modelling. 1185 23

The eukaryotic replication protein A (RPA) is a heterotrimeric protein complex. It consists of 70, 32, and 14 kDa subunits that are involved in DNA replication, repair, and genetic recombination. RPA is a 4-cysteine type zinc-finger protein. RPA's zinc-finger domain is not essential for DNA binding activity, but it is involved in the regulation of RPA's DNA binding activity through reduction-oxidation (redox). In this study, we show that yeast RPA's ssDNA binding activity is regulated by redox potential through its subcomplexes of 32 and 14 kDa subunits. In contrast, the subunits' complex, RPA70, formed a stable complex with ssDNA, even under non-reducing conditions. The addition of DTT and H202 had no effect on its DNA binding activity. In RPA70, since the addition of the subcomplexes of the 32 and 14 kDa subunits, it restored the modulating ssDNA binding activity to native RPA's DNA binding activity. These results suggest that the subcomplexes of the 32 and 14 kDa subunits may be involved in the modulating RPA's DNA binding activity through redox change. These studies, therefore, show the novel structure and function relationship of a multiprotein complex in that the role of a specific domain (or one subunit) is regulated by the other subunits.
Mol Cells 2002 Jun 30
PMID:Involvement of subcomplexes of 32 and 14 kDa subunits in RPA's DNA binding activity through redox change. 1213 92

Functional reagents known to bring about the formation of a distinct membrane molecular complex of the subunits of cytochrome b(558) (gp 91(phox) and p22(phox)) were investigated for their influence on the O2- generating capability of liposome incorporated cytochrome b(558) preparations. One, ethyleneglycolbis[sulfo-succinimidylsuccinate], (sulfo-EGS) was found to inhibit O2- generation at concentrations which are known to result in cross-linking the two subunits of cytochrome b(558). Sulfosuccinimidyl [4-azidophenyldithio] propionate, (sulfo-SADP) on the other hand, was found to be a powerful inhibitor of the cytochrome b(558) dependent O2- production at concentrations not able to result in cross linking of the two subunits. Sulfo-SADP inhibits the cytochrome b(558) O2- production 50% at 25 microM, while sulfo-EGS requires 400 microM. For these reagents, the succinimidyl group of sulfo-SADP and sulfo-EGS is the reactive group, which inhibit irreversibly, cytochrome b(558) generation of O2-. Both sulfo-SADP and sulfo-EGS have similar linker arms of 13.9 and 16.1 A, respectively. The difference, accounting for the strong inhibitory profile for sulfo-SADP as compared with sulfo-EGS, resides in the aryl group associated with the sulfo-SADP. The aryl group of sulfo-SADP has been found to be important in directing the specificity of the probe in its inhibition of O2- generation. When the disulfide bond linking the aromatic portion of the probe to the succinimidyl ring is cleaved by DTT (dithiothreitol), the product loses its specificity and has an inhibitory activity with respect to O2- generation comparable to that of sulfo-EGS. The partial protection against the inhibitory influence of sulfo-SADP by NADP(+) indicates that the reagent may interact at the pyridine nucleotide-binding domain of cytochrome b(558). Its low inhibitory titer and its water solubility suggest that sulfo-SADP reacts with a specific amine (the primary reactant for the succinimidyl group) on cytochrome b(558).
J Biochem Mol Biol Biophys 2002 Jun
PMID:Sulfo-SADP (sulfosuccinimidyl[4-azidophenyldithio]propionate) an active site directed reagent inhibiting the NADPH dependent O2- generation of leukocyte cytochrome b(558). 1218 52

We report the physiological role of OhrR as an organic peroxide sensor and transcription repressor in Xanthomonas campestris pv. phaseoli. In vivo exposure of X. campestris pv. phaseoli to either tert-butyl or cumene hydroperoxides efficiently neutralized OhrR repression of expression from the OhrR-regulated P1 promoter. H2O2 was a weak and non-physiological inducer of the system while other oxidants and metabolites of organic peroxide metabolism did not induce the expression from the P1. Northern blotting results indicated a correlation between concentrations of tert-butyl hydroperoxide used in the treatment and the induction of ohr (an OhrR-regulated gene) expression. In addition, the levels of ohr mRNA in cultures induced by various concentrations of tert-butyl hydroperoxide were reduced in cells with high levels of an organic peroxide metabolising enzyme (AhpC-AhpF) but not in cells with high catalase levels suggesting that organic peroxide interacts with OhrR. DNA band shift experiments using purified OhrR and the P1 promoter fragment showed that organic peroxide treatment prevented binding of the protein to the P1 promoter by oxidation of OhrR, as the inhibition of binding to the P1 promoter was reversed by addition of a reducing agent, DTT. The highly conserved cysteine residue C22 of OhrR is required for organic peroxide inducible gene expression. A mutant protein, OhrRC22S can repress the P1 promoter activity but is insensitive to organic peroxide treatment. Thus, OhrR is the first transcription repressor characterized that appeared to evolve to physiologically sense organic peroxides.
Mol Microbiol 2002 Sep
PMID:OhrR, a transcription repressor that senses and responds to changes in organic peroxide levels in Xanthomonas campestris pv. phaseoli. 1235 31

To study phosphorylation of D. melanogaster nuclear lamins in vivo, we used Kc tissue culture cells. Kc cells contain products of both lamin genes, the lamin Dm0 gene encoding constitutive polypeptides expressed in almost all cell types and the developmentally regulated lamin C gene. We grew Kc cells in low phosphate medium and labelled them with (32P(H3PO4. To obtain mitotic cells we used vinblastine to arrest cells in metaphase. Cells were collected, washed, lysed and resultant extracts fractionated in the presence of protein phosphatase inhibitors. D. melanogaster proteins were then denatured by boiling in SDS plus DTT, followed by immunoaffinity chromatography and SDS-PAGE purification. As anticipated, we found that a CNBr fragment derived from the N-terminal part of lamin Dm0-derivatives (amino acid residues 2-158; fragment A) was phosphorylated during both interphase and mitosis. Interphase but not mitotic phosphorylation was found on an internal CNBr fragment (derived from the end of the central rod domain and the first part of the C-terminal lamin tail; amino acid residues 385-548; fragment D). Interphase only phosphorylation was also detected on another CNBr fragment derived from the extreme C-terminal portion of lamin Dm0-derivatives (amino acid residues 549-622; fragment E). To supplement these data, we used 2-D tryptic peptide mapping followed by phosphorImager analysis. We routinely detected at least seven 'spots' derived from interphase lamins but only a single mitotic lamin phosphopeptide.
Cell Mol Biol Lett 2002
PMID:In vivo phosphorylation of Drosophila melanogaster nuclear lamins during both interphase and mitosis. 1237 69

Xenoestrogens, phytoestrogens and synthetic estrogens, are able to bind to estrogen receptors, and to mimic estrogenic activities in a cell and tissue specific manner. For the characterization of environmental estrogens mainly mammary derived and yeast based models have been used. The aim of this study was therefore to assess selected natural and synthetic compounds in an endometrial derived model. We measured the relative estrogenic potency of phytoestrogens (genistein, daidzein, coumestrol, some naringenins), synthetic estrogens (bisphenol A, octylphenol, nonylphenol, o,p'-DDT), mycoestrogen (zearalanone) as well as extracts of Cimicifuga racemosa on alkaline phosphatase (AlkP) activity in the endometrial derived adenocarcinoma cell line Ishikawa. We used a modified multiwell plate in vitro bioassay based on the estrogen-specific and dose-dependent enhancement of AlkP activity in this cell line. Estradiol, which induced AlkP at levels as low as 10(-8)M, was used as positive control. Most of the compounds studied showed a clear dose-dependent estrogenic effect. Compared to the vehicle control (ethanol) all phyto- and mycoestrogens, stimulated the AlkP activity 2-4-fold at a concentration of 10(-6)M. The synthetic chemicals bisphenol A and nonylphenol showed an effect at 10(-6)M, octylphenol at 10(-5)M. Effects of o,p'-DTT could not be measured. ICI 182,780, a pure estrogen receptor antagonist, significantly inhibited these effects. The latter result demonstrated the estrogen receptor dependency of this process. In summary, most of the phytoestrogens and industrial chemicals tested, behaved as estrogen receptor agonists in terms of the stimulation of AlkP activity.
J Steroid Biochem Mol Biol 2002 Dec
PMID:Stimulation of alkaline phosphatase activity in Ishikawa cells induced by various phytoestrogens and synthetic estrogens. 1265 Jul 20


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