30 results on '"Schwartz, R."'
Search Results
2. N-terminal processing is essential for release of epithin, a mouse type II membrane serine protease.
- Author
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Cho, E G, Kim, M G, Kim, C, Kim, S R, Seong, I S, Chung, C, Schwartz, R H, and Park, D
- Abstract
Epithin was originally identified as a mouse type II membrane serine protease. Its human orthologue membrane type-serine protease 1 (MT-SP1)/matriptase has been reported to be localized on the plasma membrane. In addition, soluble forms of matriptase were isolated from human breast milk and breast cancer cell-conditioned medium. In this paper, we report a processing mechanism that appears to be required for the release of epithin. CHO-K1 or COS7 cells transfected with single full-length epithin cDNA generated two different-sized proteins in cell lysates, 110 and 92 kDa. The 92-kDa epithin was found to be an N-terminally truncated form of the 110-kDa epithin, and it was the only form detected in the culture medium. The 92-kDa epithin was also found on the cell surface, where it was anchored by the N-terminal fragment. The results of in vivo cell labeling experiments indicate that the 110-kDa epithin is rapidly processed to the 92-kDa epithin. Using site-directed mutagenesis experiments, we identified Gly(149) of the GSVIA sequence in epithin as required for the processing and release of the protein. These results suggest that N-terminal processing of epithin at Gly(149) is a necessary prerequisite step for release of the protein.
- Published
- 2001
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3. RhoA signaling via serum response factor plays an obligatory role in myogenic differentiation.
- Author
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Wei, L, Zhou, W, Croissant, J D, Johansen, F E, Prywes, R, Balasubramanyam, A, and Schwartz, R J
- Abstract
Serum response factor (SRF) plays a central role during myogenesis, being required for the expression of striated alpha-actin genes. As shown here, the small GTPase RhoA-dependent activation of SRF results in the expression of muscle-specific genes, thereby promoting myogenic differentiation in myoblast cell lines. Co-expression of activated V14-RhoA and SRF results in an approximately 10-fold activation of the skeletal alpha-actin promoter in replicating myoblasts, while SRFpm1, a dominant negative SRF mutant, blocks RhoA dependent skeletal alpha-actin promoter activity. Serum withdrawal further potentiates RhoA- and SRF-mediated activation of alpha-actin promoter to about 30-fold in differentiated myotubes. In addition, the proximal SRE1 in the skeletal alpha-actin promoter is sufficient to mediate RhoA signaling via SRF. Furthermore, SRFpm1 and to a lesser extent dominant negative N19-RhoA inhibit myoblast fusion, postreplicative myogenic differentiation, and expression of direct SRF targets such as skeletal alpha-actin and indirect targets such as myogenin and alpha-myosin heavy chain. Moreover, RhoA also stimulates the autoregulatable murine SRF gene promoter in myoblasts, and the expression level of SRF is reduced in myoblasts overexpressing N19-RhoA. Our study supports the concept that RhoA signaling via SRF serves as an obligatory muscle differentiation regulatory pathway.
- Published
- 1998
4. Organization and myogenic restricted expression of the murine serum response factor gene. A role for autoregulation.
- Author
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Belaguli, N S, Schildmeyer, L A, and Schwartz, R J
- Abstract
Serum response factor (SRF), a member of an ancient family of DNA-binding proteins, is generally assumed to be a ubiquitous transcription factor involved in regulating growth factor-responsive genes. However, avian SRF was recently shown (Croissant, J. D., Kim, J.-H., Eichele, G., Goering, L., Lough, J., Prywes, R., and Schwartz, R. J. (1996) Dev. Biol. 177, 250-264) to be preferentially expressed in myogenic lineages and is required for regulating post-replicative muscle gene expression. Given the central importance of SRF for the muscle tissue-restricted expression of the striated alpha-actin gene family, we wanted to determine how SRF might contribute to this muscle-restricted expression. Here we have characterized the murine SRF genomic locus, which has seven exons interrupted by six introns, with the entire locus spanning 11 kilobases. Murine SRF transcripts were processed to two 3'-untranslated region polyadenylation signals, yielding 4.5- and 2.5-kilobase mRNA species. Murine SRF mRNA levels were the highest in adult skeletal and cardiac muscle, but barely detected in liver, lung, and spleen tissues. During early mouse development, in situ hybridization analysis revealed enrichment of SRF transcripts in the myotomal portion of somites, the myocardium of the heart, and the smooth muscle media of vessels of mouse embryos. Likewise, murine SRF promoter activity was tissue-restricted, being 80-fold greater in primary skeletal myoblasts than in liver-derived HepG2 cells. In addition, SRF promoter activity increased 6-fold when myoblasts withdrew from the cell cycle and fused into differentiated myotubes. A 310-base pair promoter fragment depended upon multiple intact serum response elements in combination with Sp1 sites for maximal myogenic restricted activity. Furthermore, cotransfected SRF expression vector stimulated SRF promoter transcription, whereas dominant-negative SRF mutants blocked SRF promoter activity, demonstrating a positive role for an SRF-dependent autoregulatory loop.
- Published
- 1997
5. Serum response factor mediates AP-1-dependent induction of the skeletal alpha-actin promoter in ventricular myocytes.
- Author
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Paradis, P, MacLellan, W R, Belaguli, N S, Schwartz, R J, and Schneider, M D
- Abstract
"Fetal" gene transcription, including activation of the skeletal alpha-actin (SkA) promoter, is provoked in cardiac myocytes by mechanical stress and trophic ligands. Induction of the promoter by transforming growth factor beta or norepinephrine requires serum response factor (SRF) and TEF-1; expression is inhibited by YY1. We and others postulated that immediate-early transcription factors might couple trophic signals to this fetal program. However, multiple Fos/Jun proteins exist, and the exact relationship between control by Fos/Jun versus SRF, TEF-1, and YY1 is unexplained. We therefore cotransfected ventricular myocytes with Fos, Jun, or JunB, and SkA reporter genes. SkA transcription was augmented by Jun, Fos/Jun, Fos/JunB, and Jun/JunB; Fos and JunB alone were neutral or inhibitory. Mutation of the SRF site, SRE1, impaired activation by Jun; YY1, TEF-1, and Sp1 sites were dispensable. SRE1 conferred Jun activation to a heterologous promoter, as did the c-fos SRE. Deletions of DNA binding, dimerization, or trans-activation domains of Jun and SRF abolished activation by Jun and synergy with SRF. Neither direct binding of Fos/Jun to SREs, nor physical interaction between Fos/Jun and SRF, was detected in mobility-shift assays. Thus, AP-1 factors activate a hypertrophy-associated gene via SRF, without detectable binding to the promoter or to SRF.
- Published
- 1996
6. C/EBPepsilon is a myeloid-specific activator of cytokine, chemokine, and macrophage-colony-stimulating factor receptor genes.
- Author
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Williams, S C, Du, Y, Schwartz, R C, Weiler, S R, Ortiz, M, Keller, J R, and Johnson, P F
- Abstract
C/EBPepsilon is a member of the CCAAT/enhancer binding protein family of basic region/leucine zipper transcriptional activators. The C/EBPepsilon protein is highly conserved between rodents and humans, and its domain structure is very similar to C/EBPalpha. In mice C/EBPepsilon mRNA is only detected in hematopoietic tissues, including embryonic liver and adult bone marrow and spleen. Within the hematopoietic system, C/EBPepsilon is expressed primarily in myeloid cells, including promyelocytes, myelomonocytes, and their differentiated progeny. To identify potential functions of C/EBPepsilon, cell lines over-expressing the C/EBPepsilon protein were generated in the P388 lymphoblastic cell line. In contrast to the parental cell line, C/EBPepsilon-expressing cell lines displayed lipopolysaccharide-inducible expression of the interleukin-6 and monocyte chemoattractant protein 1 (MCP-1) genes as well as elevated basal expression of the MIP-1alpha and MIP-1beta chemokine genes. In the EML-C1 hematopoietic stem cell line, C/EBPepsilon mRNA levels increased as the cells progressed along the myeloid lineage, just preceding activation of the gene encoding the receptor for macrophage-colony-stimulating factor (M-CSFR). M-CSFR expression was stimulated in C/EBPepsilon-expressing P388 cell lines, when compared with either the parental P388 cells or P388 cell lines expressing either C/EBPalpha or C/EBPbeta. These results suggest that C/EBPepsilon may be an important regulator of differentiation of a subset of myeloid cell types and may also participate in the regulation of cytokine gene expression in mature cells.
- Published
- 1998
7. The myogenic regulatory factor MRF4 represses the cardiac alpha-actin promoter through a negative-acting N-terminal protein domain.
- Author
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Moss, J B, Olson, E N, and Schwartz, R J
- Abstract
Cardiac alpha-actin is activated early during the development of embryonic skeletal muscle and cardiac myocytes. The gene product remains highly expressed in adult striated cardiac muscle yet is dramatically reduced in skeletal muscle. Activation and repression of cardiac alpha-actin gene activity in developing skeletal muscle correlates with changes in the relative content of the four myogenic regulatory factors. Cardiac alpha-actin promoter activity, assessed in primary chick myogenic cultures, was activated by endogenous myogenic regulatory factors but was inhibited in the presence of co-expressed MRF4. By exchanging N- and C-terminal domains of MRF4 and MyoD, the N terminus of MRF4 was identified as the mediator of repressive activity, revealing a novel negative regulatory role for MRF4. The relative ratios of myogenic regulatory factors may have fundamental roles in selecting specific muscle genes for activation and/or repression.
- Published
- 1996
8. Enzymatic mechanism of an RNA ligase from wheat germ.
- Author
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Schwartz, R C, Greer, C L, Gegenheimer, P, and Abelson, J
- Abstract
We have characterized the mechanism of action of a wheat germ RNA ligase which has been partially purified on the basis of its ability to participate in in vitro splicing of yeast tRNA precursors (Gegenheimer, P., Gabius, H-J., Peebles, C.L., and Abelson, J. (1983) J. Biol. Chem. 258, 8365-8373). The preparation catalyzes the ligation of oligoribonucleotide substrates forming a 2'-phosphomonoester, 3',5'-phosphodiester linkage. The 5' terminus of an RNA substrate can have either a 5'-hydroxyl or a 5'-phosphate. The 5'-phosphate, which for a 5'-hydroxyl substrate can be introduced by a polynucleotide kinase activity in the preparation, is incorporated into the ligated junction. The 3' terminus can have either a 2',3'-cyclic phosphate or a 2'-phosphate. 2',3'-Cyclic phosphates can be converted into 2'-phosphates by a 2',3'-cyclic phosphate, 3'-phosphodiesterase activity in the preparation. The 2'-phosphate of the ligated product is derived from the phosphate at the 3' terminus of the substrate. Ligation proceeds with the adenylylation of the 5'-phosphorylated terminus to form an intermediate with a 5',5'-phosphoanhydride bond.
- Published
- 1983
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9. The expression and chromatin structure of the chicken glyceraldehyde-3-phosphate dehydrogenase gene in mouse cells.
- Author
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Sen, S, Siciliano, M J, Johnston, D A, Schwartz, R J, and Kuo, T
- Abstract
Chicken glyceraldehyde-3-phosphate dehydrogenase gene (GAPD) and thymidine kinase gene (TK) were co-transfected into mouse LMTK- cells by the calcium phosphate precipitation technique. Four of the eight hypoxanthine/aminopterin/thymidine-containing medium-resistant, TK+ transfectants were shown to produce different amounts of chicken glyceraldehyde-3-phosphate dehydrogenase by zymogram analysis. Subcloning and further analysis revealed that the chicken GAPD was stably inherited and that its enzyme subunits randomly combined with mouse subunits in heterotetramers. Although the contribution of chicken enzyme varied from approximately 30 to approximately 90% of the total glyceraldehyde-3-phosphate dehydrogenase activity with a proportional increase in total activity in the different subclones, it did not appear to affect the expression of mouse endogenous glycolytic enzymes since there was no distinct change in the levels of either mouse glyceraldehyde-3-phosphate dehydrogenase mRNA nor mouse phosphoglycerate kinase enzyme activity. The levels of chicken GAPD copy number, mRNA, and enzyme apparently were generally correlated in the different subclones, suggesting that the chicken GAPD in the mouse cells were expressed constitutively. In situ hybridization revealed that the transfected genes were integrated into mouse chromosomes in one cluster, and the locations of these clusters were different in different clones. Chromatin structure analyses of the chicken GAPD in four different transfectants revealed three DNase I-hypersensitive sites located around 0.2, 2.0, and 3.4 kilobases upstream from the 5' side of the gene. These sites are also present in the same locations in chicken lymphoblastoid cells (Kuo, M. T., Iyer, B., and Schwartz, R. J. (1982) Nucleic Acids Res 10, 4565-4579), indicating the dominant transmission of DNase I-hypersensitive cleavage sites in the transfected gene.
- Published
- 1985
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10. Structure and complete nucleotide sequence of the chicken alpha-smooth muscle (aortic) actin gene. An actin gene which produces multiple messenger RNAs.
- Author
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Carroll, S L, Bergsma, D J, and Schwartz, R J
- Abstract
The alpha-smooth muscle (aortic) actin gene is a distinct member of the actin multigene family which is expressed in vascular smooth muscle cells. We have determined the complete nucleotide sequence of 11 kilobase pairs of genomic DNA encoding the chicken alpha-smooth muscle actin gene. This single copy gene specifies a protein identical in sequence to the major alpha-actin from bovine aorta. The protein-coding sequences are interrupted by seven introns which are at codons specifying amino acid residues 41/42, 84/85, 121/122, 150, 204, 267, and 327/328. An eighth intron was found in the mRNA 5' untranslated region. The 5' flanking sequences contain elements which are conserved in other chicken muscle actin genes. Additional sequences at the 5' end of the gene may be conserved in at least one human actin gene. We have identified at least four messenger RNAs ranging in size from approximately 1370 to 2700 nucleotides (excluding poly(A) tails) which are transcribed from the alpha-smooth muscle actin gene. These RNAs differ in the length of their 3' untranslated regions, probably as a result of the utilization of alternative polyadenylation signals. This is the first report of an actin gene with multiple mRNA transcripts.
- Published
- 1986
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11. Protein 4.1 in sickle erythrocytes. Evidence for oxidative damage.
- Author
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Schwartz, R S, Rybicki, A C, Heath, R H, and Lubin, B H
- Abstract
Sickle erythrocytes are known to undergo excessive auto-oxidation, resulting in the generation of increased intracellular levels of several species of free radical oxidants. This environment is likely to enhance the accumulation of oxidative lesions by membrane components, although, as yet, this has been shown directly only for the sickle membrane phospholipids. We examined the oxidative status of protein 4.1, a major component of the human erythrocyte protein skeleton. We found that protein 4.1 isolated from sickle erythrocytes bound approximately 4-fold less to protein 4.1-stripped membranes than did the normal protein. The binding defect was inherent in the sickle protein and not in its membrane-binding site(s) since normal protein 4.1 bound to sickle protein 4.1-stripped inside-out vesicles similar to normal protein 4.1-stripped inside-out vesicles. Sickle membranes, in particular spectrin-depleted inside-out vesicles, contained less protein 4.1 than normal membranes. Purified sickle protein 4.1 contained 20-40% high molecular weight aggregated protein (Mr greater than 200,000), whereas the purified normal protein contained approximately 10% high molecular weight protein. The high molecular weight protein was immunoreactive with antibodies to protein 4.1 but not with antibodies to spectrin, ankyrin, band 3, glycophorin, or hemoglobin, suggesting that the high molecular weight protein was cross-linked protein 4.1 and not a complex of protein 4.1 and some other membrane protein(s). Purified sickle protein 4.1 was eluted from an anion-exchange resin at a higher salt concentration than normal protein 4.1. Oxidizing normal protein 4.1 with diamide resulted in an anion-exchange elution pattern similar to the sickle protein, suggesting that oxidation can affect protein surface charge. Activated thiol beads bound one-half as much sickle protein 4.1 as normal protein 4.1 when both were solubilized directly from membranes, demonstrating that thiol oxidation had occurred in vivo. Direct quantification of protein thiols revealed that the sickle protein contained 1-2 mol% fewer cysteines/protein 4.1 monomer than did the normal protein. By amino acid analysis, sickle protein 4.1 was found to contain less methionine and tyrosine than did the normal protein and contained approximately 1 mol% cysteic acid, whereas the normal protein did not contain any cysteic acid. Taken together, our results strongly suggest that sickle protein 4.1 has sustained oxidative damage in vivo. This damage can alter the functional properties of the sickle protein and may be an underlying factor in the myriad of membrane abnormalities reported in sickle erythrocytes.
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- 1987
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12. Identification of novel DNA binding targets and regulatory domains of a murine tinman homeodomain factor, nkx-2.5.
- Author
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Chen, C Y and Schwartz, R J
- Abstract
A murine cardiac-specific homeodomain gene named csx (Komuro, I., and Izumo. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8145-8149) and nkx-2.5 (Lints, T. J., Parsons, L. M., Hartley, L., Lyons, I., and Harvey, R. P. (1993) Development 119, 419-431) was identified as a potential vertebrate homologue of Drosophila tinman, a mesoderm determination factor required for insect heart formation (Bodmer, R. (1993) Development 118, 719-729). Bacterial expression of the nkx-2.5 homeodomain allowed us to identify downstream DNA targets from a library of randomly generated oligonucleotides. High affinity nkx-2.5 DNA binding sites, 5'-TNNAGTG-3', represented novel binding sequences, whereas intermediate and weaker affinity sites, 5'-C(A/T)TTAATTN-3', contained the typical 5'-TAAT-3' core required by most homeodomain factors for DNA binding. We also observed that nkx-2.5 served as a modest transcription activator in transfection assays done in 10T1/2 fibroblasts with multimerized binding sites linked to a luciferase reporter gene. Functional dissection of nkx-2.5 revealed a COOH-terminal inhibitory domain composed mainly of clusters of alanines and prolines, which appeared to mask a potent activation domain composed of hydrophobic and highly charged amino acids.
- Published
- 1995
13. Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice.
- Author
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Coleman, M E, DeMayo, F, Yin, K C, Lee, H M, Geske, R, Montgomery, C, and Schwartz, R J
- Abstract
The avian skeletal alpha-actin gene was used as a template for construction of a myogenic expression vector that was utilized to direct expression of a human IGF-I cDNA in cultured muscle cells and in striated muscle of transgenic mice. The proximal promoter region, together with the first intron and 1.8 kilobases of 3'-noncoding flanking sequence of the avian skeletal alpha-actin gene directed high level expression of human insulin-like growth factor I (IGF-I) in stably transfected C2C12 myoblasts and transgenic mice. Expression of the actin/IGF-I hybrid gene in C2C12 muscle cells increased levels of myogenic basic helix-loop-helix factor and contractile protein mRNAs and enhanced myotube formation. Expression of the actin/IGF-I hybrid gene in mice elevated IGF-I concentrations in skeletal muscle 47-fold resulting in myofiber hypertrophy. IGF-I concentrations in serum and body weight were not increased by transgene expression, suggesting that the effects of transgene expression were localized. These results indicate that sustained overexpression of IGF-I in skeletal muscle elicits myofiber hypertrophy and provides the basis for manipulation of muscle physiology utilizing skeletal alpha-actin-based vectors.
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- 1995
14. Saccharomyces cerevisiae tRNA ligase. Purification of the protein and isolation of the structural gene.
- Author
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Phizicky, E M, Schwartz, R C, and Abelson, J
- Abstract
The tRNA ligase protein of Saccharomyces cerevisiae is one of the components required for splicing of yeast tRNA precursors in vitro. We have purified this protein to near homogeneity using an affinity elution chromatographic step. Purified tRNA ligase is a 90-kDa protein that, in addition to catalyzing the ligation of tRNA half-molecules in the coupled splicing reaction, will also ligate an artificial substrate. Using this artificial substrate, we provide evidence for the existence of a previously predicted activated intermediate in the ligation reaction. The amino acid sequence of the amino-terminal end of the protein was determined, and we have used this information to isolate the structural gene from a library of yeast DNA. We prove that this DNA encodes the tRNA ligase protein by DNA sequencing and by demonstrating overproduction of the protein.
- Published
- 1986
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15. Asynchronous Transport to the Cell Surface of Intestinal Brush Border Hydrolases Is Not Due to Differential Trimming of N-Linked Oligosaccharides
- Author
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Matter, K, McDowell, W, Schwartz, R T, and Hauri, H P
- Abstract
Intestinal brush border enzyme glycoproteins are transported to the microvillar membrane at different rates in the differentiated intestinal cell line Caco-2. This asynchronism is due to at least two rate-limiting events, a pre- and an intra-Golgi step (Stieger B., Matter, K., Baur, B., Bucher, K., Höchli, M., and Hauri, H. P. (1988) J. Cell Biol. 106, 1853–1861). A possible cause for the asynchronous protein transport might be differential trimming of N-linked oligosaccharide side chains. The effects of two trimming inhibitors on the intracellular transport of sucrase-isomaltase, a slowly migrating hydrolase, and dipeptidylpeptidase IV, a rapidly migrating hydrolase, are described. 1-Deoxymannojirimycin, an inhibitor of Golgi α-mannosidase I, had no influence on the rate of appearance of these hydrolases in the brush border membrane as assessed by subcellular fractionation. In the presence of N-methyl-1-deoxynojirimycin, an inhibitor of glucosidase I, 30–40% of the newly synthesized molecules appeared at the cell surface, and half-time for appearance of this pool was identical to that found in control cells. The reduced maximal transport to the cell surface observed with N-methyl-1-deoxynojirimycin may suggest that proper glycosylation is necessary for an efficient transport from the Golgi apparatus to the microvillar membrane. Inhibition of glucosidase I does not prevent the acquisition of endoglycosidase H resistance. Furthermore, evidence is presented that the processing in the presence of N-methyl-1-deoxynojirimycin leads to glucosylated endoglycosidase H-resistant glycoproteins.
- Published
- 1989
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16. Effect of estrogen on gene expression in the chick oviduct. In vitro transcription of the ovalbumin gene in chromatin.
- Author
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Harris, S E, primary, Schwartz, R J, additional, Tsai, M J, additional, O'Malley, B W, additional, and Roy, A K, additional
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- 1976
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17. Progesterone-binding components of chick oviduct. In vitro effects of purified hormone-receptor complexes on the initiation of RNA synthesis in chromatin.
- Author
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Schwartz, R J, primary, Kuhn, R W, additional, Buller, R E, additional, Schrader, W T, additional, and O'Malley, B W, additional
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- 1976
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18. Effects of estrogen on gene expression in the chick oviduct. IV. Initiation of RNA synthesis on DNA and chromatin.
- Author
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Tsai, M-J, primary, Schwartz, R J, additional, Tsai, S Y, additional, and O'Malley, B W, additional
- Published
- 1975
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19. Progesterone-binding components of chick oviduct. In vitro effect of receptor subunits on gene transcription.
- Author
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Buller, R E, primary, Schwartz, R J, additional, Schrader, W T, additional, and O'Malley, B W, additional
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- 1976
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20. Effect of estrogen on gene expression in the chick oviduct. V. Changes in the number of RNA polymerase binding and initiation sites in chromatin.
- Author
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Schwartz, R J, primary, Tsai, M-J, additional, Tsai, S Y, additional, and O'Malley, B W, additional
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- 1975
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21. Physical interaction between the MADS box of serum response factor and the TEA/ATTS DNA-binding domain of transcription enhancer factor-1.
- Author
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Gupta M, Kogut P, Davis FJ, Belaguli NS, Schwartz RJ, and Gupta MP
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- Animals, Binding Sites, Blotting, Western, COS Cells, Cell Nucleus metabolism, Collodion metabolism, Conserved Sequence, DNA metabolism, Gene Expression, Glutathione Transferase metabolism, Models, Genetic, Myocardium metabolism, Plasmids metabolism, Precipitin Tests, Protein Binding, Protein Isoforms, Protein Structure, Tertiary, Recombinant Fusion Proteins metabolism, Serum Response Factor, TEA Domain Transcription Factors, Transfection, DNA-Binding Proteins chemistry, DNA-Binding Proteins metabolism, Nuclear Proteins chemistry, Nuclear Proteins metabolism, Transcription Factors chemistry, Transcription Factors metabolism
- Abstract
Serum response factor is a MADS box transcription factor that binds to consensus sequences CC(A/T)(6)GG found in the promoter region of several serum-inducible and muscle-specific genes. In skeletal myocytes serum response factor (SRF) has been shown to heterodimerize with the myogenic basic helix-loop-helix family of factors, related to MyoD, for control of muscle gene regulation. Here we report that SRF binds to another myogenic factor, TEF-1, that has been implicated in the regulation of a variety of cardiac muscle genes. By using different biochemical assays such as affinity precipitation of protein, GST-pulldown assay, and coimmunoprecipitation of proteins, we show that SRF binds to TEF-1 both in in vitro and in vivo assay conditions. A strong interaction of SRF with TEF-1 was seen even when one protein was denatured and immobilized on nitrocellulose membrane, indicating a direct and stable interaction between SRF and TEF-1, which occurs without a cofactor. This interaction is mediated through the C-terminal subdomain of MADS box of SRF encompassing amino acids 204-244 and the putative 2nd and 3rd alpha-helix/beta-sheet configuration of the TEA/ATTS DNA-binding domain of TEF-1. In the transient transfection assay, a positive cooperative effect of SRF and TEF-1 was observed when DNA-binding sites for both factors, serum response element and M-CAT respectively, were intact; mutation of either site abolished their synergistic effect. Similarly, an SRF mutant, SRFpm-1, defective in DNA binding failed to collaborate with TEF-1 for gene regulation, indicating that the synergistic trans-activation function of SRF and TEF-1 occurs via their binding to cognate DNA-binding sites. Our results demonstrate a novel association between SRF and TEF-1 for cardiac muscle gene regulation and disclose a general mechanism by which these two super families of factors are likely to control diversified biological functions.
- Published
- 2001
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22. GATA-4 and serum response factor regulate transcription of the muscle-specific carnitine palmitoyltransferase I beta in rat heart.
- Author
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Moore ML, Wang GL, Belaguli NS, Schwartz RJ, and McMillin JB
- Subjects
- Animals, Animals, Newborn, Cells, Cultured, GATA4 Transcription Factor, Genes, Reporter, Humans, Isoenzymes genetics, Luciferases genetics, Myocardium cytology, Organ Specificity, Podophyllin metabolism, Podophyllotoxin analogs & derivatives, Promoter Regions, Genetic, Rats, Recombinant Fusion Proteins biosynthesis, Serum Response Factor, Transfection, Zinc Fingers, Carnitine O-Palmitoyltransferase genetics, DNA-Binding Proteins metabolism, Gene Expression Regulation, Enzymologic, Mitochondria, Heart enzymology, Myocardium enzymology, Nuclear Proteins metabolism, Podophyllin analogs & derivatives, Transcription Factors metabolism, Transcription, Genetic
- Abstract
Transcriptional regulation of nuclear encoded mitochondrial proteins is dependent on nuclear transcription factors that act on genes encoding key components of mitochondrial transcription, replication, and heme biosynthetic machinery. Cellular factors that target expression of proteins to the heart have been well characterized with respect to excitation-contraction coupling. No information currently exists that examines whether parallel transcriptional mechanisms regulate nuclear encoded expression of heart-specific mitochondrial isoforms. The muscle CPT-Ibeta isoform in heart is a TATA-less gene that uses Sp-1 proteins to support basal expression. The rat cardiac fatty acid response element (-301/-289), previously characterized in the human gene, is responsive to oleic acid following serum deprivation. Deletion and mutational analysis of the 5'-flanking sequence of the carnitine palmitoyltransferase Ibeta (CPT-Ibeta) gene defines regulatory regions in the -391/+80 promoter luciferase construct. When deleted or mutated constructs were individually transfected into cardiac myocytes, CPT-I/luciferase reporter gene expression was significantly depressed at sites involving a putative MEF2 sequence downstream from the fatty acid response element and a cluster of heart-specific regulatory regions flanked by two Sp1 elements. Each site demonstrated binding to cardiac nuclear proteins and competition specificity (or supershifts) with oligonucleotides and antibodies. Individual expression vectors for Nkx2.5, serum response factor (SRF), and GATA4 enhanced CPT-I reporter gene expression 4-36-fold in CV-1 cells. Although cotransfection of Nkx and SRF produced additive luciferase expression, the combination of SRF and GATA-4 cotransfection resulted in synergistic activation of CPT-Ibeta. The results demonstrate that SRF and the tissue-restricted isoform, GATA-4, drive robust gene transcription of a mitochondrial protein highly expressed in heart.
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- 2001
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23. The smooth muscle gamma-actin gene promoter is a molecular target for the mouse bagpipe homologue, mNkx3-1, and serum response factor.
- Author
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Carson JA, Fillmore RA, Schwartz RJ, and Zimmer WE
- Subjects
- Animals, Base Sequence, Binding Sites, Birds, Cell Differentiation, Cell Line, Conserved Sequence, DNA-Binding Proteins metabolism, Electrophoresis, Polyacrylamide Gel, Fibroblasts metabolism, Glutathione Transferase metabolism, Haplorhini, Homeodomain Proteins metabolism, Humans, Mesoderm metabolism, Mice, Molecular Sequence Data, Mutagenesis, Site-Directed, Nuclear Proteins metabolism, Plasmids metabolism, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Recombinant Fusion Proteins metabolism, Sequence Homology, Nucleic Acid, Serum Response Factor, Transcription Factors metabolism, Transcription, Genetic, Transcriptional Activation, Transfection, Actins genetics, DNA-Binding Proteins genetics, Drosophila Proteins, Homeodomain Proteins genetics, Muscle, Smooth metabolism, Nuclear Proteins genetics, Promoter Regions, Genetic, Transcription Factors genetics
- Abstract
An evolutionarily conserved vertebrate homologue of the Drosophila NK-3 homeodomain gene bagpipe, Nkx3-1, is expressed in vascular and visceral mesoderm-derived muscle tissues and may influence smooth muscle cell differentiation. Nkx3-1 was evaluated for mediating smooth muscle gamma-actin (SMGA) gene activity, a specific marker of smooth muscle differentiation. Expression of mNkx3-1 in heterologous CV-1 fibroblasts was unable to elicit SMGA promoter activity but required the coexpression of serum response factor (SRF) to activate robust SMGA transcription. A novel complex element containing a juxtaposed Nkx-binding site (NKE) and an SRF-binding element (SRE) in the proximal promoter region was found to be necessary for the Nkx3-1/SRF coactivation of SMGA transcription. Furthermore, Nkx3-1 and SRF associate through protein-protein interactions and the homeodomain region of Nkx3-1 facilitated SRF binding to the complex NKE.SRE. Mutagenesis of Nkx3-1 revealed an inhibitory domain within its C-terminal segment. In addition, mNkx3-1/SRF cooperative activity required an intact Nkx3-1 homeodomain along with the MADS box of SRF, which contains DNA binding and dimerization structural domains, and the contiguous C-terminal SRF activation domain. Thus, SMGA is a novel target for Nkx3-1, and the activity of Nkx3-1 on the SMGA promoter is dependent upon SRF.
- Published
- 2000
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24. Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3'-kinase/Akt signaling pathway.
- Author
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Chakravarthy MV, Abraha TW, Schwartz RJ, Fiorotto ML, and Booth FW
- Subjects
- Animals, Cell Division, Cells, Cultured, Cellular Senescence, Cyclin G, Cyclin G1, Cyclin-Dependent Kinase 2, Cyclin-Dependent Kinase Inhibitor p27, Cyclin-Dependent Kinases antagonists & inhibitors, Cyclin-Dependent Kinases metabolism, Cyclins metabolism, Enzyme Activation, G1 Phase, Hypertrophy pathology, Insulin-Like Growth Factor I genetics, Mice, Mice, Transgenic, Microtubule-Associated Proteins genetics, Microtubule-Associated Proteins metabolism, Mitogen-Activated Protein Kinases metabolism, Muscle, Skeletal enzymology, Muscle, Skeletal metabolism, Phosphoinositide-3 Kinase Inhibitors, Protamine Kinase metabolism, Protein Serine-Threonine Kinases antagonists & inhibitors, Protein Serine-Threonine Kinases metabolism, S Phase, Stem Cells enzymology, Stem Cells metabolism, Up-Regulation, CDC2-CDC28 Kinases, Cell Cycle, Cell Cycle Proteins, Insulin-Like Growth Factor I metabolism, Muscle, Skeletal cytology, Phosphatidylinositol 3-Kinases metabolism, Signal Transduction, Stem Cells cytology, Tumor Suppressor Proteins
- Abstract
Interest is growing in methods to extend replicative life span of non-immortalized stem cells. Using the insulin-like growth factor I (IGF-I) transgenic mouse in which the IGF-I transgene is expressed during skeletal muscle development and maturation prior to isolation and during culture of satellite cells (the myogenic stem cells of mature skeletal muscle fibers) as a model system, we elucidated the underlying molecular mechanisms of IGF-I-mediated enhancement of proliferative potential of these cells. Satellite cells from IGF-I transgenic muscles achieved at least five additional population doublings above the maximum that was attained by wild type satellite cells. This IGF-I-induced increase in proliferative potential was mediated via activation of the phosphatidylinositol 3'-kinase/Akt pathway, independent of mitogen-activated protein kinase activity, facilitating G(1)/S cell cycle progression via a down-regulation of p27(Kip1). Adenovirally mediated ectopic overexpression of p27(Kip1) in exponentially growing IGF-I transgenic satellite cells reversed the increase in cyclin E-cdk2 kinase activity, pRb phosphorylation, and cyclin A protein abundance, thereby implicating an important role for p27(Kip1) in promoting satellite cell senescence. These observations provide a more complete dissection of molecular events by which increased local expression of a growth factor in mature skeletal muscle fibers extends replicative life span of primary stem cells than previously known.
- Published
- 2000
- Full Text
- View/download PDF
25. The C/EBP bZIP domain can mediate lipopolysaccharide induction of the proinflammatory cytokines interleukin-6 and monocyte chemoattractant protein-1.
- Author
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Hu HM, Tian Q, Baer M, Spooner CJ, Williams SC, Johnson PF, and Schwartz RC
- Subjects
- Animals, B-Lymphocytes, Binding Sites, CCAAT-Enhancer-Binding Protein-beta, CCAAT-Enhancer-Binding Proteins, Cell Line, Chemokine CCL2 genetics, DNA-Binding Proteins genetics, Dimerization, Fungal Proteins genetics, Gene Expression Regulation, Interleukin-6 genetics, Mice, NF-kappa B metabolism, Nuclear Proteins analysis, Nuclear Proteins genetics, Promoter Regions, Genetic, Protein Isoforms metabolism, Protein Kinases genetics, Recombinant Fusion Proteins genetics, Repressor Proteins pharmacology, Transcription Factors genetics, Transcriptional Activation, Transfection, Chemokine CCL2 biosynthesis, DNA-Binding Proteins metabolism, Interleukin-6 biosynthesis, Leucine Zippers genetics, Lipopolysaccharides pharmacology, Nuclear Proteins metabolism, Saccharomyces cerevisiae Proteins, Transcription Factors metabolism
- Abstract
C/EBPalpha, beta, and delta are all expressed by bone marrow-derived macrophages. Ectopic expression of any of these transcription factors is sufficient to confer lipopolysaccharide (LPS)-inducible expression of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) to a B lymphoblast cell line, which normally lacks C/EBP factors and does not display LPS induction of proinflammatory cytokines. Thus, the activities of C/EBPalpha, beta, and delta are redundant in regard to expression of IL-6 and MCP-1. Surprisingly, the bZIP region of C/EBPbeta, which lacks any previously described activation domains, can also confer LPS-inducible expression of IL-6 and MCP-1 in stable transfectants. Transient transfections reveal that the bZIP regions of C/EBPbeta, C/EBPdelta, and, to a lesser extent, C/EBPalpha can activate the IL-6 promoter and augment its induction by LPS. Furthermore, the transdominant inhibitor, LIP, can activate expression from the IL-6 promoter. The ability of the C/EBPbeta bZIP region to activate the IL-6 promoter in transient transfections is completely dependent upon an intact NF-kappaB-binding site, supporting a model where the bZIP protein primarily functions to augment the activity of NF-kappaB. Replacement of the leucine zipper of C/EBPbeta with that of GCN4 yields a chimeric protein that can dimerize and specifically bind to a C/EBP consensus sequence, but shows a markedly reduced ability to activate IL-6 and MCP-1 expression. These results implicate the leucine zipper domain in some function other than dimerization with known C/EBP family members, and suggest that C/EBP redundancy in regulating cytokine expression may result from their highly related bZIP regions.
- Published
- 2000
- Full Text
- View/download PDF
26. Transforming growth factor-beta response elements of the skeletal alpha-actin gene. Combinatorial action of serum response factor, YY1, and the SV40 enhancer-binding protein, TEF-1.
- Author
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MacLellan WR, Lee TC, Schwartz RJ, and Schneider MD
- Subjects
- Actins biosynthesis, Animals, Animals, Newborn, Base Sequence, Binding Sites, Cells, Cultured, Chickens, Consensus Sequence, Erythroid-Specific DNA-Binding Factors, Molecular Sequence Data, Myocardium metabolism, Oligodeoxyribonucleotides metabolism, Rats, Simian virus 40 metabolism, TATA Box, TEA Domain Transcription Factors, Transfection, Transforming Growth Factor beta pharmacology, YY1 Transcription Factor, Zinc Fingers, Actins genetics, Cell Nucleus metabolism, DNA-Binding Proteins metabolism, Enhancer Elements, Genetic, Muscles metabolism, Nuclear Proteins, Transcription Factors metabolism, Transforming Growth Factor beta metabolism
- Abstract
Skeletal alpha-actin (SkA) is representative of the cardiac genes that are expressed at high levels in embryonic myocardium, down-regulated after birth, and reactivated by trophic signals including type beta-transforming growth factors (TGF beta). To investigate the molecular basis for cardiac-restricted and TGF beta-induced SkA transcription, we have undertaken a mutational analysis of the SkA promoter in ventricular myocytes, with emphasis on the role of three nominal serum response elements. Serum response factor (SRF) and the bifunctional factor YY1 are the predominant cardiac proteins contacting the proximal SRE (SRE1). Mutations of SRE1 that prevent recognition by SRF and YY1, or SRF alone, virtually abolish SkA transcription in both TGF beta- and vehicle-treated cells; mutation of distal SREs was ineffective. A mutation which selectively abrogates YY1 binding increases both basal and TGF beta-dependent expression, substantiating the predicted role of YY1 as an inhibitor of SRF effects. However, efficient SkA transcription requires combinatorial action of SRE1 with consensus sites for Sp1 and the SV40 enhancer-binding protein, TEF-1. As isolated motifs, either SRE1- or TEF-1-binding sites function as TGF beta response elements. Induction of the SkA promoter by TGF beta required SRF and TEF-1 in concert, unlike other pathways for TGF beta-dependent gene expression.
- Published
- 1994
27. The avian cardiac alpha-actin promoter is regulated through a pair of complex elements composed of E boxes and serum response elements that bind both positive- and negative-acting factors.
- Author
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Moss JB, McQuinn TC, and Schwartz RJ
- Subjects
- Animals, Base Sequence, Binding Sites, Cells, Cultured, Chickens, Helix-Loop-Helix Motifs, Mice, Mice, Inbred C3H, Molecular Sequence Data, Muscles metabolism, Serum Response Factor, Transcription, Genetic, Transcriptional Activation, Actins genetics, DNA-Binding Proteins, Gene Expression Regulation, Myocardium metabolism, Nuclear Proteins, Promoter Regions, Genetic
- Abstract
The chicken alpha-cardiac actin is one of the earliest contractile protein genes selectively expressed during embryonic skeletal and cardiac muscle differentiation. Cardiac actin promoter elements were examined in these two sarcomeric cell types. A portion of the alpha-cardiac actin promoter responsible for striated muscle specificity has been delineated (1, 2) and shown to contain four serum response elements (SRE). Previously, SRE3 was shown to be part of a complex element in conjunction with a functional E box (2), and we now show that SRE4 is also part of an upstream SRE.E box cis-element complex. The SREs function similarly, but the E boxes have dissimilar properties within and between striated muscle types. The SRE3.E1 box binds myogenic basic helix-loop-helix factors and is required for cardiac actin trans-activation in primary muscle cell cultures but functions as a negative regulatory element in cardiac muscle cells. The SRE4.E2 box, on the other hand, fails to bind basic helix-loop-helix (bHLH) factors, is negative acting in skeletal muscle cells, and is positive acting in cardiac myocytes. A DNA binding factor similar to HF1a (3) was identified that interacts specifically with the SRE4.E2 box. This study shows that the avian cardiac actin promoter elements are differentially used between skeletal and cardiac striated muscle cell lineages.
- Published
- 1994
28. Positive and negative control of the skeletal alpha-actin promoter in cardiac muscle. A proximal serum response element is sufficient for induction by basic fibroblast growth factor (FGF) but not for inhibition by acidic FGF.
- Author
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Parker TG, Chow KL, Schwartz RJ, and Schneider MD
- Subjects
- Amino Acid Sequence, Animals, Animals, Newborn, Base Sequence, Cells, Cultured, Chickens, Fibroblasts drug effects, Fibroblasts physiology, Heart drug effects, Kinetics, Molecular Sequence Data, Oligodeoxyribonucleotides, Organ Specificity, Plasmids, Protein Sorting Signals genetics, Recombinant Proteins pharmacology, Transcription, Genetic drug effects, Actins genetics, Fibroblast Growth Factor 1 pharmacology, Fibroblast Growth Factor 2 pharmacology, Heart physiology, Promoter Regions, Genetic drug effects
- Abstract
Like mechanical load in vivo, basic fibroblast growth factor (bFGF) selectively provokes cardiac expression of "fetal" genes including skeletal alpha-actin (SkA). Antithetically, acidic FGF (aFGF) suppresses SkA transcription. To define sites controlling SkA transcription in cardiac muscle cells, rat cardiac myocytes were transfected with internal-deletion and block-substitution mutations in the SkA promoter, including three motifs resembling the fos serum response element (SRE). The upstream, central, and proximal SREs each contributed to basal expression in cardiac myocytes. To determine whether identical elements mediate induction by bFGF versus inhibition by aFGF, the proximal SRE (SRE1) and fos SRE were positioned upstream from a neutral promoter. In cardiac myocytes, both the SRE1 and fos SRE were expressed at levels up to one-third that of the SkA promoter (nucleotides -202 to -11). Neither was expressed in quiescent cardiac fibroblasts. bFGF augmented SRE1-CAT activity, whereas aFGF produced no change; the fos SRE was induced by both. The transcriptional and mitogenic actions of aFGF were contingent on the presence of a putative nuclear translocation motif. Thus 1) the SkA SRE1 and fos SRE each suffice for tissue specificity in cardiac myocytes; 2) unlike the c-fos SRE, the SkA SRE1 is induced selectively by bFGF yet not aFGF; 3) sequences alternative or in addition to the SRE1 are obligatory for aFGF to suppress the SkA promoter; and 4) possible differences in intracellular localization are one basis for divergent actions of aFGF and bFGF in cardiac muscle cells.
- Published
- 1992
29. Skeletal muscle protein and amino acid metabolism in hereditary mouse muscular dystrophy. Accelerated protein turnover and increased alanine and glutamine formation and release.
- Author
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Garber AJ, Schwartz RJ, Seidel CL, Silvers A, and Entman ML
- Subjects
- Actins biosynthesis, Adenosine Triphosphate metabolism, Alanine metabolism, Animals, Aspartic Acid metabolism, Glutamates metabolism, Glutamine metabolism, Kinetics, Mice, Mice, Inbred C57BL, Mice, Inbred Strains, Phosphocreatine metabolism, Amino Acids metabolism, Muscle Proteins metabolism, Muscles metabolism, Muscular Dystrophy, Animal metabolism
- Abstract
Interactins between skeletal muscle protein and amino acid metabolism were investigated using C57BL and 129ReJ mice with hereditary muscular dystrophy. On incubation, hind limb muscle preparations from dystrophic mice released large quantities of amino acids, particularly alanine and glutamine which were increased 70% and 40% compared to muscles from carrier or control mice. The increased alanine release did not result from altered alanine oxidation to CO2 or reincorporation into protein. Alanine and glutamine formation from added amino acids were equal with dystrophic and control muscles. Incorporation in vitro of leucine, alanine, and glutamate into proteins of dystrophic muscle was 3- to 7-fold greater than control muscle, and the incorporation in vivo of [3H]- or [14C]arginine into muscle proteins was greater in extent and earlier in time with dystrophic as compared to control muscle. Proteins were also labeled in vivo using [guanido-14C]arginine. On incubation of these muscles in vitro, a 100% greater loss of label from protein was observed with dystrophic as compared to control preparations, and the appearance of label in the media was correspondingly increased. Sodium dodecyl sulfate-gel electrophoresis of dystrophic skeletal muscle showed numerous protein bands to be reduced in density, but autoradiographic studies demonstrated that these same bands were more highly labeled in vitro by [35S]methionine in dystrophic than in control muscle. Although insulin stimulation of glucose uptake was markedly blunted in dystrophic muscle, insulin inhibited alanine and glutamine release equally from both control and dystrophic muscle. These data indicate that alanine and glutamine formation and release are increased in hereditary mouse muscular dystrophy. An accelerated degradation and an increased resynthesis of many muscle proteins were also observed in dystrophic compared to control animals. This increased proteolysis may account for the increased alanine and glutamine formation in dystrophic muscle.
- Published
- 1980
30. Control of glutamine synthetase synthesis in the embryonic chick neural retina. A caution in the use of actinomycin D.
- Author
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Schwartz RJ
- Subjects
- Adenosine Triphosphate metabolism, Animals, Centrifugation, Density Gradient, Chick Embryo, Dose-Response Relationship, Drug, Electrophoresis, Polyacrylamide Gel, Enzyme Induction, Genes, Regulator, Hydrocortisone pharmacology, Organ Culture Techniques, Oxygen Consumption, Polyribosomes enzymology, Protein Biosynthesis drug effects, RNA biosynthesis, Retina cytology, Retina drug effects, Staining and Labeling, Transcription, Genetic drug effects, Tritium, Dactinomycin pharmacology, Glutamate-Ammonia Ligase biosynthesis, Retina enzymology
- Published
- 1973
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