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Y W Yang

Publications and source records attributed to Y W Yang.

65 records · Page 4Linked to original sources
PubMed1988

Transforming growth factor-beta-induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells.

We have explored the hypothesis that hypertrophy of vascular smooth muscle cells may be regulated, in part, by growth inhibitory factors that alter the pattern of the growth response to serum mitogens by characterizing the effects of the potent growth inhibitor, transforming growth factor-beta (TGF-beta), on both hyperplastic and hypertrophic growth of cultured rat aortic smooth muscle cells. TGF-beta inhibited serum-induced proliferation of rat aortic smooth muscle cells (ED50 = 2 pM); this is consistent with previously reported observations in bovine aortic smooth muscle cells (Assoian et al. 1982. J. Biol. Chem. 258:7155-7160). Growth inhibition was due in part to a greater than twofold increase in the cell cycle transit time in cells that continued to proliferate in the presence of TGF-beta. TGF-beta concurrently induced cellular hypertrophy as assessed by flow cytometric analysis of cellular protein content (47% increase) and forward angle light scatter (32-50% increase), an index of cell size. In addition to being time and concentration dependent, this hypertrophy was reversible. Simultaneous flow cytometric evaluation of forward angle light scatter and cellular DNA content demonstrated that TGF-beta-induced hypertrophy was not dependent on withdrawal of cells from the cell cycle nor was it dependent on growth arrest of cells at a particular point in the cell cycle in that both cycling cells in the G2 phase of the cell cycle and those in G1 were hypertrophied with respect to the corresponding cells in vehicle-treated controls. Chronic treatment with TGF-beta (100 pM, 9 d) was associated with accumulation of cells in the G2 phase of the cell cycle in the virtual absence of cells in S phase, whereas subsequent removal of TGF-beta from these cultures was associated with the appearance of a significant fraction of cycling cells with greater than 4c DNA content, consistent with development of tetraploidy. Results of these studies support a role for TGF-beta in the control of smooth muscle cell growth and suggest that at least one mechanism whereby hypertrophy and hyperploidy may occur in this, as well as other cell types, is by alterations in the response to serum mitogens by potent growth inhibitors such as TGF-beta.

Animals↗
PubMed1988

Synthesis of insulin-like growth factor II (IGF-II) in fetal rat tissues: translation of IGF-II ribonucleic acid and processing of pre-pro-IGF-II.

The single insulin-like growth factor II (IGF-II) gene is transcribed into multiple RNA species in most fetal and neonatal rat tissues. For IGF-II to serve as a local growth factor in fetal tissues, IGF-II RNA must be translated into pre-pro-rat (r) IGF-II, and the biosynthetic precursor processed to smaller biologically active forms. IGF-II RNA extracted from fetal rat liver, muscle, intestine, lung, and stomach, from rat placenta, and from fetal or neonatal mouse liver and lung directed the synthesis of 22,000 mol wt pre-pro-IGF-II in a reticulocyte lysate cell-free translation system. A biosynthetic precursor of this size had been observed previously in translation of RNA from BRL-3A rat liver cells and is predicted by the nucleotide sequence of cDNA clones encoding rIGF-II. Consistent with the developmental pattern of expression of IGF-II RNA observed in hybridization studies, RNA from adult rat liver, muscle, and intestine did not direct the synthesis of pre-pro-rIGF-II. To determine whether the IGF-II biosynthetic precursor was processed to smaller biologically active IGF-II, term fetal rat tissues were extracted with acid-ethanol, the extracts were fractionated by acid gel filtration, and the IGF pools were examined in a RIA specific for IGF-II. Levels of 1-2 micrograms/g were observed in liver, limb, lung, intestine, and brain; lower levels were observed in heart and kidney. In general, the levels of immunoreactive IGF-II corresponded to the levels of IGF-II mRNA. These results suggest that IGF-II mRNA is translated, and pre-pro-IGF-II processed to mature IGF-II in different fetal rat tissues. In contrast to IGF-I, in which alternative RNA splicing generates possible precursor molecules containing different COOH-terminal propeptide segments, we find no evidence for an IGF-II precursor in rat tissues other than 22,000 mol wt pre-pro-rIGF-II.

Animals↗
PubMed1987

Biosynthesis of the low molecular weight carrier protein for insulin-like growth factors in rat liver and fibroblasts.

Biosynthesis of the low mol wt (Mr) carrier protein for insulin-like growth factors (IGFs) was studied in the BRL-3A rat liver cell line, rat embryo fibroblasts (REFs), and fetal rat liver by biosynthetic labeling of intact cells and cell-free translation of extracted RNA. [35S]Cysteine-labeled carrier protein precursors were immunoprecipitated using antibodies raised to the approximately 33,000 Mr carrier protein from BRL-3A cells that recognize the IGF carrier protein present in fetal and neonatal rat serum, but not in adult rat serum. The IGF carrier protein is synthesized as a 35,000 Mr precursor in a reticulocyte lysate translation system directed by RNA from BRL-3A cells or REFs. Supplementation of the translation incubation with microsomal membranes decreases the size of the precursor to 33,000 Mr, presumably by removal of a signal peptide. In continuous labeling or pulse-chase experiments of intact BRL-3A cells or REFs, the 33,000 Mr protein is labeled within 10 min intracellularly, appears in the medium after 40 min, and persists in the medium for 24 h without a change in size. The intracellular carrier protein was biosynthetically labeled in BRL-3A cells with [3H]leucine, [3H]phenylalanine, [3H]arginine, or [35S]cysteine and purified, and its NH2-terminal amino acid sequence was determined. Eleven of 34 residues were identified and correspond to those of mature unlabeled carrier protein purified from conditioned medium, indicating that after removal of the signal peptide, the carrier protein undergoes no detectable further processing at its NH2-terminus. These results establish that although they are regulated coordinately, IGF-II and the fetal IGF carrier protein are synthesized as separate proteins. Finally, RNA extracted from fetal, but not adult, rat liver directs the synthesis of the 35,000 Mr carrier protein precursor, suggesting that the developmental regulation of the carrier protein may occur at the level of RNA abundance.

Amino Acid Sequence↗
PubMed1986

Insulin-like growth factor carrier proteins in neonatal and adult rat serum are immunologically different: demonstration using a new radioimmunoassay for the carrier protein from BRL-3A rat liver cells.

A carrier protein for insulin-like growth factors (IGFs) has been purified from serum-free medium conditioned by the Buffalo rat liver (BRL)-3A cell line and used to immunize rabbits. Purified carrier protein was 125I labeled and affinity purified on IGF-Sepharose. The major labeled protein had a mol wt of about 33,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (appropriate for the IGF carrier protein subunit) and gave a single predominant peak of radioactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and acid-urea gel electrophoresis that was immunoprecipitated by immune serum and comigrated with unlabeled proteins that bind [125I] IGF. A RIA was developed using affinity purified [125I]carrier protein and immune serum. Tracer binding was inhibited only by preparations containing IGF carrier proteins, but not by unrelated proteins or by the IGFs themselves. Carrier proteins from BRL-3A cells gave equivalent strong reactivity either after dissociation of endogenous IGF or as an IGF-carrier protein complex. The antiserum effectively recognized the approximately 40,000 mol wt (Mr approximately 40,000) carrier protein from neonatal rat serum, both as a native complex and after acid stripping. It did not effectively recognize the Mr approximately 150,000 carrier protein from adult rat serum either as endogenous complex or after acid stripping. These results suggest that the Mr approximately 40,000 carrier protein of neonatal rat serum and the Mr approximately 40,000 binding subunit of the Mr approximately 150,000 carrier protein in adult rat serum are immunologically distinct. These antisera to the BRL-3A carrier protein should be useful tools with which to dissect the relationships between different carrier protein species and to study the regulation of IGF carrier protein gene expression.

Aging↗
PubMed1985

Biosynthesis of rat insulin-like growth factor II. I. Immunochemical demonstration of a approximately 20-kilodalton biosynthetic precursor of rat insulin-like growth factor II in metabolically labeled BRL-3A rat liver cells.

BRL-3A rat liver cells synthesize mature 7484-dalton rat insulin-like growth factor II (rIGF-II) as a approximately 22-kDa precursor, presumably prepro-rIGF-II. In the present study, we have biosynthetically labeled intact BRL-3A cells with [35S]cysteine and immunoprecipitated cell lysates and media with antisera to rIGF-II. A approximately 20-kDa protein was identified in immunoprecipitates of cell lysates having properties consistent with pro-rIGF-II. The approximately 20-kDa protein is precipitated by immune sera but not by nonimmune serum. Its immunoprecipitation is specifically inhibited by unlabeled rIGF-II but not by insulin. It is not precipitated from labeled lysates of a subclone of BRL-3A cells (BRL-3A2) that does not synthesize rIGF-II. The approximately 20-kDa protein is rapidly labeled intracellularly (10 min) but is not detected in BRL-3A media. In pulse-chase experiments, radioactivity in the approximately 20-kDa protein disappears during the chase and appears, at later times, in specifically immunoprecipitated approximately 19-, approximately 10-, approximately 8-, and approximately 7-kDa proteins in media and, to a limited extent, intracellularly. A protein with electrophoretic mobility identical to that of the approximately 20-kDa protein observed in cell lysates is immunoprecipitated from 35S-proteins whose synthesis is directed by BRL-3A RNA in a reticulocyte lysate cell-free translation system supplemented with microsomal membranes, and presumably arises by cotranslational removal of the signal peptide from approximately 22-kDa prepro-rIGF-II. Processing of the approximately 20-kDa protein in intact BRL-3A cells to intermediate and mature rIGF-II species appears to occur at the time of secretion and/or shortly thereafter, with the different forms appearing at approximately the same time.

Animals↗
PubMed1985

Biosynthesis of rat insulin-like growth factor II. II. Localization of mature rat insulin-like growth factor II (7484 daltons) to the amino terminus of the approximately 20-kilodalton biosynthetic precursor by radiosequence analysis.

In previous studies, we have identified possible biosynthetic precursors of rat insulin-like growth factor II (rIGF-II) using specific immunoprecipitation, approximately 22-kDa prepro-rIGF-II and 20-kDa pro-rIGF-II. We now provide chemical evidence that amino acid sequences corresponding to mature 7484-dalton rIGF-II are present at the NH2 terminus of the putative approximately 20-kDa pro-rIGF-II. BRL-3A cultures have been labeled individually with several radioactive amino acid precursors, the cells have been lysed, and the lysates have been immunoprecipitated with antiserum to rIGF-II. Following electrophoresis of the immunoprecipitated proteins, labeled approximately 20-kDa pro-rIGF-II was eluted from the gels and subjected to automated radiosequence analysis. Discrete peaks of radioactivity were observed in 12 of the first 30 cycles of Edman degradation. The deduced partial amino acid sequence was identical at each position with that of mature 7484-dalton rIGF-II. These results directly demonstrate that mature rIGF-II sequences are present in the approximately 20-kDa protein, as required if the approximately 20-kDa protein were pro-rIGF-II. In addition, they localize the 7484-dalton rIGF-II to the NH2 terminus of the precursor molecule. A second NH2-terminal sequence differing only in the absence of the NH2-terminal residue, alanine, also was present in an approximately equal amount. Similar NH2-terminal heterogeneity has been reported for 7484-dalton rIGF-II and most likely reflects ambiguity in the cleavage sites for the signal peptidase.

Amino Acid Sequence↗
PubMed1983

Synthesis of multiplication-stimulating activity (rat insulin-like growth factor II) by rat embryo fibroblasts.

Multiplication-stimulating activity, a family of insulin-like growth factors previously identified in medium conditioned by the BRL-3A rat liver cell line, is also synthesized by third passage cultures of rat embryo fibroblasts (REFs) maintained in serum-free medium. Conditioned serum-free medium from REFs was chromatographed on Sephadex G-75 in 1 m acetic acid, and fractions in the size range of BRL-MSA contained MSA immunoreactivity. A dose-response curve of pooled G-75 fractions was parallel to BRL-MSA standard, and levels in the REF-conditioned medium were 0.5-1 microgram/ml. REF-MSA from the Sephadex G-75 pool was equipotent to BRL-MSA in stimulating [3H]thymidine incorporation into DNA in chick embryo fibroblasts and in stimulating DNA synthesis in REFs, as measured by autoradiography. In receptor binding assays using REFs, chick embryo fibroblasts, Swarm rat chondrosarcoma cells, and rat liver membranes, REF MSA was equal to BRL MSA in competition for binding of [125I]iodo-MSA. REF MSA also behaved identically to BRL MSA in a competitive binding protein assay using binding proteins in rat serum. Further characterization of REF MSA using Bio-Gel P-10 chromatography, followed by high pressure liquid chromatography or polyacrylamide gel electrophoresis, indicated that immunoreactive polypeptides in the fibroblast medium corresponded to BRL MSA II (mol wt, 8700) and BRL MSA III (mol wt, 7100). The amount of REF MSA released into the medium increased linearly over time. Cycloheximide decreased the amount of MSA in the medium, and during a recovery period, the amount of MSA returned nearly to control levels. In summary, rat embryo fibroblasts synthesize MSA which is biologically, immunologically, and chemically identical to MSA produced by the BRL-3A rat liver cell line.

Animals↗
PubMedDate not supplied

Effects of doxorubicin on pp60c-src kinase activity in polyoma virus MT antigen transformed cells.

Interaction between polyoma virus (Py) encoded middle tumor antigen (MTAg) and the cellular src gene product, pp60c-src, has been shown to be correlated with the enhancement of pp60c-src kinase activity and the transforming ability of the virus. In the present study, FR3T3 cells were transfected with plasmids encoding MTAg and used to study the effects of the antineoplastic agent, doxorubicin (DOX), on pp60c-src kinase activity. Our results showed that transfection of FR3T3 cells with MTAg results in relatively high levels of MTAg expression and enhancement of pp60c-src kinase activity several folds over that observed in the control cells. Treatment of the transformed MTF4 cells with DOX up to 5 x 10(-6) M was shown to have no effect on the in vitro phosphorylation of MTAg and pp60c-src in the drug-treated cell lysates. On the contrary, treatment of src immunocomplexes with DOX resulted in a reduction in kinase activity. The inhibition appears to be a dose-dependent competition with respect to ATP concentrations.

Animals↗
PubMedDate not supplied

Inhibition of pp60c-src protein kinase by herbimycin A in polyomavirus middle tumor antigen-transformed cells.

The middle T antigen (MTAg) encoded by polyomavirus plays an important role in virus-mediated tumorigenesis. The activated protein kinase activity of MTAg-associated pp60c-src has been shown to be necessary for cell transformation by polyomavirus. In this study, the effects of herbimycin A on the pp60c-src kinase activities in the polyomavirus- and MTAg-transformed cells were studied. Phosphorylation of src and MTAg is reduced in polyomavirus and MTAg- transformed cells pretreated with herbimycin A. Inactivation of the enzymatic activity by herbimycin A was found to be dependent on the time of incubation and the drug concentration. In contrast, src immunoprecipitates from untreated MTAg-transformed cells appeared to be resistant to inhibition by herbimycin A. Herbimycin A does not affect the synthesis of MTAg and pp60c-src in the MTAg-transformed cells. These results suggest that pp60c-src kinase activity in the drug-treated cell lysates is more sensitive to herbimycin A inhibition than the same activity in the immunoprecipitates.

3T3 Cells↗