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)

Cloning of the genes encoding distinct subtypes of human alpha 2-adrenergic receptors (alpha 2-AR) allows the separate recombinant expression of each individual subtype in heterologous systems. We report here the transfection, selection and preliminary pharmacological characterization of two mammalian cell lines, adherent Shionogi S115 mouse mammary tumour cells and human B-lymphoblastoid IBW4 cells growing in suspension, expressing the human alpha 2-AR subtypes alpha 2-C4 and alpha 2-C10 at densities of approx. 2 x 10(5) receptors/cell. Transfection of the subtype genes was verified using a specific RNase protection assay. Pharmacological characterization was carried out with [3H]rauwolscine binding, which was inhibited by oxymetazoline and prazosin in a subtype-selective manner. The sensitivity of (-)-noradrenaline binding to the GTP-analogue 5'-guanylylimidodiphosphate suggested that the receptors are coupled to G-proteins. This was verified in S115 cells by efficient inhibition of forskolin-stimulated cAMP production by the alpha 2-AR agonists, (-)-noradrenaline and clonidine. These cell lines thus appear to be suitable for pharmacological studies on receptor function and ligand binding.
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PMID:Stable expression of recombinant human alpha 2-adrenoceptor subtypes in two mammalian cell lines: characterization with [3H]rauwolscine binding, inhibition of adenylate cyclase and RNase protection assay. 131 4

Northern blot and ribonuclease protection assay were used to identify alpha 2-adrenoceptor subtypes in human colonic adenocarcinoma (HT29), neuroblastoma x glioma rat-mouse hybrid NG108-15 (NG108) and opossum kidney (OK) cell lines. Radioligand binding studies showed that the alpha 2-adrenoceptor expressed in HT29, NG108 and OK cells represent the pharmacological alpha 2A, alpha 2B and alpha 2C subtypes respectively. In our Northern blot analysis, hybridization of poly(A)+ RNA from HT29, NG108 and OK cells with human kidney alpha 2-adrenoceptor cDNA probe (alpha 2-C4) identified a single band of 4.4, 4.2 and 4.4 kb respectively in each cell line. Hybridization with a human platelet alpha 2-adrenoceptor genomic probe (alpha 2-C10) resulted in two bands for HT29 cells with the size of 4.4 kb and 3.9 kb. No bands were seen for HT29, NG108 and OK cells when hybridized with a third alpha 2-adrenoceptor human genomic DNA probe which is localized in chromosome 2 (alpha 2-C2). For the HT29 cells, the 3.9 kb band was seen only when using the alpha 2-C10 probe. Thus, this band probably represents alpha 2-C10 mRNA. To further characterize the alpha 2-adrenoceptor mRNA expressed in HT29, NG108 and OK cells, the sensitive ribonuclease protection assay was performed. A single band about 900 bp was protected when the poly(A)+ RNA from NG108 and OK cells was hybridized with an alpha 2-C4 RNA probe and digested with RNAases. Hybridization of mRNA from HT29 cells with alpha 2-C10 RNA probe and digestion with RNAases protected a 500 bp fragment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Northern blot and ribonuclease protection study of alpha 2-adrenoceptor subtypes in cultured cell lines. 132 48

The density of the alpha 2A-adrenergic receptor in the HT29 cell line, a human colonic adenocarcinoma, increases when the cells are placed in fetal calf serum (FCS)-free culture medium and decreases again, in a concentration-dependent manner, when they are re-exposed to FCS. In an attempt to identify the FCS components responsible for this phenomenon, we examined the effect of insulin and of various growth factors on receptor expression. Incubation of HT29 cells with insulin resulted in a time- and dose-dependent lowering of the alpha 2-adrenergic receptor number. The decrease of [3H] RX821002 binding sites after a 48-h period of treatment reached 70-75% with 170 nM insulin, and a half-maximal effect was observed at 2.6 nM. This value is in agreement with the EC50 of the hormone for stimulating the glycolytic activity of HT29 cells (8 nM) and is sufficiently low to indicate that the decrease of alpha 2-adrenergic receptor number is mediated through stimulation of insulin receptors. Direct quantification of [3H] UK14304 binding sites and the study of the inhibition of [3H]RX821002 binding by (-)-epinephrine indicated that the degree of receptor coupling to Gi protein was not affected when the receptor number was decreased by insulin treatment. The reduction in receptor number did result in an attenuation of the inhibitory effect of UK14304 on forskolin-induced cAMP accumulation in a manner which was consistent with the existence of a large population of spare receptors in untreated cells. The action of insulin is not due to an accelerated rate of receptor degradation and can be mimicked by other growth factors (epidermal growth factor and insulin-like growth factors I and II) acting through stimulation of tyrosine kinase receptors. RNase mapping experiments with a 0.35-kilobase riboprobe prepared from the human alpha 2 C10-adrenergic receptor gene demonstrated that the decrease of receptor number induced by the different treatments is a reflection of changes occurring at the level of its mRNA. The use of cycloheximide indicated that the effect of insulin on alpha 2-adrenergic receptor mRNA does not require protein synthesis. The half-life of the alpha 2-adrenergic receptor mRNA measured after the addition of actinomycin D was unchanged by insulin which suggests that a decrease in the transcription rate is the predominant factor responsible for the observed regulation of receptor expression.
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PMID:Regulation of the alpha 2A-adrenergic receptor in the HT29 cell line. Effects of insulin and growth factors. 167 44

The complete amino acid sequence of ribonuclease N1 (RNase N1), a guanine-specific ribonuclease from a fungus, Neurospora crassa, was determined by conventional protein sequencing, using peptide fragments obtained by tryptic digestion of cyanogen bromide-treated RNase N1 and by Staphylococcus aureus V8 protease digestion of heat-denatured RNase N1. The results showed that the protein is composed of a single polypeptide chain of 104 amino acid residues cross-linked by two disulfide bonds and has a molecular weight of 11,174: (sequence; see text) (Disulfide bonds: C2-C10, C6-C103) The amino acid sequence was homologous with those of RNase T1 (65% identity) and related microbial RNases.
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PMID:The amino acid sequence of ribonuclease N1, a guanine-specific ribonuclease from the fungus Neurospora crassa. 297 30

Combined action of polyornithine and lecithin modified tobacco mosaic virus (TMV) virions making them sensitive to ribonuclease (RNase), pronase or Triton X-100. Sedimentational analysis and examination of the fluorescence spectrum revealed that the reaction product obtained after RNase treatments of modified TMV was a three-component complex made of coat protein, polyornithine and lecithin. The minimum requirement for the modification was completely fulfilled by cetyltrimethylammonium bromide, suggesting that a positively charged nitrogen group and an alkyl group of moderate size, C10--18, are necessary components. These components react with the surface region of TMV which is considered to have an important role in connecting coat protein subunits in TMV virions.
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PMID:Modification of tobacco mosaic virus by polyornithine and lecithin. 741 96

Escherichia coli ribonuclease HI mainly recognizes the DNA/RNA hybrid regions preceding the cleavage site. To understand the interaction between the enzyme and the substrate in more detail, the kinetic properties of the enzyme, as well as its variant with mutations in the basic protrusion, were studied using a series of oligomeric DNA/RNA hybrids as substrates. These substrates were prepared by hybridizing a 12-b RNA (5'-CGGAGAUGACGG-3') with DNA oligomers varying in size and sequence. The 12-b RNA hybridized to the complementary 12-b DNA was primarily cleaved at A9-C10. Since an increase in the length of the RNA between the cleavage site and the 5' end of the DNA/RNA hybrid, achieved using a longer DNA/RNA substrate, did not seriously affect the kinetic parameters of the enzyme, the 12-bp DNA/RNA hybrid seems to be large enough to contact the entire substrate-binding site of the enzyme. The kinetic data presented here suggest that the DNA residues complementary to the RNA residues located six or seven residues upstream from the cleavage site interact with the basic protrusion of the enzyme, regardless of whether or not it is hybridized to the RNA strand. Such an interaction is permitted only when the conformation of either the enzyme or the substrate, or both, is changed upon binding.
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PMID:Kinetic analysis of Escherichia coli ribonuclease HI using oligomeric DNA/RNA substrates suggests an alternative mechanism for the interaction between the enzyme and the substrate. 754 80

Sequences from cDNA molecules encoding alpha 2-adrenoceptor subtype genes were subcloned into prokaryotic vectors and riboprobes generated to hybridise selectively with each of the human alpha 2C2-, alpha 2C4- and alpha 2C10-adrenoceptor subtype mRNA species. The riboprobes were labelled with either 32P or digoxigenin and used to study the expression of alpha 2-adrenoceptor subtypes in sections of human pancreas, in isolated human islets of Langerhans and in clonal HIT-T15 pancreatic beta-cells. Using a ribonuclease protection assay protocol, expression of mRNA species encoding both alpha 2 C2 and alpha 2 C10 was demonstrated in preparations of isolated human islets of Langerhans. mRNA encoding alpha 2C4 was also detected in human islet RNA, using reverse transcription coupled with the polymerase chain reaction. In situ hybridisation was then employed to examine the distribution of each alpha 2-adrenoceptor subtype in sections of human pancreas. All three subtypes of alpha 2-adrenoceptor mRNA were identified in sections of formalin-fixed, paraffin-embedded human pancreas using riboprobes labelled with digoxigenin. Although some labelling of the three alpha 2-adrenoceptor mRNA subtypes was seen in the islets, the labelling was most intense in the exocrine tissue of the pancreas for each receptor subtype. The specificity of the digoxigenin-labelled RNA probes was confirmed in several control tissues and by in situ hybridisation studies using sense probes in the pancreas. The integrity of the pancreas sections was confirmed by in situ hybridisation with an antisense riboprobe derived from human insulin cDNA. The results demonstrate that multiple alpha 2-adrenoceptor subtypes are expressed in human pancreas. Both the exocrine and endocrine cells express more than one receptor subtype, although the islets stain less intensely than the bulk of the tissue suggesting that the islet cells may have lower levels of expression than the acinar tissue. The presence of alpha 2-adrenoceptor subtype mRNA species in pancreatic beta-cells was confirmed by Northern blotting of RNA extracted from the clonal beta-cell line, HIT-T15. Transcripts encoding each of the three cloned alpha 2-adrenoceptor subtypes were detected in HIT-T15 cells. Hybridisation of sections of human pancreas with oligodeoxynucleotide probes designed to hybridise with beta 2-adrenoceptor mRNA revealed expression of this species in islet beta-cells but not in the exocrine tissue of the pancreas.
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PMID:Expression of alpha 2- and beta-adrenoceptor subtypes in human islets of Langerhans. 877 32

Transgenic plant experiments have great potential for extending our understanding of the role of specific genes in controlling pollination. Often, the intent of such experiments is to over-express a gene and test for effects on pollination. We have examined the efficiency of six different S-RNase constructs in Nicotiana species and hybrids. Each construct contained the coding region, intron, and downstream sequences from the Nicotiana alata S(A2)-RNase gene. Among the six expression constructs, two utilized the cauliflower mosaic virus (CaMV) 35S promoter with duplicated enhancer, and four utilized promoters from genes expressed primarily in pistils. The latter included promoters from the tomato Chi2;1 and 9612 genes, a promoter from the N. alata S(A2)-RNase gene, and a promoter from the Brassica SLG-13 gene. Some or all of the constructs were tested in N. tabacum, N. plumbaginifolia, N. plumbaginifolia x SI N. alata S(C10)S(c10) hybrids, N. langsdorffii, and N. langsdorffii x SC N. alata hybrids. Stylar specific RNase activities and S(A2)-RNase transcript levels were determined in transformed plants. Constructs including the tomato Chi2;1 gene promoter or the Brassica SLG-13 promoter provided the highest levels of S(A2)-RNase expression. Transgene expression patterns were tightly regulated, the highest level of expression was observed in post-anthesis styles. Expression levels of the S(A2)-RNase transgenes was dependent on the genetic background of the host. Higher levels of S(A2)-RNase expression were observed in N. plumbaginifolia x SC N. alata hybrids than in N. plumbaginifolia.
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PMID:Expressing foreign genes in the pistil: a comparison of S-RNase constructs in different Nicotiana backgrounds. 961 22

Although S-locus RNases (S-RNases) determine the specificity of pollen rejection in self-incompatible (SI) solanaceous plants, they alone are not sufficient to cause S-allele-specific pollen rejection. To identify non-S-RNase sequences that are required for pollen rejection, a Nicotiana alata cDNA library was screened by differential hybridization. One clone, designated HT, hybridized strongly to RNA from N. alata styles but not to RNA from Nicotiana plumbaginifolia, a species known to lack one or more factors necessary for S-allele-specific pollen rejection. Sequence analysis revealed a 101-residue ORF including a putative secretion signal and an asparagine-rich domain near the C terminus. RNA blot analysis showed that the HT-transcript accumulates in the stigma and style before anthesis. The timing of HT-expression lags slightly behind S(C10)-RNase in SI N. alata S(C10)S(C10) and is well correlated with the onset of S-allele-specific pollen rejection in the style. An antisense-HT construct was prepared to test for a role in pollen rejection. Transformed (N. plumbaginifolia x SI N. alata S(C10)S(C10)) hybrids with reduced levels of HT-protein continued to express S(C10)-RNase but failed to reject S(C10)-pollen. Control hybrids expressing both S(C10)-RNase and HT-protein showed a normal S-allele-specific pollen rejection response. We conclude that HT-protein is directly implicated in pollen rejection.
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PMID:A small asparagine-rich protein required for S-allele-specific pollen rejection in Nicotiana. 1055 58

Fibrosis is a common outcome of chronic inflammation or injury. Pulmonary fibrosis may be the result of abnormal repair after an acute inflammatory response. The process of repair initiated by a tissue insult is largely a function of the activation of cells to produce important biological mediators such as cytokines, growth factors and chemokines, which orchestrate most aspects of the inflammatory response. Consequently, altered regulation of the production of inflammatory cell cytokines and chemokines after injury and repair likely contributes to the fibrosis. Our hypothesis is that chronic expression of specific chemokine and chemokine receptors during the fibrotic phase induced by thoracic irradiation may perpetuate the recruitment and activation of lymphocytes and macrophages, which may contribute to the development of fibrosis. Fibrosis-sensitive (C57BL/6) and fibrosis-resistant (C3H/HeJ) mice were irradiated with a single dose of 12.5 Gy to the thorax. Total lung RNA was prepared and hybridized using microarray analysis and RNase protection assays. At 26 weeks postirradiation, messages encoding the chemokines BLC (now known as Scyb13), C10 (now known as Scya6), IP-10 (now known as Scyb10), MCP-1 (now known as Scya2), MCP-3 (now known as Scya7), MIP-1gamma (now known as Scya9), and RANTES (now known as Scya5) and the chemokine receptors Ccr1, Ccr2, Ccr5 and Ccr6 were elevated in fibrosis-sensitive (C57BL/6) mice. In contrast, only the messages encoding SDF-1alpha (now known as Sdf1) and Ccr1 were elevated 26 weeks postirradiation in fibrosis-resistant (C3H/HeJ) mice. Our results point to the CC and CCR family members as the predominant chemokine responders during the development of fibrosis. These studies suggest that monocyte/macrophage and lymphocyte recruitment and activation are key components of radiation-induced fibrosis.
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PMID:Radiation-induced pulmonary fibrosis: examination of chemokine and chemokine receptor families. 1183 87


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