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)

Alkaline ribonuclease (RNase) from polyribosomes derived from experimental granulation tissue has been purified 1900-fold through affinity chromatography. The preparation was homogeneous in sodium dodecyl sulfate (SDS) polyacrylamide-gel electrophoresis with an estimated molecular weight of 15 000. Purified RNase was completely inhibited in the presence of divalent ions Mg2+(100 mM) and Ca2+(100 mM) but activated slightly with Na+(50 mM). The enzyme is an endonuclease and the best substrates were poly(U), mixed RNA from yeast, rRNA from granulation tissue and poly(C). The estimated apparent Km-values were 0.037, 0.064, 0.13 and 0.27 g1-1, respectively. In polyribosomes RNase occurred in both free and p-chloromercuribenzoate (pCMB)-liberated forms. The total activity was at the highest but the proportion of the free activity minimal in the granulation tissue during the maximal synthesis of collagen.
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PMID:Alkaline ribonuclease associated with polyribosomes in fibroblasts of experimental granulation tissue. 3 15

Macromolecules from rat peritoneal macrophage culture media were separated into 30 fractions by flat bed isoelectric focusing (IEF). The fractions were tested for their influence on thymidine and proline incorporation into cultured rat granuloma fibroblasts. Three fractions, stable after freezing and lyophilization, were of interest: one inhibiting thymidine incorporation (focusing at pH 7.3-7.6), another stimulating thymidine incorporation (focusing at pH 4.4-5.3), and the third stimulating proline incorporation into cells and medium collagen (focusing at pH 10.2-10.7). The last also exhibited a ribonuclease (RNase) activity with a pH-optimum of 7.0-7.5.
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PMID:Isoelectric focusing of macrophage culture media and the effect of the fractions on the synthesis of DNA and collagen by fibroblasts. 4 72

Collagen-synthesizing polysomes were isolated by low-speed centrifugation of the post-mitochondrial supernatant of chick homogenates. Electron microscopy of the fraction thus isolated shows it to be exclusively composed of ribosomes. Amino acid incorporation in vitro showed that these particles were efficient in the incorporation of proline, but not tryptophan, as opposed to ribosomes obtained from the supernatant of the low-speed centrifugation. The incorporation process was highly dependent on GTP, and exibited an optimal Mg2+concentration of 5.6mM. The reaction was inhibited by RNase, elongation inhibitors as anysomycin, sparsomycin, fusidic acid and GDPCP. It was also moderately inhibited by initiation inhibitors such as aurintricarboxilic acid and pyrocatechol violet. The product of the incorporation was characterized as collagen by its sensitivity towards purified collagenase, lack of tryptophan, chromatography in CM-cellulose and molecular sieve chromatography in Sephadex G-200.
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PMID:Isolation and characterization of collagen-synthesizing polysomes from chick embryos. 16 69

The medium of cultured, SiO2-treated peritoneal macrophages contained a factor which enhances the incorporation of labelled proline to collagen and other proteins in granulation tissue slices, cells and polysomes. Simultaneously, the activity of alkaline RNase in the whole medium was decreased in comparison with the corresponding control. Polyvinylpyridine-N-oxide, PVNO, protected the macrophages against SiO2. Latex-particles and E. coli lipopolysaccharide decreased the RNase activity in the macrophage medium, but unlike SiO2 did not cause liberation of the collagen synthesis-stimulating factor. Fractionation of the medium by gel filtration chromatography showed the SiO2-pretreatment to have caused a very significant decrease in the aggregation state of RNase. The fraction from gel filtration chromatography that contained the SiO2-liberated factor stimulating collagen synthesis also contained the disaggregated RNase. There was no RNase-activity in the control sample. A homogenous protein (mol. wt. 14,300) was isolated with repeated gel filtrations from the medium of silica-treated macrophages. It increased the incorporation of 3H proline and 3H thymidine into cultured granuloma cells.
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PMID:Fractionation of connective-tissue-activating factors from the culture medium of silica-treated macrophages. 22 24

The effects of a mild zinc-deficient state in humans were studied. Four male volunteers received restricted zinc intake for several weeks under strict metabolic conditions. As a result of dietary zinc restriction, a decrease in zinc concentration of plasma, erythrocytes, leukocytes, and urine was observed. Changes in the activities of zinc-dependent enzymes in the plasma such as alkaline phosphatase and ribonuclease were also related to the dietary zinc status. An adverse effect of zinc restriction on total protein, total collagen, ribonucleic acid, and the activity of deoxythymidine kinase (a zinc-dependent enzyme) in the sponge connective tissue of the two volunteers in whom this test was done was noted. During the zinc restriction period, the ammonia level in the plasma was elevated. Weight loss occurred in all subjects as a result of dietary zinc restriction. Inasmuch as the zinc-deficient state was mild, this study provides a basis for developing diagnostic criteria for zinc deficiency in humans.
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PMID:Experimental zinc deficiency in humans. 69 27

Antibodies against embryonic chick bone collagen were prepared in rabbits and were purified by affinity and ion exchange chromatography until collagen-specific and RNase-free. 125I-anti-collagen antibodies were used to locate the collagen-synthesizing polysomes of 8-day chick embryo wings and legs on sucrose gradients by measuring the polysome associated radioactivity. The 125I-anti-collagen antibodies bound predominantly to polysomes in the heavy region of sucrose gradients. These binding sites could only be saturated with homologous anti-collagen antibodies. Further evidence for the specificity of this reaction was provided by a correlation of the amount of anti-collagen antibodies bound in the heavy regions of sucrose gradients with the amount of collagen being synthesized by a particular tissue. The validity of this immunochemical method was confirmed by localizing collagen-synthesizing polysomes by an independent method which utilizes their ability to incorporate [3H]proline into collagen peptides in a cell-free system. The collagen-synthesizing polysomes are found in a single, rather broad peak in these gradients. The results of shortening the centrifugation time indicate that larger species of collagen-synthesizing polysomes are not present in these tissues. Partial purification of the collagen-synthesizing polysomes may be achieved by specifically sedimenting them after treatment with anti-collagen antibodies followed by goat anti-rabbit antibodies.
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PMID:Identification and purification of collagen-synthesizing polysomes with anti-collagen antibodies. 116 62

The amino- and carboxyl-terminal globular domains of type VI collagen are composed of several homologous modules similar to the type A collagen-binding modules present in von Willebrand factor. The human alpha 3(VI) chain that contributes most of the amino-terminal globule appears heterogeneous in size as a result of alternative splicing of two exons (Stokes D. G., Saitta, B., Timpl, R., and Chu, M.-L. (1991) J. Biol. Chem. 266, 8626-8633). In the present study, we report a further characterization of the 5'-end of the gene of the human alpha 3(VI) chain and show that transcription initiates at multiple sites. Southern blotting and DNA sequencing indicate that there is an additional type A exon (A9/N10) at about 1.8 kilobase pairs downstream of the exon coding for the signal peptide. The open reading frame of this additional exon reveals 1 cysteine and three potential N-glycosylation sites. Polymerase chain reaction, Northern blotting, and RNase protection assays demonstrate that exon A9/N10 is subject to alternative splicing in normal and tumor cell lines and that this generates more protein variants of the alpha 3(VI) chain than expected before. A comparison with the corresponding amino-terminal globule of the chicken alpha 3(VI) chain shows the presence of 1 additional cysteine in this portion of the molecule and suggests that human type VI collagen has more possibilities for structural and functional variations compared to chicken type VI collagen.
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PMID:The human type VI collagen gene. mRNA and protein variants of the alpha 3 chain generated by alternative splicing of an additional 5-end exon. 133 40

We sought to determine the cis-acting elements responsible for the pattern of tissue specific expression of the mouse alpha 2(I) collagen gene. Using an RNase protection assay we first verified that expression of the alpha 2(I) collagen gene is mainly confined to tendons, bone, and skin in mice. Both transgenic mice and DNA transfection of tissue culture cells were used as experimental approaches. Transgenic mice lines were generated harboring chloramphenicol acetyltransferase (CAT) chimeric genes that contained either (a) 2000 base pairs (bp) of 5'-flanking sequences of the mouse alpha 2(I) collagen gene plus additional sequences between +418 and +1524 of the first intron of this gene or (b) the same promoter sequences without intron sequences or (c) the 350-bp proximal promoter sequences. Transgenic mice containing both types of 2000-bp promoters showed a pattern of CAT expression that was tissue specific. The presence of sequences of the first intron in the transgene did not increase the level of promoter activity. Transgenic mice harboring the 350-bp alpha 2(I) collagen promoter also showed a pattern that was tissue-specific except that high level expression also occurred in the brain. This suggests that negative regulation is an important component of tissue-specific expression. In order to analyze the first 350 bases in detail, we performed transient expression experiments, using promoter fragments attached to the luciferase reporter gene. Fibroblasts, which show a high level expression of the endogenous alpha 2(I) collagen gene, and B cells, in which the gene is silent, were transfected with a series of deletions and substitution mutations within the proximal 350-bp promoter. These experiments were unable to define unique cell-specific cis-acting elements. However, when the sequence between -315 and -284 was tandemly repeated upstream of a minimal alpha 2(I) collagen promoter (-41 to +54), the activity of this construction was considerably higher in fibroblasts than in B cells when compared with the minimal promoter itself. In gel retardation assays, the levels of complexes that bind to this sequence were higher in fibroblast nuclear extracts than in myeloma nuclear extracts. Our results are consistent with the hypothesis that the -315 to -284 DNA sequence participates in the cell-specific control of the alpha 2(I) collagen gene in fibroblasts.
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PMID:Tissue-specific expression of the mouse alpha 2(I) collagen promoter. Studies in transgenic mice and in tissue culture cells. 152 81

In mammals, the periodontal ligament (PDL) is a highly specialized tissue which facilitates tooth eruption and lends mechanical support to the tooth once in occlusion. The PDL extracellular matrix fibers play a major role in such functions. During its development, the spatial arrangement of the PDL extracellular matrix undergoes rapid changes. So that it could be determined whether the structural alteration in the PDL is associated with changes in the expression of collagenous proteins with different functional properties, the transcriptional patterns of collagens I and XII were examined. The maxillary dento-alveolar segments, each containing three molars, from 25-day-old and 40-day-old Sprague-Dawley rats were selected as being representative of developing and matured tissues, respectively. Rat alpha 2(I) collagen cDNA and rat alpha 1(XII) collagen cDNA were used as molecular probes for identification of the corresponding mRNAs by RNA transfer blot analysis, RNase protection assay, and in situ hybridization. The results showed that alpha 2(I) collagen mRNA was expressed in both developing and matured tissues. However, the level of expression decreased with maturity. In contrast, the expression of alpha 1(XII) collagen was increased in the matured tissue as compared with the developing tissue. In situ hybridization in these tissues indicated that the expression of alpha 1(XII) collagen mRNA was limited to the mature stage of PDL development. It is suggested that collagen fibril arrangement during PDL development may be related to the expression of collagen XII.
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PMID:Site-specific expression of collagen I and XII mRNAs in the rat periodontal ligament at two developmental stages. 162 50

A critical process during early heart development is the formation of mesenchymal cells which will contribute to valves and septa of the mature heart. These cells arise by an epithelial-mesenchymal transformation of endothelial cells in the atrioventricular (AV) canal and outflow tract areas of the heart. Adjacent endothelial cells in the atrium and ventricle remain epithelial. A three-dimensional collagen gel culture system has been exploited to examine the interactions that mediate this transformation. The AV canal myocardium produces a stimulus that is transmitted through an intervening extracellular matrix to the AV canal endothelium. This interaction is regionally specific, such that ventricular myocardium does not provide an adequate stimulus and ventricular endothelium does not respond to the AV canal myocardial stimulus. Exogenous TGF-beta 1 (or TGF-beta 2) can complement ventricular myocardium to produce transformation by AV canal endothelium. A blocking antibody, effective against several TGF-beta, prevents cell transformation. To identify the specific member of the TGF-beta family that functions in situ, antisense oligonucleotides for each of the numbered TGF-beta were topically added to AV canal explant cultures. Only the oligonucleotide targeted to TGF-beta 3 was an effective inhibitor of mesenchymal cell formation. Studies have been undertaken to localize specific mRNas by in situ hybridization and RNase protection assays. These assays have concentrated on the regional and temporal appearance of TGF-beta 2 and 3. Surprisingly, RNase protection assays with a TGF-beta 3 sense probe showed the presence of a transcript complementary to TGF-beta 3.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:TGF-beta 3-mediated tissue interaction during embryonic heart development. 163 53


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