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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A capillary electrophoresis (CE) method using acidic buffers and capillaries coated with Polybrene, a cationic polymer has been developed for the separation of glycoproteins and glycopeptides. Electrophoretic conditions have been optimized to provide resolution of individual glycoforms observed for different glycoprotein preparations. These conditions were found to be entirely compatible with the operation of electrospray mass spectrometry (ESMS), which facilitated the assignments of possible carbohydrate compositions of glycopeptides arising from digests of glycoproteins. By using operating conditions enhanced the formation of oxonium fragment ions prior to mass spectral analysis, selective identification of glycopeptides was achieved for complex samples such as those from proteolytic digests or chemical cleavages. Examples of applications are presented for ribonuclease B, ovalbumin, horseradish peroxidase, and a lectin from Erithrina corallodendron using both CE-ESMS and CE with ultraviolet detection (CE-UV).
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PMID:Development of electrophoretic conditions for the characterization of protein glycoforms by capillary electrophoresis-electrospray mass spectrometry. 860 Dec 4

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found only in R. catesbeiana (bullfrog) oocytes, but not in other organs. The protein is localized in the yolk granules of oocytes but not in other organelles, as detected by immunohistochemistry. More than 99% of RC-RNase was found in the yolk granule pellet when a mild separation method was employed under physiological conditions. The ribonuclease was purified by precipitation of yolk granules, extraction of RC-RNase with 0.09 M NaCl, selective removal of impurities by Hepes buffer, and chromatographies on phosphocellulose and carboxymethyl cellulose columns. Three milligrams of RC-RNase was purified from a 1-g pellet of yolk granules prepared from 2 g of ovary tissue. Therefore, 150 milligrams of RC-RNase could be obtained from a mature female bullfrog (600 g in weight) which had 100 g of ovary tissue. The properties of RC-RNase isolated from yolk granules tested so far are identical to those of RC-RNase isolated from the cytosolic fraction and similar to those of a sialic acid-binding lectin from bullfrog oocytes. To investigate the possible role of RC-RNase in the regulation of cell growth and differentiation during embryogenesis, its cytotoxic activity against various cell lines was examined. The degradation of ribosomal RNA was found in RC-RNase-treated HeLa cells. However, both events were not found in RNase A-treated HeLa cells. Therefore, RC-RNase is proposed to have both ribonucleolytic and cytotoxic activity and a specific receptor on the tumor cell surface is suspected to be involved in the recognition and binding, and possibly entry of RC-RNase.
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PMID:Large-scale preparation of a ribonuclease from Rana catesbeiana (bullfrog) oocytes and characterization of its specific cytotoxic activity against tumor cells. 881 61

RC-RNase is a pyrimidine-guanine sequence-specific ribonuclease and a sialic-acid-binding lectin purified from Rana catesbeiana (bullfrog) oocytes. This 111-amino acid protein exhibits cytotoxicity toward several tumor cell lines. In this paper we report the assignments of proton NMR resonances and the identification of the secondary structure deduced from NOE constraints, chemical shift index, 3JNH alpha and amide proton exchange rates. The protein was directly isolated from bullfrog oocytes; we were able to assign all but five of the amino acid backbone protons of the unlabeled protein by analyzing a large set of two-dimensional proton NMR spectra obtained at several temperatures and pH conditions. Our results indicate that the structure of RC-RNase is dominated by the presence of two triple-stranded antiparallel beta-sheets and three alpha-helices, similar to those of the pyrimidine family ribonucleases. Two sets of resonances were observed for 11 amide protons and 8 alpha-protons located in the loop-1 region, an alpha 2 helix, and three beta-strands, (beta 1, beta 3 and beta 4), suggesting the presence of nonlocalized multiple conformations for RC-RNase.
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PMID:The secondary structure of a pyrimidine-guanine sequence-specific ribonuclease possessing cytotoxic activity from the oocytes of Rana catesbeiana. 895 20

Calnexin and calreticulin are lectin-like molecular chaperones that promote folding and assembly of newly synthesized glycoproteins in the endoplasmic reticulum. While it is well established that they interact with substrate monoglucosylated N-linked oligosaccharides, it has been proposed that they also interact with polypeptide moieties. To test this notion, glycosylated forms of bovine pancreatic ribonuclease (RNase) were translated in the presence of microsomes and their folding and association with calnexin and calreticulin were monitored. When expressed with two N-linked glycans in the presence of micromolar concentrations of deoxynojirimycin, this small soluble protein was found to bind firmly to both calnexin and calreticulin. The oligosaccharides were necessary for association, but it made no difference whether the RNase was folded or not. This indicated that unlike other chaperones, calnexin and calreticulin do not select their substrates on the basis of folding status. Moreover, enzymatic removal of the oligosaccharide chains using peptide N-glycosidase F or removal of the glucoses by ER glucosidase II resulted in dissociation of the complexes. This indicated that the lectin-like interaction, and not a protein-protein interaction, played the central role in stabilizing RNase-calnexin/calreticulin complexes.
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PMID:N-linked oligosaccharides are necessary and sufficient for association of glycosylated forms of bovine RNase with calnexin and calreticulin. 900 68

The activation of transcriptional factor c-Fos/c-Jun AP-1 is essential for normal T cell responsiveness and is often impaired in T cells during aging. In the present study, we investigated whether aberrancies in the regulation of c-fos/c-jun at the mRNA or protein level might underlie the age-associated impairments of AP-1 in human T cells. Whereas T cells from young subjects stimulated with cross-linked anti-CD3epsilon mAb OKT3 plus PMA or with the lectin PHA plus PMA demonstrated considerable increases in c-Fos protein expression, the expression of c-Fos but not c-Jun was markedly reduced in stimulated T cells from certain elderly subjects. In addition, RNase protection assays revealed that anti-CD3/PMA-stimulated T cells from a substantial proportion of elderly subjects exhibited decreased levels of c-fos and/or c-jun mRNA compared to T cells from young subjects. Using electrophoretic mobility shift assays, the levels of nuclear regulatory proteins recognizing the AP-1 consensus TRE motif, the proximal c-jun TRE-like promoter element, and the c-fos serum response element (SRE) were determined in resting and stimulated T cells. Although the stimulation of T cells from young subjects resulted in coordinated increases of nuclear protein complexes binding the AP-1 TRE, c-jun TRE, and c-fos SRE DNA sequence motifs, age-related reductions in the activation of AP-1 were accompanied by decreased levels of c-jun TRE and c-fos SRE binding complexes. Furthermore, the nuclear protein complexes binding the SRE motif induced in activated T cells of young and elderly subjects contained serum response factor and Elk-1 pointing toward age-related defects in the activation of transcriptional regulatory proteins distinct from c-jun/AP-1. These results suggest that underlying aberrancies in the induction of c-fos/c-jun as well as their nuclear regulatory proteins may contribute to the age-related impairments of AP-1 activation in human T cells.
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PMID:Impaired induction of c-fos/c-jun genes and of transcriptional regulatory proteins binding distinct c-fos/c-jun promoter elements in activated human T cells during aging. 901 87

Calnexin is a membrane protein of the endoplasmic reticulum that associates transiently with newly synthesized N-linked glycoproteins in vivo. Using defined components, the binding of ribonuclease B (RNase B) Man7-Man9 glycoforms to the luminal domain of calnexin was observed in vitro only if RNase B was monoglucosylated. Binding was independent of the conformation of the glycoprotein. Calnexin protected monoglucosylated RNase B from the action of glucosidase II and PNGase F but not from that of Endo H, which completely released the protein from calnexin. These observations directly demonstrate that calnexin can act exclusively as a lectin.
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PMID:Conformation-independent binding of monoglucosylated ribonuclease B to calnexin. 901 2

Galectin-3 is a beta-galactoside-specific lectin that is a pre-mRNA splicing factor. Here we report the genomic organization of the human LGALS3 (galectin-3) gene and functional characterization of the promoter. Southern blot analysis of genomic DNA revealed that galectin-3 is coded by a single gene in the human genome. The gene is composed of six exons and five introns, spanning a total of approximately 17 kilobases (kb). Based on primer extension and ribonuclease protection analyses, there are two transcription initiation sites located 52 and 50 nucleotides (nt) upstream of the exon I-intron 1 border, and defined here as +1a and +1b, respectively. The translation start site is in exon II. The ribonucleoprotein-like N-terminal domain, containing the proline-glycine-alanine-tyrosine (PGAY) repeat motif, is found entirely within exon III. The carbohydrate recognition sequence is found entirely within exon V. Genomic fragments encompassing -836 to +141 nt (relative to +1a) have significant promoter activity when linked to the luciferase reporter gene and transiently transfected into HeLa cells or human diploid fibroblasts. Quiescent fibroblasts have low promoter activity but the activity increases 100-fold following serum addition. Serum responsive activation regions in the promoter are located between -513 and -339 nt and between -339 and -229 nt; an additional activation region may be located between -105 and -15 nt. Because galectin-3 is an immediate-early gene whose expression is dependent on the proliferative state of the cell, this study provides the basis for determining the molecular mechanisms of transcriptional regulation in neoplasia or cellular senescence.
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PMID:The human LGALS3 (galectin-3) gene: determination of the gene structure and functional characterization of the promoter. 943 77

RC-RNase is a pyrimidine-guanine sequence-specific ribonuclease and a lectin possessing potent cell cytotoxicity. It was isolated from the oocytes of Rana catesbeiana (bull frog). From analysis of an extensive set of 1H homonuclear 2D NMR spectra we have completed the resonance assignments. Determination of the three-dimensional structure was carried out with the program X-PLOR using a total of 951 restraints including 814 NMR-derived distances, 61 torsion angles, and 76 hydrogen bond restraints. In the resultant family of 15 best structures, selected from a total of 150 calculated structures, the root-mean-square deviation from the average structure for the backbone heavy-atoms involved in well-defined secondary structure is 0.48 A, while that for all backbone heavy-atoms is 0.91 A. The structure of RC-RNase consists of three alpha-helices and two triple-stranded anti-parallel beta-sheets and folds in a kidney-shape, very similar to the X-ray crystal structure of a homolo gous protein, onconase isolated from Rana pipiens. We have also investigated the interaction between RC-RNase and two inhibitors, cytidylyl(2'-->5')guanosine (2',5'-CpG) and 2'-deoxycytidylyl(3'-->5')-2'-deoxyguanosine (3',5'-dCpdG). Based on the ligand-induced chemical shift changes in RC-RNase and the NOE cross-peaks between RC-RNase and the inhibitors, the key residues involved in protein-inhibitor interaction have been identified. The inhibitors were found to bind in a "retro-binding" mode, with the guanine base bonded to the B1 subsite. The His103 residue was found to occupy the B state with the imidazole ring pointing away from the active site. The structure coordinates and the NMR restraints have been deposited in the Brookhaven Protein Data Bank (1bc4 and 1bc4mr, respectively).
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PMID:The solution structure of a cytotoxic ribonuclease from the oocytes of Rana catesbeiana (bullfrog). 976 86

Gamma delta T lymphocytes are thought to play a role in the pathogenesis of multiple sclerosis (MS) contributing to demyelinization and fibrosis in the central nervous system. In this study, we show that, in MS patients with active disease, the percentage of circulating V delta 2+ gamma delta T cells coexpressing NKRP1A is significantly increased compared with healthy donors. V delta 2+ and V delta 1+ T cells were sorted from MS patients and healthy volunteers and cloned. At variance with V delta 1+ clones, all V delta 2+ clones expressed NKRP1A, which was strongly up-regulated upon culture with IL-12; this effect was neutralized by specific anti-IL-12 Abs. No up-regulation of NKRP1A by IL-12 was noted on V delta 1+ clones. RNase protection assay showed that IL-12R beta 2 subunit transcript was significantly less represented in V delta 1+ than V delta 2+ clones. This finding may explain the different effect exerted by IL-12 on these clones. In transendothelial migration assays, V delta 2+ NKRP1A+ clones migrated more effectively than V delta 1+ clones, and this migratory potential was enhanced following culture with IL-12. Migration was strongly inhibited by the F(ab')2 of an anti-NKRP1A Ab, suggesting that this lectin is involved in the migration process. We also show that, in freshly isolated PBMC from MS patients, the migrated population was enriched for V delta 2+ NKRP1A+ cells. We conclude that the expression of NKRP1A on V delta 2+ cells is associated with increased ability to migrate across the vascular endothelium and that this phenomenon may be regulated by IL-12 present in the microenvironment.
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PMID:IL-12-mediated NKRP1A up-regulation and consequent enhancement of endothelial transmigration of V delta 2+ TCR gamma delta+ T lymphocytes from healthy donors and multiple sclerosis patients. 1020 68

A novel glycoprotein designated glycolactin, with a molecular weight of 64 kDa, a sequence hitherto unknown in the literature and capable of inhibiting the hemagglutinating activities of soybean lectin and Ricinus communis agglutinin 120, was isolated from bovine milk. Its lectin-inhibiting activity differed from that of lactoferrin, another milk protein. Like other milk proteins, glycolactin inhibited superoxide formation in vitro. Glycolactin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of about 31 nM. It exhibited ribonucleolytic (RNase) activity towards yeast transfer RNA with a pH optimum of 7.5, and specific RNase activity towards poly C. The purification protocol of glycolactin involved removal of globulin from the acid whey fraction of bovine milk by precipitation with 1.8 M (NH4)2SO4, and adsorption on the ion exchangers CM-Sepharose and Mono S. Deglycosylation of glycolactin using glycopeptidase F produced only a slight decrease of 4 kDa in the molecular weight of glycolactin.
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PMID:Purification and characterization of glycolactin, a novel glycoprotein from bovine milk. 1073 13


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