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)

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

Growth factor-depleted Swiss 3T3 cells responded to basic fibroblast growth factor (bFGF) with a burst of mitogenesis and with a rapid and marked increase in thrombospondin (TS) mRNA levels. mRNA levels for the alpha 1 chain of type I collagen and for fibronectin were unaffected. At early times following stimulation (0-2 h), "superinduction" of TS mRNA by inhibition of protein synthesis with cycloheximide was not observed, and the increase in TS mRNA could be attributed primarily to an increase in transcription rate of the TS gene. However, at later times (4-8 h) the combination of cycloheximide and bFGF superinduced TS mRNA levels, suggesting the existence of a labile inhibitor of transcription or a short-lived RNase that might be produced in response to prolonged treatment with bFGF. In contrast to its stimulatory effect on 3T3 cells, bFGF did not stimulate the proliferation of mouse muscle BC3H1 cells nor did it cause an increase in TS mRNA levels, but BC3H1 cells do respond to bFGF by inhibition of myogenic differentiation. We propose, on the basis of these and other findings, that TS facilitates the progression of some anchorage-dependent cells through the cell cycle.
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PMID:Thrombospondin gene expression is associated with mitogenesis in 3T3 cells: induction by basic fibroblast growth factor. 221 38

Calf aortic smooth muscle cell cultures produce both type III and type I collagen. Polyadenylated mRNA species purified from these cells direct the synthesis of prepro-alpha 1(III), prepro-alpha 1(I), and prepro-alpha 2(I) in a rabbit reticulocyte cell-free system. These polypeptides were identified by specific immunoprecipitation, cyanogen bromide peptide mapping, and bacterial collagenase digestion. Lower molecular weight collagenase susceptible polypeptides were also produced in translation reactions incubated under conditions optimized for incorporation of radiolabeled amino acids. Their presence did not appear to result from ribonuclease or protease involvement or from premature termination. Increasing the Mg2+ concentration in the translation system significantly reduced the production of these lower molecular weight species. Pulse-chase experiments indicate that the time required for completion of full length preprocollagen at the high Mg2+ concentration is greatly decreased compared to the low concentration. Additional experiments suggest that the incomplete collagen polypeptides result from pausing of ribosome movement during elongation. The relative synthesis of type III and type I chains was examined as a function of mRNA concentration in the cell-free system. At levels of RNA above saturation, the relative production of type III decreased with respect to type I. These data suggest that the ability of the alpha 1(III) mRNA to initiate translation is less efficient than the mRNAs of alpha 1(I) and alpha 2(I).
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PMID:Cell-free translation of calf type III collagen. Effect of magnesium on ribosome movement during elongation. 661 53

This study tested the hypothesis that the remodeling processes of adult periodontal ligament (PDL) reiterate the cellular and molecular events that occur sequentially during development. Type XII collagen has been implicated in the three-dimensional organization of the PDL extracellular matrix, and its expression has been restricted to the terminally differentiated stages. This study focused on the examination of the temporal and spatial expression of type XII collagen during experimental PDL remodeling in the rat. The temporal expressions of types I and XII collagen mRNAs were examined by RNA transfer blot and RNase protection assays, respectively, and were found to be relatively stable in the control group throughout the experimental period. In the tooth movement group, the expression of type I collagen increased at 72 hours and sustained the high level of expression at one week, while an increase in the expression of type XII collagen was first noted at the one-week period. The temporal activation of types I and XII collagen expression in the remodeling occurred in a pattern similar to that found during the development of the PDL. The spatial expression of type XII collagen mRNA was examined by in situ hybridization in the one-week-tooth-movement specimens. Labeled cells, which were more evident in the tension side, typically exhibited a spindle shape and were surrounded by the mature PDL matrix. Our data suggest that the type XII collagen expression may be closely associated with the functional regeneration of the PDL.
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PMID:Temporal and spatial expressions of type XII collagen in the remodeling periodontal ligament during experimental tooth movement. 787 23

Cirrhosis is characterized by a marked increase in the deposition of type I collagen and in the expression of the type I collagen genes alpha 1(I) and alpha 2(I). Although alpha 1(I) gene regulation has been extensively studied in cultured cells, these results may not be applicable to hepatic fibrogenesis in vivo. Therefore the regulation of the alpha 1(I) endogenous gene and an alpha 1(I) transgene was studied in a transgenic mouse model that has a single copy of a human alpha 1(I) gene segment containing the structural gene and 1.6 Kb of 5' DNA and 20 Kb of 3' DNA. To initiate hepatic fibrogenesis, we treated mice with the hepatotoxin carbon tetrachloride, either in a single dose or in biweekly doses for a period of 3 to 8 wk. Subsequently, hepatic alpha 1(I) messenger RNA levels were determined by a species-specific RNase protection assay. Carbon tetrachloride injections coordinately increased the messenger RNA levels of the alpha 1(I) endogenous gene and the transgene, both immediately and after 8 wk. These experiments demonstrate that this alpha 1(I) transgene fragment contains information sufficient for appropriate basal and carbon tetrachloride-stimulated hepatic expression. They further demonstrate that sufficient homology exists between the human and mouse regulatory elements for the recognition of human cis-acting elements by mouse trans-acting factors. Thus transgenic mice provide a unique model in which to characterize the collagen alpha 1(I) regulatory elements that are required in vivo for pathophysiological responses.
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PMID:Stimulation of the collagen alpha 1 (I) endogenous gene and transgene in carbon tetrachloride-induced hepatic fibrosis. 842 27

Type I collagen synthesis and deposition is generally indicative of irreversible damage in alcohol-induced cirrhosis in humans. However, in rodents, ethanol alone does not readily cause hepatic fibrosis. To determine whether this is because of a lack of ethanol-responsive elements, an artificial enhancer construct controlling rat type I collagen gene transcription was prepared in transgenic mice. The gene construct, ColCAT3.6, was a chimeric sequence containing the marker chloramphenicol acetyltransferase (CAT) gene linked to 3.5 kb of the rat alpha 1(I) 5'-flanking DNA, and 115 base pairs (bp) of transcribed collagen gene. Groups of transgenic mice were given 4 g/kg ethanol orally, twice daily for 4 weeks. As a positive control for hepatic fibrosis, transgenic mice were given intraperitoneal injections of CCl4, twice weekly for 4 weeks. Livers were assayed for CAT activity. Endogenous mouse collagen alpha 1(I) messenger RNA (mRNA) and transgene CAT mRNA were measured by RNase protection assays. Collagen synthesis in livers from the transgenic mice treated with ethanol were increased over controls, but the levels were not significantly different. Endogenous collagen alpha 1(I) steady-state mRNA levels in ethanol-treated mice were not significantly different compared with saline-treated controls. However, the transgene mRNA levels in ethanol-treated animals increased approximately 21-fold compared with saline-treated controls, as measured by RNase protection assays. Furthermore, the transgene product as measured by CAT activity in ethanol-treated mice was significantly increased threefold over saline-treated controls. We conclude that the 5'-flanking region of the rat alpha 1(I) collagen gene does contain regulatory elements that are strongly responsive to ethanol administration.
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PMID:A collagen enhancer-promoter construct in transgenic mice is markedly stimulated by ethanol administration. 859 57

Reexpression of aggrecan and type II collagen genes in dedifferentiated adult human articular chondrocytes (AHAC) in suspension culture varied widely depending on the specific lot of bovine serum used to supplement the culture medium. Some lots of serum provided strong induction of aggrecan and type II collagen expression by AHAC while others did not stimulate significant production of these hyaline cartilage extracellular matrix molecules even following several weeks in culture. Addition of 50 ng/ml insulin-like growth factor-I (IGF-I) to a deficient serum lot significantly enhanced its ability to induce aggrecan and type II collagen mRNA. Given this observation, IGF-I and other growth factors were tested in defined serum-free media for their effects on the expression of these genes. Neither IGF-I nor insulin nor transforming growth factor beta (TGF-beta) alone stimulated induction of aggrecan or type II collagen production by dedifferentiated AHAC. However, TGF-beta 1 or TGF-beta 2 combined with IGF-I or insulin provided a strong induction as demonstrated by RNase protection and immunohistochemical assays. Interestingly, type I collagen, previously shown to be downregulated in serum supplemented suspension cultures of articular chondrocytes, persisted for up to 12 weeks in AHAC cultured in defined medium supplemented with TGF-beta and IGF-I.
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PMID:Synergistic action of transforming growth factor-beta and insulin-like growth factor-I induces expression of type II collagen and aggrecan genes in adult human articular chondrocytes. 943 27

The role of the first intron of the Col1A1 gene in the regulation of type I collagen synthesis remains uncertain and controversial despite numerous studies that have made use of transgenic and transfection experiments. To examine the importance of the first intron in regulation of the gene, we have used the double-replacement method of gene targeting to introduce, by homologous recombination in embryonic stem (ES) cells, a mutated Col1A1 allele (Col-IntDelta). The Col-IntDelta allele contains a 1. 3-kb deletion within intron I and is also marked by the introduction of a silent mutation that created an XhoI restriction site in exon 7. Targeted mice were generated from two independently derived ES cell clones. Mice carrying two copies of the mutated gene were born in the expected Mendelian ratio, developed normally, and showed no apparent abnormalities. We used heterozygous mice to determine whether expression of the mutated allele differs from that of the normal allele. For this purpose, we developed a reverse transcription-PCR assay which takes advantage of the XhoI polymorphism in exon 7. Our results indicate that in the skin, and in cultured cells derived from the skin, the intron plays little or no role in constitutive expression of collagen I. However, in the lungs of young mice, the mutated allele was expressed at about 75% of the level of the normal allele, and in the adult lung expression was decreased to less than 50%. These results were confirmed by RNase protection assays which demonstrated a two- to threefold decrease in Col1A1 mRNA in lungs of homozygous mutant mice. Surprisingly, in cultured cells derived from the lung, the mutated allele was expressed at a level similar to that of the wild-type allele. Our results also indicated an age-dependent requirement for the intact intron in expression of the Col1A1 gene in muscle. Since the intron is spliced normally, and since the mutant allele is expressed as well as the wild-type allele in the skin, reduced mRNA stability is unlikely to contribute to the reduction in transcript levels. We conclude that the first intron of the Col1A1 gene plays a tissue-specific and developmentally regulated role in transcriptional regulation of the gene. Our experiments demonstrate the utility of gene-targeting techniques that produce subtle mutations for studies of cis-acting elements in gene regulation.
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PMID:A gene-targeting approach identifies a function for the first intron in expression of the alpha1(I) collagen gene. 958 77

Wnt glycoproteins mediate short range intracellular communication that facilitates morphogenesis and, in some settings, promotes tumor formation. Although the involvement of the Drosophila homolog wingless in ectodermal patterning is well established, the role that Wnt genes play in mammalian skin biology is not defined. We detected Wnt-4 and Wnt-10b mRNA in adult murine epidermis using degenerate primers and reverse transcriptase PCR, and confirmed expression by RNase protection. Normal murine keratinocytes and a melanocyte cell line (melan-A) propagated in vitro also contained Wnt-4 mRNA, whereas dermal fibroblasts and Langerhans cell-like dendritic cells did not. Because Wnt-4 mRNA was more abundant than Wnt-10b mRNA in epidermis and Wnt-10b trancripts were not detected in cells propagated in vitro, additional studies emphasized Wnt-4 exclusively. Wnt-4 mRNA levels were increased in cultured keratinocytes as they approached confluence and were strikingly downregulated by mitogenic growth factors. Although Wnt-4 mRNA levels were not modulated during calcium-induced keratinocyte differentiation in vitro, assessment of Wnt-4 transcripts in keratinocyte cell lines suggested that loss of Wnt-4 gene expression was associated with a less differentiated, more malignant, phenotype. Despite this, epidermal abnormalities were not identified in newborn Wnt-4 null (-/-) skin, or in full-thickness -/- skin that was engrafted to nude or athymic mice and allowed to mature for as long as 3 months. However, histologic examination of newborn Wnt-4 null skin did reveal fibroplasia involving the dermis with increased accumulation of type I collagen fibrils. These results indicate that several Wnt genes are expressed in adult murine epidermis and suggest that Wnt-4 proteins may be involved in epidermal-dermal interactions in mammalian skin.
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PMID:Characterization of Wnt gene expression in murine skin: possible involvement of epidermis-derived Wnt-4 in cutaneous epithelial-mesenchymal interactions. 971 59

Angiotensin II is an established regulator of vascular tone and smooth muscle cell (SMC) growth. However, there are little data about its effect on collagen synthesis by SMCs and none regarding the mechanism of such an effect. We studied the effect of angiotensin II on collagen production by human arterial SMCs, using uptake of [(3)H]proline into collagenase-digestible proteins, and by ribonuclease protection assay for mRNA encoding the proalpha1 chain of type I collagen, the major collagen in arteries. This revealed a dose-dependent increase in relative collagen synthesis rate and a dose-dependent increase in proalpha1(I) collagen mRNA abundance, with the half-maximal effect at 1.7 nmol/L. Angiotensin II-stimulated collagen expression was associated with a 6-fold increase in transforming growth factor-beta (TGF-beta) production and was inhibited by a neutralizing antibody to TGF-beta. Both collagen production and TGF-beta release were inhibited by the AT(1)-specific antagonist, losartan, but not by the AT(2) receptor antagonist, PD123319. To determined if tyrosine phosphorylation was functionally linked to collagen synthesis, we studied the effect of 2 mechanistically distinct inhibitors of tyrosine kinase, genistein, and tyrphostin A25. These inhibitors abrogated angiotensin II-mediated procollagen mRNA expression and angiotensin II-mediated TGF-beta production, whereas the inactive homolog tyrphostin A1 had no effect. We conclude that angiotensin II stimulates collagen production in human arterial SMCs via the AT(1) receptor and an autocrine loop of TGF-beta, induction of which requires tyrosine phosphorylation.
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PMID:Angiotensin II stimulates collagen synthesis in human vascular smooth muscle cells. Involvement of the AT(1) receptor, transforming growth factor-beta, and tyrosine phosphorylation. 1044 62


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