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A R Strauch

Publications and source records attributed to A R Strauch.

32 records · Page 2Linked to original sources
PubMed1992

Activation of a muscle-specific actin gene promoter in serum-stimulated fibroblasts.

Treatment of AKR-2B mouse fibroblasts with serum growth factors or inhibitors of protein synthesis, such as cycloheximide, results in a stimulation of cytoskeletal beta-actin transcription but has no effect on transcription of muscle-specific isotypes, such as the vascular smooth muscle (VSM) alpha-actin gene. Deletion mapping and site-specific mutagenesis studies demonstrated that a single "CArG" element of the general form CC(A/T)6GG was necessary and possibly sufficient to impart serum and cycloheximide-inducibility to the beta-actin promoter. Although the VSM alpha-actin promoter exhibits at least three similar sequence elements, it remained refractory to serum and cycloheximide induction. However, deletion of a 33 base pair sequence between -191 and -224 relative to the transcription start site resulted in the transcriptional activation of this muscle-specific promoter in rapidly growing or serum-stimulated fibroblasts. Although the activity of this truncated promoter was potentiated by cycloheximide in a manner indistinguishable from that of the beta-actin promoter, this was dependent on a more complex array of interacting elements. These included at least one CArG box and a putative upstream activating element closely associated with the -191 to -224 inhibitory sequences. These results demonstrate that the expression of a muscle-specific actin gene in fibroblasts is suppressed by a cis-acting negative control element and that in the absence of this element, the promoter is responsive to growth factor-induced signal transduction pathways.

Actins↗
PubMed1991

Induction of the proliferative phenotype in differentiated myogenic cells by hypoxia.

The effect of cellular differentiation on the response of cells to hypoxic stress has been evaluated using the myogenic cell line BC3H1. Aerobic myocytes were predominantly in G0/G1 of the cell cycle and could be induced into S and G2/M of the cell cycle only by replating in high serum-containing medium at subconfluent cell density. In contrast, hypoxic myocytes demonstrated marked progression into S and G2/M upon reoxygenation without replating in the presence of serum. This modulation of myocytes by hypoxia was suggested further by the induction of 100-kDa and 9-kDa proteins (PSP 100 and PSP 9) which were otherwise only detectable in myoblasts. Two-dimensional gel analysis of newly synthesized proteins demonstrated that the five major glucose/oxygen-regulated proteins (GRP/ORP 260, 150, 100, 80, and 33) were induced in hypoxic myogenic cells independent of their state of differentiation. In addition to the GRP/ORPs, synthesis of 20 and 23 other major proteins was influenced in myocytes and myoblasts, respectively. The bulk of these alterations in myoblasts (70%) were inhibitions. In contrast, 75% of the alterations in myocyte protein synthesis were either enhancements or inductions. The data show that hypoxia can modulate the myocyte phenotype and invoke proliferative characteristics. Moreover, the data suggest that ischemia will have a different effect on and prognosis for tissues with a high mitotic index compared with differentiated tissues.

Animals↗
PubMed1991

Substrate-associated macromolecules promote cytodifferentiation of BC3H1 myogenic cells.

Differentiated mouse BC3H1 myogenic cells secrete substrate-associated macro-molecules (SAM) which restrict the proliferation of undifferentiated cells and promote both cell shape changes and expression of predominantly the vascular smooth muscle (VSM)-specific isoform of the contractile protein alpha-actin. While we previously reported that high cell density was required for stimulating maximal expression of VSM alpha-actin in BC3H1 cells (Strauch and Reeser: Journal of Biological Chemistry 264:8345-8355, 1989), the permissive effect of SAM on myoblast cytodifferentiation was not at all dependent on the formation of cell to cell contacts. This observation suggests that biogenesis of an extracellular matrix rather than the formation of physical contacts between cells may be the rate-limiting step for induction of VSM alpha-actin expression at high cell density. The biologically active moieties in SAM that promote cytodifferentiation also are expressed by mouse embryonic fibroblast cell lines and are distinctly different from a class of adheron-like macromolecules released by differentiated BC3H1 myocytes directly into the culture medium. While SAM was cell growth restrictive, reconstituted particulate material (RPM) prepared from myocyte-conditioned medium promoted the adhesion and proliferation of growth-arrested myoblasts. SAM and RPM are composed of different polypeptide subunits which collectively may establish microenvironmental conditions that are permissive for BC3H1 myogenic cell differentiation.

Actins↗
PubMed1990

The 5'-flanking region of the mouse vascular smooth muscle alpha-actin gene contains evolutionarily conserved sequence motifs within a functional promoter.

The 5'-flanking, 5'-untranslated, and amino-terminal protein coding regions of the single-copy 13-kilobase mouse vascular smooth muscle (VSM) alpha-actin gene have been cloned and sequenced. Respectively, there is 73 and 89% homology from the start of transcription (+1) to a point 206 base pairs upstream when comparing mouse to chicken and mouse to human VSM alpha-actin 5'-flanking region sequences. Two proximal 16-base pair motifs containing putative cis-acting regulatory elements having the configuration CC(A/T)6GG were found to be 100% conserved and present in the same position upstream from the transcription start site in all three species. A third more distal CC(A/T)6GG-like motif was 100% conserved between only the mouse and human genes whereas a fourth motif was unique to the mouse gene. The two upstream motifs may be important in controlling VSM alpha-actin gene transcription in mammals. Cell transfection assays using hGH reporter gene fusion plasmids showed that all four CC(A/T)6GG elements were required for tissue-specific, core promoter activity and were able to direct hGH expression in both mouse BC3H1 myogenic cells and early-passage rabbit aortic smooth muscle cells. The core promoter was not active in mouse fibroblasts suggesting that the region between -372 and -143 may mediate tissue-restrictive expression of the VSM alpha-actin gene. A putative "cell density responsive element" may be located between -1074 and -372 since fusion plasmids containing this portion of the VSM alpha-actin 5'-flanking region were significantly more active in promoting hGH expression in inducible, density-activated BC3H1 myoblasts compared to aortic smooth muscle cells which are largely constitutive for VSM alpha-actin expression.

Actins↗
PubMed1989

Sequential expression of smooth muscle and sarcomeric alpha-actin isoforms during BC3H1 cell differentiation.

High cell density and cell cycle withdrawal stimulate the differentiation of BC3H1 smooth muscle-like cells. The differentiation process is accompanied by extensive changes in cell shape and the increased expression of a variety of muscle-specific proteins including the vascular smooth muscle-specific isoform of the contractile protein, alpha-actin. Results of actin peptide map analyses described in this report now indicate that a second, sarcomeric muscle-specific alpha-actin isoform is expressed in serum-deprived BC3H1 myocytes and that the induction of this actin isoform occurs late in differentiation well after the observed upregulation of vascular alpha-actin synthesis. The sarcomeric alpha-actin was identified in myocytes on the basis of the unique electrophoretic mobility of its NH2-terminal tryptic peptide, the distribution of cleavage products that were obtained when the NH2-terminal tryptic peptide was subjected to secondary proteolytic cleavage with thermolysin and Staphylococcus aureus V8 protease, and the presence of an additional cysteine residue at the NH2 terminus of the biosynthetic precursor of this novel alpha-actin. While expression of vascular alpha-actin was stimulated when myoblasts reached confluence, a 6-day post-confluent treatment with serum-free medium was required to induce maximal expression of the sarcomeric alpha-actin. Blot hybridization analysis of total BC3H1 myocyte RNA using actin gene-specific cDNA probes indicated that the sarcomeric alpha-actin corresponds to the skeletal muscle-specific isoform. This is the first report describing dual expression of smooth muscle and sarcomeric muscle alpha-actins in a clonal myogenic cell line. The results indicate the potential usefulness of the BC3H1 cell line for studying relationships between divergent muscle alpha-actin gene sequences and transcriptional and translational controls during myogenesis.

Actins↗
PubMed1986

Characterization of actin mRNA levels during BC3H1 cell differentiation.

The expression of a vascular smooth muscle specific alpha-actin isoform can be induced in mouse BC3H1 smooth muscle cells by treating confluent monolayers with serum-free medium (Strauch, A. R., and Rubenstein, P. A. (1984) J. Biol. Chem. 259, 3152-3159; 7224-7229). Using blot hybridization techniques, two size classes of actin RNA were identified in BC3H1 cells with the relative amount of RNA in each size class varying according to the developmental state of the cells; a 2100-nucleotide actin RNA was most abundant in myoblasts, whereas a smaller 1500-nucleotide actin RNA was found predominantly in fully differentiated myocytes. Results of in vitro translation experiments suggested that the 2100-nucleotide actin RNA on blots of myoblast total RNA corresponded to a mixture of similar size transcripts encoding both beta- and gamma-actin, while the 1500-nucleotide actin RNA in myocytes was an alpha-actin mRNA. Cell-cell contact and serum withdrawal initiated a 6-fold increase in the level of alpha-actin mRNA in BC3H1 cells that was followed by a 3-fold decrease in the amount of beta- and gamma-actin mRNA when confluent cells were exposed to serum-free medium for prolonged periods. Vascular smooth muscle alpha-actin was the major alpha-actin isoform synthesized in L-[35S]cysteine-labeled BC3H1 myocytes, indicating that the 1500-nucleotide actin mRNA size class in these cells may be enriched for vascular smooth muscle alpha-actin transcripts.

Actins↗
PubMed1984

A vascular smooth muscle alpha-isoactin biosynthetic intermediate in BC3H1 cells. Identification of acetylcysteine at the NH2 terminus.

A fully translated actin biosynthetic intermediate containing N-acetylcysteine at the NH2 terminus has been identified in homogenates of differentiated mouse BC3H1 cerebrovascular smooth muscle cells labeled with L-[35S]cysteine. Thermolysin digestion of the highly acidic NH2-terminal tryptic peptide of this intermediate and electrophoretic analysis of the resulting fragments indicated that the intermediate was a precursor of smooth muscle alpha- isoactin , the major isoactin species in vascular smooth muscle. Carboxypeptidase A digestion of the thermolysin cleavage product corresponding to the first eight amino acid residues of the NH2-terminal tryptic peptide demonstrated an acetylcysteine-glutamate residue at the NH2 terminus. These results imply that the gene for smooth muscle alpha- isoactin , like genes coding for skeletal and cardiac alpha- isoactins , contains a cysteine codon immediately following the initiator methionine codon. Both the methionine and cysteine residues must be removed from the NH2 terminus of the intermediate to yield the mature form of smooth muscle alpha- isoactin . The removal of the cysteine residue in vivo is not direct but apparently involves acetylation of the cysteine and subsequent post-translational cleavage of the resulting acetylcysteine. Such an acetylation-dependent pathway has been demonstrated for removal of cysteine from the NH2 terminus of Drosophila actin synthesized in a cell-free translation system ( Rubenstein , P. A., and Martin, D. J. (1983) J. Biol. Chem. 258, 11354-11360). In vivo pulse-chase experiments indicate that the smooth muscle alpha- isoactin intermediate in BC3H1 cells turns over much more slowly than nonmuscle actin intermediates previously identified in mouse L-cells.

Acetylcysteine↗
PubMed1984

Induction of vascular smooth muscle alpha-isoactin expression in BC3H1 cells.

An isoactin analysis was performed on L-[35S]cysteine labeled BC3H1 cells to determine if these smooth muscle-like cells synthesize vascular smooth muscle actin. Three different NH2-terminal peptides were identified on thin layer electrophoretograms of DNase I-purified and trypsin-digested BC3H1 cell actin. Results obtained from secondary digestion with thermolysin or Staphylococcus aureus V8 protease showed that the most acidic NH2-terminal peptide was derived from vascular smooth muscle alpha-isoactin. Treatment of cell monolayers with serum-free medium caused a 3-fold increase in the level of alpha-isoactin expression and a concomitant decrease in the level of non-muscle beta- and gamma-isoactin. Cell-cell contact was required for induction of alpha-isoactin, and the effects of serum depletion on isoactin expression and cell growth were reversible. The intensity of about 11 out of 500 polypeptide spots on two-dimensional gels of BC3H1 cell polypeptides also was influenced by the culture conditions. The finding that smooth muscle isoactin expression was coupled to cell growth conditions indicate the potential usefulness of BC3H1 cells in studies of isoactin expression and utilization during vascular smooth muscle development.

Actins↗
PubMed1984

Cerebral microvascular smooth muscle in tissue culture.

Cerebral endothelium is being studied rather extensively in tissue culture, but no reports are available describing the tissue culture of cerebral microvascular smooth muscle. The present paper describes for the first time the isolation and culture of non-neoplastic mouse cerebral vascular smooth muscle. Microvessels from a dounce homogenate of mouse brain are plated onto plastic culture dishes in Dulbecco's modified Eagle media plus 20% fetal bovine serum and treated briefly with collagenase. Cells migrate from vessels and proliferate sufficiently to be transferred out of primary culture in 2 to 3 wk. Light microscopy reveals generally broad, polygonal cells that grow collectively in a "hill and valley" pattern. By transmission electron microscopy the cells possess many characteristics of smooth muscle: basal laminas, clusters of pinocytotic vesicles, and bundles of thin filaments. Several ill-defined cell-to-cell junctions are also present. Isoelectric focusing and sodium dodecyl sulfate-electrophoresis of cellular proteins on polyacrylamide gels after pulse labeling cultures with [S-35]methionine demonstrate that these cells actively synthesize a smooth-muscle-specific isoactin, alpha-actin. The identity of alpha-actin is confirmed by analysis of NH2-terminal peptides after actin digestion with trypsin and subsequent peptide cleavage with thermolysin. Both their morphology and active synthesis of alpha-actin strongly suggest that these cells are of smooth-muscle origin. Future studies of their metabolism and interactions with endothelium and astrocytes should provide a better understanding of the cerebral microcirculation.

Actins↗
PubMed1982

Centrifugation shearing exposes filamentous networks in cortical regions of crane-fly spermatocytes.

Spermatocytes of the crane-fly, Nephrotoma suturalis, were attached to electron microscope grids and then sheared by applying centrifugal force. Transmission electron microscopy of exposed regions of the cell cortex revealed networks containing arrays of filamentous structures. Networks were present in sheared spermatocytes at all stages of meiosis. The networks of dividing spermatocytes (meta- through telophase) were denser and appeared to contain more aggregated material then networks of prophase cells. The appearance of networks in spermatocytes resembled actin-containing networks of sheared and detergent-extracted human erythrocytes. Networks treated with myosin subfragment 1 under conditions in which muscle F-actin was clearly decorated were not distinguishable from those of untreated cells. Exposure to deoxyribonuclease-1 caused the disruption of networks in sheared spermatocytes as well as in erythrocytes. The results of deoxyribonuclease experiments are interpreted as an indication that actin is a component of the cell cortex in crane-fly spermatocytes.

Actins↗
PubMed1980

Biochemical analysis of actin in crane-fly gonial cells: evidence for actin in spermatocytes and spermatids--but not sperm.

A biochemical assay employing DNase-I affinity chromatography, two-dimensional peptide analysis and SDS polyacrylamide gel electrophoresis was used to isolate, identify, and assess the amount of actin from gonial cells of the crane fly, Nephrotoma suturalis. Based on the analysis of cell homogenates under conditions in which all cellular actin is converted to the monomeric DNase-binding form, actin comprises approximately 1% of the total protein in homogenates of spermatocytes and spermatids. SDS gel analysis of mature sperm reveals no polypeptides with a molecular weight similar to that of actin. Under conditions that preserve native supramolecular states of actin, approximately 80% of the spermatocyte actin is in a sedimentable form whereas only approximately 30% of the spermatid actin is sedimentable. These differences could be meaningful with regard to strutural changes that occur during spermiogenesis. A comparative analysis of two-dimensional peptide maps of several radioiodinated actins reveals similarities among spermatocyte, spermatid, and human erythrocyte actins. The results suggest the general applicability of this approach to other cell types that contain limited amounts of actin.

Actins↗