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 convenient precolumn labeling method was developed for the analysis of neutral and sialic acid-containing oligosaccharides in glycoproteins using 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). PMPMP reacts with a reducing oligosaccharide under slightly alkaline conditions (pH 8.3) to form a 2:1 adduct (bis-PMPMP derivative). Sialic acid residues in the oligosaccharides remain intact during the reaction. Tryptic glycopeptides digested with glycopeptidase A for oligosaccharide liberation can be directly derivatized with PMPMP without prior treatment. Separation of the labeled oligosaccharides was performed by reverse-phase high-performance liquid chromatography on a C-18 column with aqueous acetonitrile, and positional isomers such as isomeric triantennary tetradecasaccharides from bovine fetuin were completely resolved. The bis-PMPMP derivatives were labile in alkaline media to form mono-PMPMP derivatives; however, the mono-PMPMP derivatives could be easily reconverted to the original bis-PMPMP derivatives. The proposed method is simpler than the reductive pyridylamination method, and detection sensitivity could reach subnanomole range with a uv detector. Oligosaccharides from ribonuclease B (bovine pancreas), ovalbumin, thyroglobulin (porcine thyroid), fetuin (bovine), and transferrin (human) have been successfully analyzed to demonstrate the usefulness of this method as an alternative to the existing methods.
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PMID:Precolumn labeling of reducing carbohydrates with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone: analysis of neutral and sialic acid-containing oligosaccharides found in glycoproteins. 172 51

A highly purified thyroglobulin mRNA was isolated from human nodal euthyroid goiter. A full-length cDNA was synthesized from 33S RNA by using reverse transcriptase in the presence of human placenta ribonuclease inhibitor. A DNA complementary to human Tg mRNA was used in liquid hybridization experiments to quantify Tg mRNA. The amount of Tg mRNA in euthyroid nodal and congenital goiter was reduced. In thyroid cancer Tg specific mRNA was absent. Direct correlation between Tg gene expression in thyroid cells and DNAase-I hypersensitivity of chromatin from the thyroid gland nucleus was revealed.
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PMID:[Changes in the chromatin structure of the thyroid cells related to the expression of the thyroglobulin gene]. 197 92

After exposure to ligand at 0-4 degrees C, estrogen receptors from mouse uteri characteristically eluted between thyroglobulin (Mr 669,000) and ferritin (Mr 443,000) during size-exclusion HPLC. However, when preparations were warmed with ligand under mild activating conditions, most or all of the receptor was observed as a much larger complex, which eluted between dextran blue 2000 and thyroglobulin. Formation of the large complex required ligand, was inhibited by molybdate, and occurred even in 0.4 M KCl. Slower ligand dissociation characterized the large complex, indicating that activated receptors were included preferentially. This large complex did not form when charged cytosols were aged, concentrated, or precipitated, indicating that formation was not the result of random aggregation. After exposure to conditions commonly used for activation (25 degrees C, 60 min), most receptor existed as a very large, monodisperse complex of finite size, predicting an ordered structure for these large complexes that should be useful for defining the types of proteins which can interact with estrogen receptors. Formation of the large complex was not impeded or disrupted by EDTA, RNase, DNase I, thiourea, or mercaptoethanol; however, the capacity to form this large complex was not demonstrated by preparations that had been exposed to trypsin or by the small receptor forms obtained after salt extraction. Proteolytic sensitivity and lack of sensitivity to RNase or DNase indicate that interactions between receptors and other proteins are involved in peak A formation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Intermolecular engagement of estrogen receptors indicated by the formation of a high molecular weight complex during activation. 251 8

Highly purified thyroglobulin mRNA was isolated from human nodal euthyroid goiter. Full length cDNA was synthesized from 33S RNA by using reverse transcriptase in the presence of human placenta ribonuclease inhibitor. DNA complementary to human Tg mRNA was used in liquid hybridization experiments to determine the quantity of Tg mRNA. The amount of Tg mRNA in euthyroid nodal and congenital goiter was reduced. Tg specific mRNA was absent in thyroid cancer cells.
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PMID:[Thyroglobulin gene expression in human thyroid cells in various types of thyroid pathology]. 258 2

Human thyroglobulin mRNA was isolated from Graves' goitres by size selection of total poly(A)-rich RNA in a sucrose gradient. It sedimented at 33 S, as in other mammalian species, and showed a single component of approximately 8500 bases by gel electrophoresis. cDNA was synthesized from the 33-S RNA by using reverse transcriptase in the presence of human placenta ribonuclease inhibitor and in conditions allowing the formation of long transcripts. The latter was made double-stranded using reverse transcriptase and blunt-ended with nuclease S1. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved at the endonuclease PstI site and tailed with dGTP. The resulting plasmids were used to transform Escherichia coli C600 cells and four cloned recombinants were selected. Each plasmid DNA was shown to contain a sequence complementary to human thyroglobulin mRNA by hybridization with a labeled 33-S mRNA, visualization of cDNA . mRNA hybrids by electron microscopy and filter hybridization selection of mRNA directing the synthesis of immunologically related thyroglobulin peptides in the reticulocyte lysate. The four inserted DNA sequences were 1400 - 1800 base pairs long, two of them showing an homologous sequence of 1100 base pairs. Together, the four cloned DNA fragments represented 63% of the 8500 bases of human thyroglobulin mRNA.
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PMID:Cloning of four DNA fragments complementary to human thyroglobulin messenger RNA. 617 25

TTF-1 is a homeodomain-containing transcription factor mainly expressed in the thyroid where it controls the tissue-specific expression of the thyroglobulin, thyroperoxidase and TSH receptor genes. It is therefore potentially implicated in the hormonal control exerted by thyrotropin via the second messenger cyclic AMP on the transcription of these genes in thyrocytes. In order to investigate whether there exists a relationship between the stimulation of the cAMP pathway and TTF-1 gene expression in these cells, we have compared the amounts of TTF-1 protein, its state of phosphorylation and its subcellular distribution in control and cAMP-stimulated dog thyrocytes in primary culture. Dog TTF-1 was expressed in bacteria as a fusion protein and antibodies were raised against the dog TTF-1 moiety. Stimulation of the thyrocytes by cyclic AMP agonist only marginally increased TTF-1 gene expression as shown for the mRNA by RNase protection assay and for the protein by immunoblotting and immunoprecipitation of extracts from 35S-methionine labelled cells. The phosphorylation state of TTF-1 was investigated by immunoprecipitation of extracts from 32P-labelled thyrocytes. Phosphorylation level appeared to be essentially unaffected by forskolin treatment of the cells. We also looked for differences in the use of phosphorylation sites by partial proteolytic digestion of immunoprecipitated 32P-labelled TTF-1 with Glu-C and Asp-N endoproteases. Comparison of radioactivity distribution amongst the generated fragments did not reveal any difference in the pattern of TTF-1 phosphorylation in control and forskolin conditions. Lastly, in situ detection of TTF-1 by immunofluorescence demonstrated that the protein was localized in the nucleus of the cells, irrespective of the culture conditions. No major change in TTF-1 gene expression upon stimulation of the thyrocyte with a cAMP agonist could thus be detected in this study. The absence of an obvious modification of the TTF-1 protein itself in response to cAMP stimulation may indicate that other transcription factor(s) or co-factor(s) are involved in the control exerted by cAMP on the expression of thyroid-specific genes.
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PMID:Study of TTF-1 gene expression in dog thyrocytes in primary culture. 758 89

Alternative splicing of primary transcripts from the calcitonin/alpha calcitonin gene-related peptide (alpha CGRP) gene result in mature mRNAs encoding either calcitonin or alpha CGRP. We have produced sequence-specific, synthetic, biotinylated oligodeoxynucleotide probes that recognize calcitonin (exon 4), and alpha CGRP (exon 6) sequences as well as sequences common to both splice variants (exon 3) of this gene. Probes to exons 4 and 3 revealed strong cytoplasmic signals in rat parafollicular cells. In addition, a punctate nuclear signal was obtained with these probes. The alpha CGRP-specific (exon 6) probe resulted in weak cytoplasmic labelling of parafollicular cells, but produced a punctate nuclear labelling similar to that seen with the exon 4 and 3 probes. RNase digestion removed all the cytoplasmic and nuclear signals obtained with all probes. Hybridization with a thyroglobulin-specific probe failed to label parafollicular cells. A control (human enterovirus) probe yielded negative results, while a probe to rat somatostatin produced cytoplasmic labelling of a small subpopulation of parafollicular cells. Finally, a probe specific for beta CGRP mRNA labelled most, if not all, parafollicular cells. Fluorescent alkaline phosphatase development of in situ hybridizations could be combined with indirect immunofluorescence for CGRP. Analysis by fluorescence and confocal microscopy revealed that CGRP immunoreactive cells contained calcitonin, alpha CGRP and beta CGRP hybridization signals. Our results demonstrate that all three genes may be simultaneously expressed by thyroid parafollicular cells and show that synthetic biotinylated oligonucleotide probes can be used for highly precise localizations of primary transcripts in the nuclei of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Detection of primary and mature transcripts of calcitonin-gene-related peptide genes in rat parafollicular cells by light, fluorescence and confocal microscopy. 773 76

Metabolic abnormalities in thyroid hormonogenesis cause congenital goiter. Here we studied a case of mild hypothyroidism caused by a novel missense mutation in the thyroglobulin (TG) gene. A female patient underwent thyroidectomy twice at the age of 27 and 43 years because of gradual enlargement of the thyroid. By RNase cleavage assay and PCR direct sequencing we identified a thymine to cytosine transition at nucleotide 3828 (from the transcription start site) which causes amino acid change from cysteine to arginine at codon 1263. A pedigree study suggested autosomal recessive inheritance due to consanguineous marriage of her parents. Immunohistochemical study suggested impaired intracellular transport of the mutant TG. Sensitivity to endoglycosidase H confirmed that the mutant TG failed to reach the Golgi compartment. Native polyacrylamide gel electrophoresis and Western blot analyses showed that formation of monomers and homodimers was defective with abundant high molecular-weight aggregates which are normally formed transiently after translation. To examine if the mutant TG is functionally defective, we separated thyroid tissue extract on a Biogel A5m column and measured T4 and T3 released from proteins in each fraction by treatment with proteinase K. Although thyroid hormones released per mole of the mutant TG protein did not decrease, those released per mg of total protein decreased. In conclusion, the missense mutation in the TG gene caused congenital goiter with mild hypothyroidism due to an altered protein structure which resulted in defective intracellular processing and premature degradation by "quality control" mechanisms. Although the tissue TG content was greatly reduced, the hypothyroidism was mild with slow progression of the goiter, because the mutant TG was a relatively good substrate for the synthesis of the thyroid hormones.
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PMID:Missense mutation (C1263R) in the thyroglobulin gene causes congenital goiter with mild hypothyroidism by impaired intracellular transport. 979 Feb 65

The efficiencies in derivatization of reducing carbohydrates were compared by capillary electrophoresis using maltose as a model with nine monoaminobenzene derivatives by reductive amination in the presence of sodium cyanoborohydride. We found that aminobenzene derivatives substituted at the 3-position showed good reactivity with reducing carbohydrates as expected from the reaction mechanism, although the fluorescence intensities and molar absorptivities of these derivatives were not as high as those of 2- and 4-aminobenzene derivatives. The reagents, 3-aminobenzamide and 3-aminobenzoic acid, which showed the highest reactivity, were applied to the labeling of carbohydrate chains obtained from some sialic acid-containing glycoprotein samples, and also high-mannose and hybrid-type oligosaccharides. Capillary electrophoresis of these labeled carbohydrate chains in an inner surface-modified capillary with (50% phenyl)methylpolysiloxane allowed excellent separation of sialic acid-containing carbohydrate chains derived from fetuin and thyroglobulin as well as high mannose-type and hybrid-type carbohydrates derived from bovine pancreas ribonuclease B, soybean agglutinin and hen ovalbumin. The lower limit of calibration was as low as the 10(-16) mol (injected amount) with helium-cadmium laser induced detection.
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PMID:3-Aminobenzamide and 3-aminobenzoic acid, tags for capillary electrophoresis of complex carbohydrates with laser-induced fluorescent detection. 1059

A major difficulty with isolating enzymatically or chemically released oligosaccharides from large-scale glycoprotein deglycosylation reactions is the time-consuming chromatography, desalting, and concentration steps required to prepare a glycan fraction of manageable proportions. To overcome these time and preparative chromatography equipment requirements, we have developed a rapid organic solvent precipitation/extraction procedure that allows sequential isolation of endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96)-released high-mannose and hybrid, peptide-N(4)-(N-acetyl-beta-glucosaminyl) Asn amidase (EC 3.5.1. 52)-released complex, and beta-eliminated O-linked glycans without the need for intermediate chromatography, desalting, or concentration steps. The method involves precipitation of protein and released glycans at -20 degrees C in 80% acetone and extraction of the glycans from the pellet with 60% aqueous methanol after each deglycosylation step. Three pools of essentially salt- and detergent-free oligosaccharides (high-mannose/hybrid, complex, and O-linked) can be isolated in a high yield in 4 days with this protocol, which has been extensively tested using bovine RNase B, human bile salt-stimulated lipase expressed in Pichia pastoris, hen ovalbumin, bovine fetuin, bovine thyroglobulin, and several invertase preparations from wild-type and mutant yeast strains.
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PMID:Selective organic precipitation/extraction of released N-glycans following large-scale enzymatic deglycosylation of glycoproteins. 1066 Apr 52


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