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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Murine NIH3T3 cells were used to study the effect of ribozymes on H-ras-mediated transformation. Parental 3T3 cells were transfected with the activated H-ras gene. H-ras-transformed cells had altered morphology and increased colony formation in soft agar in contrast to untransfected 3T3 cells. A hammerhead ribozyme (site-specific ribonuclease) designed to cleave codon 12 (GUC) of the activated H-ras RNA was expressed in transformed cells. 3T3 clones expressing the ras ribozyme displayed decreased expression of activated H-ras RNA. The ras ribozyme reversed the transformed phenotype to resemble that of untransfected 3T3 cells. Furthermore, 3T3 cells containing the ras ribozyme were shown to suppress transformation when they were subsequently transfected with activated H-ras. Insertion of a mutant ribozyme largely devoid of cleaving capacity into H-ras-transformed cells resulted in smaller reductions in H-ras gene expression and colony formation in soft agar when compared with the ras ribozyme. Finally, the ras ribozyme alone did not perturb normal 3T3 cell growth. This study suggests the possible utility of anti-oncogene ribozymes as suppressors of tumor cell growth as well as inhibitors of cellular transformation.
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PMID:Suppression of H-ras-mediated transformation in NIH3T3 cells by a ras ribozyme. 794 47

Lysyl oxidase, an extracellular enzyme involved in the maturation of collagen and elastin, also appears to function as a phenotypic suppressor of transformation by the ras gene product, p21. Genomic clones of the mouse lysyl oxidase gene have been isolated, analyzed, and sequenced. Lysyl oxidase appears to be a single-copy gene, organized into seven exons and six introns, and spans approximately 14 kb of the mouse genome. The gene encodes two messages, sized at about 4.8 and 3.8 kb, that differ in the length of the untranslated sequence at the 3' end of the gene. All of the 3' untranslated sequence and the polyadenylation signals are contained in exon VII; there is no evidence of alternate splicing. Primer extension and ribonuclease protection experiments revealed two sites of transcription initiation in a region with sequence motifs characteristic of a promoter, which was upstream and adjacent to the 5' untranslated sequence found in the cDNA.
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PMID:Structure of the mouse lysyl oxidase gene. 810 Feb 14

In this study, the efficacy of an anti-ras ribozyme in reversing a transformed phenotype was investigated. A murine NIH/3T3-derived cell line, designated 2-12, contains an inducible Ha-ras oncogene, which is regulated by the Escherichia coli (E. coli) lac operator/repressor system, and displays a transformed phenotype after isopropyl-beta-D-thiogalactoside induction. To reverse the transformed characteristics, the ribozyme, which specifically targets the Ha-ras oncogene at the codon 12 mutation site (GGC to GUC), was transfected into 2-12 cells. Two (ribZ4 and ribZ7) clones were subsequently selected and analyzed for their transforming features. Our results show that, in the transfectants, ribozyme gene expression was detected, and the target Ha-ras transgene was expressed at basal levels. Their phenotypic responses, including morphology, cell growth rate, colony-formation efficiency and tumorigenicity in mice with severe combined immunodeficiency were more similar to those of NIH/3T3 than 2-12 transformed cells. Directly injecting the ribozyme DNA into tumors induced by transformed 2-12 cells in BALB/c mice also caused tumor regression. The enzymatic cleavage products of the ribozyme acting on mutant Ha-ras mRNA in vivo were detected by primer-extension analysis. These results indicate that the ribozyme were designed exhibits a site-specific ribonuclease function that effectively abrogates Ha-ras-oncogene-induced transformation, and this unique anti-Ha-ras property should shed light on the development of strategies against the Ha-ras-oncogene-initiated malignancy.
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PMID:A ribozyme specifically suppresses transformation and tumorigenicity of Ha-ras-oncogene-transformed NIH/3T3 cell lines. 903 Feb 47

Ras associated with diabetes (Rad), a new ras-related GTPase, was recently identified by subtractive cloning as an mRNA in skeletal muscle that is overexpressed in NIDDM. To better understand its metabolic significance, we measured skeletal muscle Rad expression in well-characterized insulin sensitive (IS) and insulin resistant (IR) subjects with normal glucose tolerance and in untreated NIDDM patients. We found no differences in expression of Rad mRNA levels among IS, IR, and NIDDM groups using a ribonuclease protection assay (0.22 +/- 0.06, 0.13 +/- 0.01, and 0.16 +/- 0.02 relative units, respectively; NS) and no differences in Rad protein expression using a specific anti-peptide Rad antibody (1.05 +/- 0.18, 1.14 +/- 0.08, and 1.08 +/- 0.21 units/mg protein, respectively; NS). However, Rad protein levels were positively correlated with BMI (r = 0.43, P = 0.03) and percentage body fat (r = 0.55, P < 0.005), two independent measures of obesity, and negatively correlated with resting metabolic rate (r = 0.49, P = 0.01). In multiple regression analyses, percentage body fat and resting metabolic rate independently accounted for 30 and 10% of individual variability in muscle Rad protein expression. In conclusion, Rad expression in skeletal muscle is not altered as a function of insulin resistance or NIDDM in humans. However, these data, for the first time, implicate a role for Rad in regulating body composition and energy expenditure and provide a framework for studies designed to elucidate Rad's cellular functions.
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PMID:Muscle Rad expression and human metabolism: potential role of the novel Ras-related GTPase in energy expenditure and body composition. 903 1

Proto-oncogenes are involved in the regulation of gene expression, for example after ligand binding to growth factor receptors. Expression of the proto-oncogenes c-fos, c-jun, c-ha-ras and c-myc was studied in in vivo grown and in vitro cultured bovine preimplantation blastocysts employing RT-PCR, ribonuclease protection assay and immunohistochemistry. Thirteen- and 14- day-old preimplantation blastocysts, i.e. stages before and during trophoblast elongation, were used. In in vivo-grown blastocysts c-fos, c-jun and c-ha-ras transcripts as well as c-Fos, c-Jun and c-Myc proteins were detected in all stages studied. Cultured blastocysts were treated with 10 nM epidermal growth factor and 10 nM transforming growth factor-alpha simultaneously. Epidermal growth factor and transforming growth factor-alpha treatment induced c-fos mRNA and c-Myc protein expression. The induction of downstream targets of the epidermal growth factor receptor by epidermal growth factor and transforming growth factor-alpha indicates a functional epidermal growth factor signal transduction pathway in elongating bovine blastocysts.
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PMID:Expression of proto-oncogenes in bovine preimplantation blastocysts. 1083 31

The antiproliferative action of the guanine-specific ribonuclease secreted by Bacillus intermedius (binase) was studied in different chicken and mouse cell lines. The proliferation rate of chicken embryo fibroblasts, either normal or Rous sarcoma virus-transformed, was significantly reduced by binase treatment. Among mouse fibroblasts, v-ras-transformed NIH3T3 cells were sensitive to binase, whereas the growth of non-transformed, v-src-transformed or v-fms-transformed NIH3T3 cells was not affected. A 48 h treatment with binase inhibited the Ca2+-dependent K+ current of v-ras-transformed NIH3T3 cells but had no effect on this membrane current in non-transformed and in v-src- or v-fms-transformed NIH3T3 cells. Our results suggest that mammalian cells expressing the ras-oncogene are a potential target for the antiproliferative action of binase.
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PMID:Bacillus intermedius ribonuclease as inhibitor of cell proliferation and membrane current. 1116 12

An inducible in vitro cell culture system was developed to assay HCV replication by direct biochemical means. A transcription plasmid containing a T7 promoter at the 5' end, full-length cDNA of the HCV genome, a ribozyme sequence from the antigenomic strand of hepatitis delta virus and a T7 terminator was prepared. To facilitate high-level transcription of HCV RNA, HepG2 cells were infected with replication deficient adenovirus containing the T7 RNA polymerase gene and later transfected with the transcription plasmid containing the full-length HCV genome. This transfection-based cell culture system expressed high levels of HCV structural (core, El and E2) and non-structural proteins (NS3 and NS5B) detectable by Western blot and immunofluorescence assays. Production of HCV RNA transcripts and presence of replicative negative strand of HCV was confirmed by ribonuclease protection assay indicating replication of HCV in the transfected HepG2 cell. The transfected HepG2 cells assembled 50-60 nm virus-like particles, which could be aggregated by anti-E2 antibodies. This model can be utilized for studying mechanisms of HCV replication, assembly of HCV particles and to test potential anti-HCV compounds.
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PMID:Inducible model to study negative strand RNA synthesis and assembly of hepatitis C virus from a full-length cDNA clone. 1133 40

Ribonuclease Sa (pI = 3.5) from Streptomyces aureofaciens and its 3K (D1K, D17K, E41K) (pI = 6.4) and 5K (3K + D25K, E74K) (pI = 10.2) mutants were tested for cytotoxicity. The 5K mutant was cytotoxic to normal and v-ras-transformed NIH3T3 mouse fibroblasts, but RNase Sa and 3K were not. The structure, stability, and activity of the three proteins are comparable, but the net charge at pH 7 increases from -7 for RNase Sa to -1 for 3K and to +3 for 5K. These results suggest that a net positive charge is a key determinant of ribonuclease cytotoxicity. The cytotoxic 5K mutant preferentially attacks v-ras-NIH3T3 fibroblasts, suggesting that mammalian cells expressing the ras-oncogene are potential targets for ribonuclease-based drugs.
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PMID:Changing the net charge from negative to positive makes ribonuclease Sa cytotoxic. 1223 73

Previously, we designed a ribozyme that targets the H-ras oncogene at the 12th codon mutation site (Chang et al., 1997). Ribozymes have antisense molecule and site-specific ribonuclease potential. In this study, an adenoviral vector was used to transduce the H-ras ribozyme into laryngeal cancer cells (HEp-2). This served to downregulate the H-ras gene expression in which this ribozyme performed antisense activity due to HEp-2 cells containing wild-type alleles in the 12th H-ras codon. Together, our data demonstrated that the recombinant adenovirus encoding H-ras ribozyme can be broadly regarded as a cytotoxic gene therapy in laryngeal cancer cells regardless of containing wild-type or mutant ras gene. In addition, the mechanism through which the H-ras ribozyme inhibited tumor growth was apoptosis and involved both caspase- and mitochondria-mediated pathways. The activators caspase-8 and -9 as well as the effector caspase-3 in the induction phase of apoptosis and the substrate PARP of caspase-3 in the execution phase were activated 48h following the H-ras ribozyme treatment. Mitochondrial events characterized by the production of superoxide anion and the release of cytochrome c started at 24h. Mitochondrial transmembrane potential loss occurred 48h after the ribozyme treatment. However, Bcl-2 delayed cytochrome c release to the cytosol, but it could not protect the apoptosis effect, suggesting that cytochrome c release from mitochondria may not play a role in H-ras ribozyme-induced apoptosis.
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PMID:Recombinant adenovirus encoding H-ras ribozyme induces apoptosis in laryngeal cancer cells through caspase- and mitochondria-dependent pathways. 1241 27

Hepatitis C virus (HCV) infection is usually treated with the combination of interferon and ribavirin, but only a small fraction of patients develop a sustained remission. There is need for the development of specific molecular approaches for the treatment of chronic HCV infection. We propose that RNA interference is highly effective antiviral strategy that offers great potential for the treatment of HCV infection. Three plasmid constructs expressing small interfering RNAs (siRNAs) targeted to sequences encoding the structural gene (E2) and non-structural genes (NS3, NS5B) of HCV1a genome were prepared. Antiviral properties of siRNAs against the HCV1a strain were studied in a transient replication model that involved the use of a transcription plasmid containing the full-length HCV genome and an adenovirus expressing T7 RNA polymerase. We found that siRNAs targeted to the E2, NS3 and NS5B regions of the HCV genome efficiently inhibited expression of the HCV core and NS5A protein measured by Western blot analysis and immunocytochemical staining. Intracytoplasmic immunization of siRNAs in HCV-transfected cells efficiently degraded genomic positive strand HCV RNA, as shown by ribonuclease protection assay (RPA). All three siRNAs efficiently inhibited synthesis of replicative negative strand HCV RNA in the transfected cells. A control siRNA plasmid against a Epstein--Barr virus latency gene did not inhibit protein expression and negative strand HCV RNA. These results suggest that RNAi is an effective and alternative approach that can be used to inhibit HCV expression and replication.
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PMID:Small interfering RNA effectively inhibits protein expression and negative strand RNA synthesis from a full-length hepatitis C virus clone. 1597 38


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