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R Raghow

Publications and source records attributed to R Raghow.

68 records · Page 4Linked to original sources
PubMed1986

Pretranslational regulation of type I collagen, fibronectin, and a 50-kilodalton noncollagenous extracellular protein by dexamethasone in rat fibroblasts.

The effect of dexamethasone on the synthesis of total cellular and extracellular proteins and specifically on the synthesis of type I procollagen chains, fibronectin, and a 50-kDa extracellular noncollagenous polypeptide was examined in cultured rat dermal fibroblasts. A slight but consistent inhibition of total protein synthesis by dexamethasone was dose and time dependent. Treatment of cells with 1 microM dexamethasone for 24 h while abolishing procollagen synthesis nearly completely (less than 95%) had the opposite effect (5-7-fold increase) on the synthesis of an extracellular noncollagenous 50-kDa polypeptide. Dexamethasone did not significantly affect the rates of synthesis of fibronectin. Cell-free translation of mRNA from dexamethasone-treated cells revealed corresponding changes in the steady-state levels of functional mRNAs coding for procollagens, the 50-kDa polypeptide, and fibronectin. Northern blot hybridization using nick-translated cDNA plasmids coding for pro-alpha 1(I), fibronectin, and cytoplasmic beta-actin mRNA corroborated the data obtained from cell-free translation experiments. Run-off transcription assays using nuclei from cells treated with 1 microM dexamethasone for 24 h revealed that glucocorticoid treatment did not significantly affect the rate of transcription of type I collagen genes; similarly, the rate of transcription of fibronectin and cytoplasmic beta-actin genes also remained unchanged under these conditions. An analysis of the kinetics of decay of radiolabeled mRNA coding for pro-alpha 1(I), pro-alpha 2(I), and fibronectin in dexamethasone-treated cells revealed that procollagen mRNAs were turned over at an accelerated rate in glucocorticoid-treated cells. These data suggest that dexamethasone regulates type I collagen gene expression by preferentially decreasing the stability of pro-alpha 1(I) and pro-alpha2(I) mRNAs. Although dexamethasone increased the levels of translatable mRNAs coding for a 50-kDa polypeptide, the molecular mechanism(s) of how hormone exerts this effect remains unknown.

Animals↗
PubMed1985

Profiles of steady state levels of messenger RNAs coding for type I procollagen, elastin, and fibronectin in hamster lungs undergoing bleomycin-induced interstitial pulmonary fibrosis.

We have characterized the messenger RNAs (mRNAs) coding for procollagen alpha 1(I), elastin, fibronectin, and actin in the lungs of Syrian golden hamsters by Northern blot analyses. While elastin, fibronectin, and beta-actin were each coded for by a single mRNA species of 4.1 kilobases (kb), 9.1 kb, and 2.1 kb in size, respectively, we identified a major (5.4 kb) and a minor (6.5 kb) procollagen alpha 1(I) mRNA species in the hamster lungs. The mRNAs for the three extracellular matrix proteins showed increased accumulation followed by steady decline in the bleomycin-treated lungs. There were significant differences among the three mRNAs in the relative increase and the time of maximum accumulation. After reaching the peak levels between 2-3 wk posttreatment, the levels of procollagen alpha 1(I) and elastin mRNAs declined to near normal values around the fourth week. In contrast, the accumulation of fibronectin mRNA was maximum in the first week after bleomycin treatment. The procollagen alpha 1(I) mRNA accumulated most dramatically (sevenfold above the levels in the untreated animals) compared with a five-fold increase in mRNA coding for fibronectin. Elastin mRNA increased approximately twofold above the control values. Nuclear runoff transcription experiments demonstrated a selective increase in the rates of transcription of genes coding for procollagen alpha 1(I), fibronectin, and elastin; the extent of transcriptional stimulation of procollagen alpha 1(I) and fibronectin genes was significantly greater than that of elastin. Since the amount of actin mRNA, as well as the rate of transcription of actin gene(s), varied only slightly after bleomycin treatment, we conclude that the metabolism of mRNAs coding for extracellular matrix proteins may be preferentially perturbed during pulmonary fibrosis.

Actins↗
PubMed1984

Transcriptional regulation of type I collagen genes in cultured fibroblasts by a factor isolated from thioacetamide-induced fibrotic rat liver.

Recently Hatahara and Seyer (Hatahara, T., and Seyer, J.M. Biochim. Biophys. Acta (1982) 716, 377-382) isolated a factor from fibrotic rat liver which stimulates collagen synthesis in cultured fibroblasts without affecting their rate of proliferation. To investigate the mechanism of fibrogenic factor-mediated enhancement of type I collagen synthesis, we quantitated the levels of mRNAs coding for pro-alpha 1(I) and pro-alpha 2(I) chains in rat dermal fibroblasts. Cell-free translation experiments revealed that the fibrogenic factor caused greater than 5-fold increase in the translatable levels of type I mRNAs. We also quantitated collagen mRNAs by techniques of Northern blotting of glyoxylated poly(A+) RNA followed by hybridization to nick-translated human cDNA clones containing the coding sequence of pro-alpha 1(I) and pro-alpha 2(I) chains. Furthermore, we investigated the relative rates of collagen mRNA transcription in the isolated nuclei of treated and control fibroblasts. Similar quantitation of beta-actin mRNA transcription, which remains unaffected by the treatment with fibrogenic factor, was used as an internal control. We demonstrate that the fibrogenic factor causes a 4-6-fold increase in the rate of transcription of pro-alpha 1(I) and pro-alpha 2(I) genes. Finally, we also show that the rate of intracellular degradation of collagen is not significantly altered in cells treated with fibrogenic factor. These results combined with data on cell-free translation strongly suggest that the increased accumulation of type I collagen mRNA in fibrogenic factor-treated fibroblasts is a consequence of enhanced rates of collagen mRNA transcription.

Acetamides↗
PubMed1983

Cell-free translation of frog virus 3 messenger RNAs. Initiation factors from infected cells discriminate between early and late viral mRNAs.

Cell-free protein-synthesizing extracts prepared from rabbit reticulocytes, wheat germ, or cultured baby hamster kidney cells efficiently translated frog virus 3 early mRNAs; in contrast, late mRNAs were translated poorly under similar conditions. However, the translational efficiency of the late viral mRNAs was markedly enhanced in cell-free extracts prepared from frog virus 3 (FV 3)-infected baby hamster kidney cells and in nuclease-treated rabbit reticulocyte extracts by the addition of a 0.5 M KCl wash from FV 3-infected cell ribosomes; the 0.5 M KCl wash (initiation factors) from uninfected cells had no such effect. Total cytoplasmic RNA from infected cells was fractionated according to size on sucrose gradients and fractions containing different concentrations, and relative proportions of early and late mRNAs were translated in either native or initiation factor-supplemented extracts. Under these conditions, the translation efficiency of early mRNAs was unchanged, while the translation of late mRNAs increased 2-7-fold. Thus, the in vitro discriminatory activity of the 0.5 M wash was not dependent on the complexity of the mRNAs present in the translation mixture. We show also that in native extracts, under conditions of blocked polypeptide chain elongation, early mRNAs are initiated preferentially. However, late as well as early mRNAs are initiated equally well in reticulocyte extracts under similar experimental conditions when supplemented with crude initiation factors from infected cells. These data support the conclusion that the translational enhancement of FV 3 mRNAs in vitro is mediated by a virus-specified or virus-modified initiation factor(s) and likely represents a regulatory mechanism of protein synthesis operative in vivo (Willis, D. B., Goorha, R., Miles, M., and Granoff, A. (1977) J. Virol. 24, 326-342).

Animals↗
PubMed1979

Topography of a flexible ribonucleoprotein helix: protein-protein contacts in Sendai virus nucleocapsids.

Contacts among the three polypeptide species in the flexible helical nucleocapsids of a paramyxovirus were examined with bifunctional protein cross-linking reagents. Polypeptides L and P, minor components of Sendai virus nucleocapsids implicated in viral RNA polymerase activity, were efficiently cross-linked into large complexes, indicating that they enjoy abundant contacts with neighboring protein molecules in the helix. Less reactivity was found in the case of the major structural polypeptide, NP; about half of all molecules of NP formed large cross-linked complexes, most of the rest remaining as monomers along with a small proportion of homodimers and low-order oligomers. Marked heterogeneity in the cross-linking reactivity of NP molecules, which may reflect the conformational quasi-equivalence inherent in a flexible helix, was indicated by the production of several conformers of homodimers and other low-order oligomers of NP, and by failure of the kinetics of NP cross-linking to conform to a simple statistical model of random polmerization. The validity of the statistical model was shown by cross-linking experiments with the rigid helical virus, tobacco mosaic virus.

Capsid↗
PubMed1977

Regulation of Sendai virus transcription: evidence for a single promoter in vivo.

The synthesis of Sendai virus RNA species was examined after UV irradiation of cells late in infection. Compared with the inactivation of 50S genomic RNA synthesis, the synthesis of the group of mRNA species that sediments at 18S was inactivated at an average rate consistent with a process of sequential transcription from a single promoter. The rates of inactivation of the synthesis of individual mRNA's separated by polyacrylamide gel electrophoresis confirmed this and, with the aid of additional data, suggested that the order of genes in the Sendai virus genome is: 3'-NP-Fo-M-P-HM-L-5'.

Culture Techniques↗
PubMed1977

Structural polypeptides of Sendai virus: analysis by tryptic peptide mapping.

Tryptic peptide patterns identified Sendai virus polypeptides polypeptides HN, HN, P,F0, NP, and M as unique gene products, and confirmed the precursor-product relationship of polypeptides F0 and F. By the same criterion, the major nucleocapsid polypeptides, P and NP, in virions were derived from the same genes as their counterparts in nucleocapsids obtained from infected cells.

Culture Techniques↗