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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cell-type-specific expression of human O6-methylguanine-DNA methyltransferase (O6-MT) was determined in paraffin-embedded sections of human kidney. A 39 base oligomer complementary to O6-MT cDNA was labeled with digoxigenin and visually detected in situ using an alkaline phosphatase-conjugated anti-digoxigenin antibody. This allowed direct determination of O6-MT-specific mRNA levels, while simultaneously identifying the structures and cell types in the kidney section. Expression of O6-MT was high in distal tubular and glomerular epithelial cells and low in the cells of the Bowman's capsule, collecting and proximal tubular cells. Hybridization of the oligomer was specific to RNA, since RNase and not DNase eliminated the signal. Expression was uniform in all the cell types, except the glomerular cells exhibited varying levels of high intensity. Cell-specific expression was constant between tissue sections from the same and different kidney tissues. These data may help explain the differential response of various cell types to alkylating agents.
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PMID:In situ hybridization of human kidney tissue reveals cell-type-specific expression of the O6-methylguanine-DNA methyltransferase gene. 154 38

The F1-20 protein is a novel neuronal-specific, synapse-associated protein that is expressed nonuniformly in mouse brain. Expression of the F1-20 protein is developmentally regulated in a pattern coincident with active synaptogenesis and synaptic maturation. Here we report the cloning of the cDNA sequence for the F1-20 protein. We found two distinct isoforms of F1-20 cDNA that differed by the presence of 15 additional nucleotides, which does not interrupt the open reading frame. RNase protection analysis and PCR amplification of mouse brain RNA revealed that both isoforms are present in cellular RNA. It is likely that the two F1-20 mRNA isoforms are derived from RNA splicing events utilizing alternative 3' acceptor sites. Analysis of the deduced amino acid sequence for the complete open reading frame revealed that the predominant F1-20 mRNA encodes an 896 amino acid polypeptide with a molecular weight of 91,319 Da. The deduced amino acid sequence does not contain a signal sequence, or any extensive hydrophobic regions. The deduced amino acid sequence does contain a number of consensus sequences for protein kinases. Searches of the protein and nucleic acid sequence data bases revealed that the F1-20 protein has not been previously characterized at the primary structure level, although a weak similarity was found between rabbit calpastatin and the C-terminal portion of the F1-20 protein. We then determined biochemically that the F1-20 protein is a substrate for Ca(2+)-dependent proteolysis, which is specifically inhibited by calpain inhibitors in vitro. This indicates that the F1-20 protein is a substrate for neuronal calpain. We observed that treatment of a synaptosomal lysate with alkaline phosphatase led to an increase in the electrophoretic mobility of the F1-20 protein, as well as to an increase in the sharpness of the electrophoretic band. This indicates that the F1-20 protein is phosphorylated in vivo.
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PMID:Characterization of a novel synapse-specific protein. II. cDNA cloning and sequence analysis of the F1-20 protein. 160 33

This study compares the synthesis of mutant type I collagen in cultured dermal fibroblasts and trabecular osteoblasts that were isolated from a patient with moderately severe osteogenesis imperfecta (type IV). Previous study of this patient's dermal fibroblasts revealed a 2000 dalton deletion located in cyanogen bromide peptide 4 of alpha 2(I)-collagen. The phenotype of the bone cell cultures was defined by a 3-4 day logarithmic phase doubling time, predominantly type I collagen production over type III and alkaline phosphatase activity 13.5 times dermal fibroblast levels. The current study revealed that both fibroblasts and osteoblasts synthesized a normal and a shortened alpha 2(I) chain, each as the product of separate alleles. Following pepsin treatment of the procollagens, a shortened alpha 1(I) chain was also seen in both cell types. Cyanogen bromide peptide mapping of osteoblast alpha-chains demonstrated the same deletions in the cyanogen bromide peptide 4 as observed in the fibroblast cyanogen bromide maps. PAGE analysis of oligonucleotide-specific cDNA that was reverse transcribed from RNA isolated from fibroblasts and osteoblasts also demonstrated the presence of two bands, one the normal size of alpha 2(I) cDNA and a second species that was smaller by 54 base pairs. Sequencing of polymerase chain reaction-amplified cDNA fragments revealed an in-frame deletion of exon 12. This finding was confirmed by the RNase protection method. Genomic DNA sequencing detected a T----G point mutation in the second position of the 5' splice donor site of intron 12. Therefore, in this patient with osteogenesis imperfecta there was no qualitative alteration in the osteoblast-specific expression of this mutant alpha 2(I)-collagen allele compared to dermal fibroblasts.
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PMID:Expression of mutant alpha (I)-procollagen in osteoblast and fibroblast cultures from a proband with osteogenesis imperfecta type IV. 164 48

The activities of 5-nucleotidase (Ec.3.1.3.5), alkaline phosphatase (Ec.3.1.3.1), glucose-6-phosphatase (Ec.3.1.3.9), and ribonuclease (Ec.3.1.13) had been measured in tissue homogenate and in haemolymph of Biomphalaria alexandrina, the specific intermediate host for the parasitic disease schistosomiasis, induced by the parasite Schistosoma mansoni.
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PMID:Activity of some hydrolytic enzymes in tissue homogenates and haemolymph of fresh water snails, intermediate hosts in schistosomiasis. 165 90

The nucleosomes of transcriptionally active genes can be separated from those of inactive genes by affinity chromatography on organomercury-agarose (Hg-agarose) columns. The basis for this separation is the difference in accessibility of the sulfhydryl groups of histone H3 and certain non-histone proteins in active and inactive chromatin. A new procedure distinguishing between different modes of binding of transcriptionally active nucleosomes to the Hg-agarose column has been applied to study several factors which might influence the binding reaction. Nucleosomes that bind to the column because of salt-labile associations with SH-reactive non-histone proteins, such as the high-mobility-group proteins, HMG-1 and HMG-2, were released by adding 0.5 M NaCl to the eluting buffer. The remaining nucleosomes, in which reactive histone H3 thiol groups can bind covalently to the organomercury, were then displaced from the column by 10 mM dithiothreitol. Both Hg-agarose-bound fractions contain the transcriptionally active DNA sequences of the cell, but inactive nucleosomes, such as those containing alpha-globin DNA, pass through the column. The histones of both Hg-agarose-bound fractions have significantly higher levels of acetylation than do histones of the unbound fraction, but the content of tri- and tetra-acetylated H3 and H4 is significantly higher in the nucleosomes with reactive H3 thiols. The rate of turnover of histone N-acetyl groups is also far greater in the Hg-agarose-bound nucleosomes than in the unbound nucleosomes. Although the overall levels of histone acetylation can be increased significantly by incubating HeLa cells in the presence of the deacetylase inhibitor, 5 mM sodium butyrate, this treatment has little if any effect on the total number of nucleosomes retained on the Hg-agarose column. However, the ability of Hg-agarose chromatography to detect localized changes in chromatin structure is evidenced by an 11-fold increase in the Hg-agarose binding of nucleosomes containing the DNA of the butyrate-inducible alkaline phosphatase gene, compared to the Hg-agarose-bound nucleosomes of control cells. Although nascent RNA chains are present in the Hg-agarose-bound nucleosomes released by dithiothreitol, binding of the SH-reactive nucleosomes to the Hg-agarose column is not dependent on the presence of proteins associated with nascent RNA chains, since binding does not decrease following removal of the nascent transcripts by ribonuclease treatment.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Factors affecting nucleosome structure in transcriptionally active chromatin. Histone acetylation, nascent RNA and inhibitors of RNA synthesis. 170 16

The steroid-binding capacity of the adrenocortical pregnenolone-binding protein (PBP) is effectively destroyed by extreme temperature (boiling water for 2-5 min); however, the boiled preparation contains a factor that potentiates ligand binding when readded to native PBP. Treatment of the boiled fraction with calf intestinal alkaline phosphatase at pH 9 reverses the stimulatory effect on PBP activity. Additionally, if native PBP is first incubated with alkaline phosphatase, which converts it to a nonbinding form, activity can be fully restored in a dose-dependent manner by the addition of the boiled preparation. The factor (itself devoid of binding capacity) can also be generated by exposing native PBP to acidic conditions (pH 4). The molecule is small (mol wt, less than 2000), as judged by Sephadex G-25 gel filtration and equilibrium dialysis. It is not retained on Concanavalin-A-Sepharose and is not extractable with a variety of organic solvents. The factor remains active after lyophilization and has a net negative charge at pH 7.4 (determined by DEAE-cellulose chromatography). While the binding capacity of native PBP is destroyed by a variety of proteases, the heat-stable factor is unaffected by similar treatment. Additionally, factor activity is not susceptible to RNase, DNase, or lipase digestion. Thus, the protein moiety of the PBP has an absolute requirement for a distinct phosphorylated heat-stable factor for expression of ligand-binding activity, and it may be through this factor that binding activity is regulated. It is not yet known whether the factor is acting allosterically or actually functions as part of the steroid-binding site.
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PMID:Adrenocortical pregnenolone-binding protein activity requires a small heat-stable factor: evidence that regulation by phosphorylation/dephosphorylation occurs at the level of the factor, not the protein. 177 Sep 49

An alkaline phosphatase-labelled anti-sense oligonucleotide probe specific for tyrosine hydroxylase (TOH) mRNA has been used for visualisation of TOH mRNA in the rat brain and adrenal gland. Both ribonuclease pre-treatment and the use of excess non-labelled probe abolished the specific hybridization signal. Furthermore the TOH mRNA-positive signal was only found in cells known from earlier studies to react with anti-TOH antibodies. To determine if the alkaline phosphatase-labelled probe could be used in a semiquantitative manner for measurement of the density of TOH mRNA signal, we used reserpine pre-treatment which induces TOH mRNA expression. The results revealed a significant increase in TOH mRNA signal in locus coeruleus and substantia nigra neurons, and in adrenal medulla chromaffin cells. The increased signal in these areas agreed with the increase in TOH mRNA signal previously observed by Northern analysis and suggests that this type of alkaline phosphatase-labelled probe allows sensitive detection of changes in TOH gene expression.
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PMID:Sensitive non-radioisotopic in situ hybridization histochemistry: demonstration of tyrosine hydroxylase gene expression in rat brain and adrenal. 197 Aug 45

tolA mutants of Escherichia coli K-12 release periplasmic proteins into the extracellular medium; they are sensitive to growth inhibitors such as cholic acid and tolerant to group A colicins and filamentous bacteriophage. Suppressor mutants of the tolA-876 allele were isolated by selecting for cholic acid resistant clones that did not release periplasmic ribonuclease I. One class of tolA suppressor strains carried mutations in the staA gene (for suppressor of tolA) located a 41 min. tolA-876 staA strains partially recovered a wild-type phenotype: they exported alkaline phosphatase and beta-lactamase into the periplasm and only released very low amounts of periplasmic proteins; moreover, they were sensitive to E1 and A colicins and more resistant than tolA-876 staA+ strains to various growth inhibitors. Furthermore, tolA-876 staA-2 and tolA+staA-2 mutants were 10- to 2700-times more resistant than staA+ strains to bacteriophages TuIa, TuIb and T4, and TuII whose receptors are major outer membrane proteins OmpF, OmpC and OmpA, respectively. SDS-PAGE analysis suggested that cell envelopes of staA or staA+ strains contained similar amounts of these proteins but characterization of strains carrying ompF (or C or A)-phoA gene fusions showed that mutation stA-2 reduced ompF gene expression by a factor of two. Analysis of double mutants strains carrying mutation staA-2 and a tolA, tolB, excC or excD periplasmic-leaky mutation showed that staA suppression was allele specific which suggested that proteins TolA and StaA might directly interact.
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PMID:Isolation and characterization of extragenic suppressor mutants of the tolA-876 periplasmic-leaky allele in Escherichia coli K-12. 204 Apr 38

RNP, Sm and SS-B nuclear antigens from calf thymus were studied with respect to the size distribution on sucrose gradients as well as to the molecular integrity and related structural changes when they were subjected to enzymatic digestions under different conditions. Making a difference with RNP particles, the Sm size distribution is concentration dependent, a property in accordance with the complexity of the Sm particles in comparison with the RNPs. The use of combined effects of temperature, endogenous proteases and RNase A, allowed us to gain insight into the limits of stability of the three antigenic particles. Following treatments in the absence of RNAse A, the degradation products (32-38 Kd molecular weight) of the 70 Kd RNP polypeptide remain stable and associated with other molecules within the RNP particle. It was also found that the phosphate groups of the SS-B protein moiety are only accessible to alkaline phosphatase if the RNA of the SS-B particle is degraded by the action of RNAse A.
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PMID:RNP, Sm and SS-B antigens from calf thymus: molecular stability upon enzymatic digestion. 209 30

Data from differential scanning calorimetry (DSC) may be used to estimate very large binding constants that cannot be conveniently measured by more conventional equilibrium techniques. Thermodynamic models have been formulated to describe interacting systems that involve either one thermal transition (protein-ligand) or two thermal transitions (protein-protein) and either 1:1 or higher binding stoichiometry. Methods are described for obtaining binding constants and heats of binding by two different methods: calculation or simulation fitting of data. Extensive DSC data on 2'CMP binding to RNase are presented and analyzed by the two methods. It is found that the methods agree when binding sites are completely saturated, but substantial errors arise in the calculation method when site saturation is incomplete and the transition of liganded molecules overlaps that of unliganded molecules. This arises primarily from an inability to determine TM (i.e., the temperature where concentrations of folded and unfolded protein are equal) under weak-binding conditions. Results from simulation show that the binding constants and heats of binding from the DSC method agree quantitatively with corresponding estimates obtained from equilibrium methods when extrapolated to the same temperature. It was also found from the DSC data that the binding constant decreases with increasing concentration of ligand, which might arise from nonideality effects associated with dimerization of 2'CMP. Simulations show that the DSC method is capable of estimating binding constants for ultratight interactions up to perhaps 10(40) M-1 or higher, while most equilibrium methods fail well below 10(10) M-1. DSC data from the literature on a number of interacting systems (trypsin-soybean trypsin inhibitor, trypsin-ovomucoid, trypsin-pancreatic trypsin inhibitor, chymotrypsin-subtilisin inhibitor, subtilisin BPN-subtilisin inhibitor, RNase S protein-RNase S peptide, avidin-biotin, ovotransferrin-Fe3+, superoxide dismutase-Zn2+, alkaline phosphatase-Zn2+, and assembly of regulatory and catalytic subunits of aspartate transcarbamoylase) were analyzed by simulation fitting or by calculation. Apparent single-site binding constants ranged from ca. 10(5) to 10(20) M-1, while the interaction constant for assembly of aspartate transcarbamoylase was estimated as 10(37) in molarity units. For most of these systems, the DSC interaction constants compared favorably with other literature estimates, for some it did not for reasons unknown, while for still others this represented the first estimate. Simulations show that for proteins having two binding sites for the same ligand within a single cooperative unit, ligand rearrangement will occur spontaneously during a DSC scan as the transition temperature of the unliganded protein is approached.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Study of strong to ultratight protein interactions using differential scanning calorimetry. 220 24


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