Studies on actin-azomercurial complexes.
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Azo Compounds↗
Biomedical subjects
R Zak
Publications and source records attributed to R Zak.
Sympathetic control of cardiac myosin heavy chain gene expression.
Several neuroendocrine factors have been shown to influence the muscle phenotype. Various physiological reports have suggested the role of adrenergic nervous system for cardiac myosin heavy chain (MHC) expression. We have used cultured fetal rat heart myocytes to investigate the role of cAMP on the alpha- and beta-MHC gene expression. In low density cultures, addition of 1 mM 8 Br cAMP resulted in up regulation of alpha-MHC and down regulation of beta-MHC mRNA. This antithetic effect of cAMP depends on the basal expression of both expression of both MHC transcripts. In transient transfection analysis employing a series of alpha-MHC gene promoter/reporter constructs, we identified a 13 bp E-box M-CAT hybrid motif (EM element) which conferred a basal muscle specific and cAMP-inducible expression of the alpha-MHC gene. Data obtained from the mobility gel-shift analysis indicated that one of the factor(s) binding to the EM element is related to troponin T M-CAT binding factor (TEF-1). To test whether the protein binding to this sequence could be a substrate for cAMP-dependent phosphorylation, the cardiac nuclear proteins were preincubated in a kinase reaction buffer either with a catalytic subunit of PKA (CatPKA) or with cAMP, and binding activity of proteins to the EM element was evaluated by mobility gel shift assay. In a concentration dependent manner, a twofold increase in the intensity of the retarded band was observed. Furthermore, at 100 units of CatPKA, an additional band of faster mobility was observed which was not present either when phosphorylated nuclear extract was incubated with alkaline phosphatase or when ATP was absent in kinase reaction buffer. These results strongly suggest that factor(s) binding to the EM element is a substrate for cAMP dependent phosphorylation.
8-Bromo Cyclic Adenosine Monophosphate↗
Catecholamines and cardiac growth.
The present knowledge concerning the alpha- and beta-adrenergic systems in the regulation of cardiac growth and gene expression is reviewed. To investigate the mechanism by which cAMP regulates the expression of cardiac genes we have used cultured myocytes derived from fetal rat hearts. We have shown previously that the addition of Br cAMP to the culture medium produced an increase in alpha-myosin heavy chain (alpha-MHC) mRNA level, in its rate of transcription as well as in the amount of V1 isomyosin. To characterize the promoter element(s) involved in cAMP responsive regulation of alpha-MHC expression we performed transient transfection analysis with a series of alpha-MHC gene promoter-CAT constructs. We have identified a 13 bp E-box/M-CAT hybrid motif (EM element) which conferred a basal muscle specific and cAMP inducible expression of the alpha-MHC gene. Using mobility shift assay we have documented that one of the EM element binding protein is TEF-1. Moreover, by incubating cardiac nuclear extracts with the catalytic subunit of PK-A we have found that factor(s) binding to the EM element is a substrate for cAMP dependent phosphorylation.
8-Bromo Cyclic Adenosine Monophosphate↗
Metabolism of myofibrillar proteins in the normal and hypertrophic heart.
The pathways of myofibrillar assembly and degradation were studied in normal heart and during developing hypertrophy by two independent methods: amino acid incorporation kinetics and the double isotope technique. The validity and sensitivity of both methods were evaluated by computer analysis of data for which leucyl-tRNA was used as a protein precursor. The data obtained indicate that the myofibrillar proteins turn over at nonuniform rates. The half-lives of the proteins studied increase as follows: myosin HC = alpha-actin = tropomyosin greater than LC1 = LC2 greater than actin. In the case of light chains, a macromolecular precursor pool was detected which contributes to the observed lower labeling with 3H-leucine. During developing hypertrophy, the rate of light-chain labeling is increased relative to that of heavy chains.
Actins↗
Mitochondrial proliferation in cardiac hypertrophy.
Mitochondrial proliferation was studied in mature female rats following aortic constriction. Mitochondrial DNA (mtDNA) was assayed by a fluorometric method. The conditions for removal of nuclear DNA were developed and verified by assessment of molecular conformation of DNA. The mtDNA concentration in mitochondria increased 2,4, and 7 days post-operatively by 11, 72 and 117% respectively. Comparison with the rates of accumulation of cytochrome c, b, and aa3 indicates that during the first 24 hours of cardiac enlargement the inner mitochondrial components accumulate faster then mtDNA, but during the six subsequent days the rate of mtDNA increment far outstrips that of the cytochromes. These data indicate that the amount of available mtDNA templates is not the only factor regulating the transcriptional and translational processes in the enlarging myocardium. The analysis of population of replicative intermediates of mtDNA have shown dramatic decrease in the frequency of D-loops in preparations obtained from hypertrophied hearts. This observation indicates that the increase in replicative flux of mtDNA is associated with the removal of a block in the conversion of D-loops to other intermediates.
Animals↗
Carbon monoxide inhalation: effect on heart cytochrome c in the neonatal and adult rat.
Male rats, 5 and 90 d of age, continuously inhaled 500 ppm CO [40% carboxyhemoglobin (COHb)] for 5.5-8.0 wk. Cardiomegaly and polycythemia developed as previously reported. Heart weight gain in young and old rats treated with CO was accompanied by increased cytochrome c content (nmol) in both left ventricle (LV) and right ventricle (RV) relative to controls. Cytochrome c concentration (nmol/g wet weight), however, was significantly depressed in LV and RV of young CO-exposed rats relative to controls, while there was no change in LV and RV of the old CO-exposed group. LV cytochrome c concentration was significantly higher than that of RV in both young and old, CO-exposed and control rats. On the other hand, cytochrome c concentration in young and old control LVs was similar, as was cytochrome c concentration in RVs of young and old control rats. Three additional experiments were carried out with 5-d-old rats inhaling 500 ppm CO, for 47 and 25 d. These also showed the increase in myocardial cytochrome c content and compromise of cytochrome c concentration, and both changes were greater with longer exposure.
Aging↗
DNA vaccine against oncogenic hamster cells transformed by HPV16 E6/E7 oncogenes and the activated ras oncogene.
The capability of DNA to elicit anti-tumour immunity was studied using human papillomavirus type 16 (HPV16)-transformed Syrian hamster cells denoted K3/II. These cells had been derived after cotransfection of primary kidney cell cultures with p16HHMo plasmid containing E6/E7 oncogenes of HPV16 and pEJ6.6 plasmid containing the activated human H-ras oncogene; they express both the HPV16 and activated H-ras genes. As a DNA vaccine, the p16HHMo plasmid was used. Three doses of the plasmid (either 100 microg or 10-15 microg per dose) were administered intramuscularly at 3-week intervals. The animals were challenged with four different doses (10(3)-10(6) per animal) of K3/II cells 10 days after the last plasmid injection. In one experiment the lower dose of plasmid DNA was also given in a mixture with the cationic lipid DOTAP. In another experiment, the pEJ6.6 plasmid (100 microg per dose) was used either alone or in combination with p16HHMo. In all experiments animals inoculated with the same doses of pBR322 plasmid served as controls. A moderate protective effect was observed in animals inoculated with the 100-microg doses of p16HHMo, but not in those inoculated with 10-15 microg of the same plasmid, whether given with or without DOTAP. A protective effect was also observed after administration of the pEJ6. 6 plasmid. At the time of challenge a portion of the p16HHMo-immunized, but not the pBR322-treated, animals possessed antibodies reactive in ELISA with peptides derived from the N-terminal portion of HPV16 E7 protein and with one peptide derived from E6 protein, while two other E6 peptides exhibited non-specific reactivity.
Animals↗
The pathways of protein synthesis and degradation in normal heart and during development and regression of cardiac hypertrophy.
The half-life of cardiac myosin heavy chains (HC) was determined, with leucyl-tRNA as precursor, to be 5.4 days. Myosin HC are labeled more rapidly than actin; myosin light chains (LC1 and LC2) are labeled more slowly than HC. The observed differences are attributable to heterogeneity in the half-lives, e.g., actin, and to the effect of dilution by the existing macromolecular precursor pool (LC1 and LC2). Cardiac and skeletal muscle contain a population of filaments that can be released from myofibrils by ATP-relaxing solution. The easily released filaments (ERF) are devoid of alpha-actinin and M-protein. Labeling of ERF is more rapid than that of residual myofibrils. Cardiac and skeletal muscle contains calcium-activated neutral protease, which selectively removes alpha-actinin when incubated with isolated myofibrils. During development of pressure-induced cardiac hypertrophy, the labeling of LC2 is increased. In regressing cardiac hypertrophy the activities of free and total cathepsin D and of acidic RNase are unaltered.
Actins↗
[Coincidence of disorders of hair growth and follicular melanogenesis in various pathological conditions].
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Alopecia↗