Murray Rabinowitz

Actinomycin D Inhibition of Deoxyribonucleic Acid-Dependent Synthesis of Ribonucleic Acid

Minute amounts of actinomycin D inhibit the synthesis of ribonucleic acid by nuclear extracts of HeLa cells in a ribonucleic acid-synthesizing system that is dependent on deoxyribonucleic acid and requires the presence of all four ribonucleoside triphosphates. The inhibition can be reversed by adding deoxyribonucleic acid to the enzymatic reaction. These findings support the work of others on the mode of action of actinomycin D in vivo.

Biochemical Correlates of Cardiac Hypertrophy I. Experimental Model; Changes in Heart Weight, RNA Content, and Nuclear RNA Polymerase Activity

Cardiac hypertrophy occurred in mature rats after producing supravalvular aortic stenosis with a specially designed silver clip. For 2 weeks following this procedure, heart weight, body weight, and RNA content of the myocardium were serially determined. Heart weight and RNA content increased within 24 hours of aortic banding, reaching a maximal level in 2 days and remaining elevated during the 2 weeks of observation. Nuclei were isolated and purified from heart muscle homogenates, and changes in RNA polymerase activity following aortic banding were determined. The nearest neighbor frequency of the bases of the RNA synthesized by the polymerase from nuclear preparations was identical in both the banded animals and the sham-operated controls. Both groups could thus be compared on the basis of the enzyme assay. RNA polymerase activity in nuclei from the hearts of banded rats rose rapidly when compared with the activity in sham-operated rats; peak values were reached on the second day, the earliest detectable change being around 12 hours. The increase in RNA polymerase activity represents one of the earliest biochemical events that take place in the myocardium following aortic banding.

Correlation of Visco-Elastic Properties of Large Arteries with Microscopic Structure

The media of 14 regions of the aorta and 3 regions of the pulmonary artery of dogs were subjected to a step-function circumferential stretch taking 20 msec to complete. The tension rose synchronously with the increase in circumference, then dropped exponentially to a reasonably steady state within 2 sec. A mathematical model, developed consistent with this stress-relaxation curve, showed how to use the tension curves to measure a viscous, a serieselastic, and a parallel-elastic constant unique for a given curve. These constants were compared with the microscopic structure of the same or similar segments; collagen was determined as hydroxyproline in a water soluble fraction, elastin as hydroxyproline in the residue and from the width and number of elastic lamellae, and muscle from the nitrogen content of a nonfibrous fraction, from cell counts and from contractility. The constituents varied widely and independently enough to permit correlating viscous and elastic constants with microscopic structure. The viscous and series-elastic constants were higher where muscle content was high, and increased markedly when the muscle was tonically contracted. The parallel-elastic constant was high when elastin was high and in the presence of contracted muscle, but seemed independent of collagen content, at the moderate tensions tested.

Mitochondrial-satellite and circular DNA filaments in yeast.

Mitochondrial DNA of Saccharomyces cerevisiae contains a satellite DNA (density, 1.682) that appears to exist as open-ended filaments at least 5 microns long. DNA from intact cells contains circular filaments whose lengths vary from 0.5 to 7 microns, with a great majority at 1.95 microns. The circular DNA has a density similar to that of the major nuclear peak (1.697). When heat-denatured mitochondrial-satellite DNA is renatured, it cross-links to form a molecule that is larger than the native molecule. The formation of cross-links results in hypersharpening of the density profiles in cesium chloride and also leads to failure to pass Millipore filter paper.

Mitochondria and Cardiac Hypertrophy

• Normally, myocardial metabolism is almost exclusively aerobic. The substantial quantities of adenosine triphosphate (ATP) required for muscle contraction and the much smaller amounts necessary for the maintenance of functions such as ion transport, rhythmicity, and conduction and the synthesis of membrane and protein constituents of the myocardium are supplied almost exclusively by mitochondrial oxidation of fatty acid and carbohydrate substrates. Even under conditions of stress such as those that exist during severe exercise, when the cardiac ATP requirement may be increased considerably, the capacity of the mitochondria for oxidative phosphorylation appears to be adequate to meet the requirements. The high level of and the capacity for oxidative metabolism in the heart are reflected morphologically in the remarkable observation that mitochondria constitute more than 35% of the cardiac cell volume as measured by quantitative electron microscopic stereological procedures (1). Acute changes in energy requirements such as those that occur during strenuous exercise appear to be effectively met by increased mitochondrial synthesis of ATP, and the synthesis is finely adjusted to ATP requirements by the respiratory control mechanism. Increased utilization of ATP results in transient accumulation of adenosine diphosphate (ADP), which acts as a phosphate acceptor and stimulates the mitochondrial oxidative rate by decreasing the level of mitochondrial high-energy intermediates (2) or the proton-motive force across the inner mitochondrial membrane (3), according to the chemical or chemiosmotic theories of oxidative phosphorylation, respectively. Sustained levels of increased preload or afterload, however, activate another more slowly re

Hybridization of mitochondrial transfer RNA's with mitochondrial and nuclear DNA of grande (wild type) yeast☆

Abstract The hybridization of mitochondrial leucyl- (mit-leucyl-) tRNA and mit-valyl-tRNA with mitochondrial and nuclear DNA of grande yeast has been studied. The tRNAs were charged in vitro with amino acids of high specific radioactivity and hybridization carried out at 33 °C in formamide at pH 5.0 to minimize deacylation of the tRNA. The mit[ 3 H]leucyl- and mit[ 3 H]valyl-tRNAs hybridized with mitochondrial DNA but not with yeast nuclear DNA or Escherichia coli DNA. Apparent hybridization-saturation curves were obtained. The melting profile of the hybrid was sharp and equivalent to a T m of 71 °C in 2 × standard saline citrate. Unlabeled yeast mitochondrial tRNA competed for the hybridization, but yeast supernatant and E. coli tRNAs did not. RNase and alkaline digests of the mit[ 3 H]valyl-tRNA-mit-DNA hybrid yielded [ 3 H]valyladenosine and [ 3 H]valine, respectively, providing further evidence for the validity of the hybridization system.

Biochemical Correlates of Cardiac Hypertrophy V. LABELING OF COLLAGEN, MYOSIN, AND NUCLEAR DNA DURING EXPERIMENTAL MYOCARDIAL HYPERTROPHY IN THE RAT

The incorporation of [2, 3-3H]proline into collagen hydroxyproline and into noncollagenous protein was measured during development of cardiac hypertrophy produced by surgical constriction of the ascending aorta in rats. Heart weight increased sharply during the first 4 days after aortic banding and then slowly rose to a plateau. Free intracellular proline concentration remained unaltered 2 days after banding and increased by 38% on the fifth postoperative day. Specific radioactivity of free proline 3 hours after injection of [3H]proline was elevated by 40% on the second postoperative day but was unchanged on the fifth day. Incorporation of [3H]proline into collagen hydroxyproline peaked sharply on the second day after aortic constriction (550% above control) in one experiment, and on the fourth day (330% above control) in another. The peak labeling of noncollagenous protein by [3H]proline (100% over control) was less than that of collagen. To further evaluate the differences in synthetic response of muscle cells and interstitial cells to increased work load, incorporation of radioactive precursors into myosin, collagen, nuclear DNA, and noncollagenous protein were measured simultaneously, after aortic constriction. Collagen labeling was maximal either on day 2 or 4, depending on the degree of hypertrophy. Labeling of myosin and noncollagenous protein reached a plateau on day 4. Incorporation of [3H]thymidine into nuclear DNA peaked on day 7. Thus both muscle cells and connective tissue cells respond independently during cardiac hypertrophy with an increased synthesis of specific proteins.

Protein synthesis and turnover in normal and hypertrophied heart

Abstract This review deals with some aspects of the synthesis, assembly and turnover of mitochondrial and myofibrillar proteins in normal and hypertrophied hearts. The dynamic state of the myocardium, in which intracellular proteins and organelles are constantly being destroyed and synthesized, is emphasized. In normal cardiac muscle, mitochondrial inner membrane cytochromes turn over synchronously with half-lives of 5 to 6 days. The various myofibrillar proteins also turn over; the fact that they do so with identical apparent half-lives of 8 to 10 days suggests that these proteins are assembled and degraded synchronously. In cardiac hypertrophy produced by constriction of the ascending aorta in rats, inner mitochondrial membrane cytochromes increase in parallel. Early in hypertrophy (1 day after aortic constriction) cytochrome content per gram of heart weight increases, indicating a disproportionate increase in mitochondrial cytochrome mass. By the third postoperative day there already is a decrease in cytochrome content per gram of heart weight. The increase in mitochondrial cytochrome content observed in the hypertrophied heart appears to be due to a decreased rate of cytochrome destruction as well as to an increased rate of cytochrome synthesis. The data on myosin accumulation are consistent with either decreased destruction of myosin or increased efficiency of reutilization of labeled amino acids.

Electron microscopic and renaturation kinetic analysis of mitochondrial DNA of cytoplasmic petite mutants of Saccharomyces cerevisiae

Mitochondrial DNA isolated from a series of nine petite yeast strains and from the parent grande strain was characterized by electron microscopic and renaturation kinetic analysis. The mtDNA† from all strains contained a variety of branched molecules which may be intermediates of replication or recombination. Although no circles were observed in the grande mtDNA, all the petites contained circular mtDNA molecules. The size distribution of the circles conformed to an oligomeric series that was characteristic for each strain. In seven petites, the length series could be related to a single circle monomer size, ranging from 0.13 μm to 5.5 μm; and in two petites to two or more circular monomer lengths. In contrast to circular mtDNA, linear molecules showed no unique size distribution. Circle monomer lengths were linearly related to the kinetic complexity (κ2 or C0t12) of sheared total mtDNA in the seven petite strains that contained a predominant single series of circle lengths. Thus in each of these petite strains the circle monomer length defined the same DNA sequence present in the linear DNA molecules of non-unique length.

Deletion mapping of mitochondrial transfer RNA genes in Saccharomyces cerevisiae by means of cytoplasmic petite mutants

SummaryMitochondrial transfer RNA genes have been ordered relative to the position of five mitochondrial drug resistance markers, namely, chloramphenicol (C), erythromycin (E), oligomycin I and II (OI, OII), and paromomycin (P). Forty-six petite yeast clones that were genetically characterized with respect to these markers were used for a study of these relationships. Different regions of the mitochondrial genome are deleted in these individual mutants, resulting in variable loss of genetic markers. Mitochondrial DNA was isolated from each mutant strain and hybridized with eleven individual mitochondrial transfer RNAs. The following results were obtained: i) Of the seven petite clones that retained C, E, and P resistance markers (but not OI or OII), four carried all eleven transfer RNA genes examined; the other three clones lost several transfer RNA genes, probably by secondary internal deletion; ii) Prolyl and valyl transfer RNA genes were located close to the P marker, whereas the histidyl transfer RNA gene was close to the C marker; iii) Except for a glutamyl transfer RNA gene that was loosely associated with the OI region, no other transfer RNA genes were found in petite clones retaining only the OI and/or the OII markers; and iv) Two distinct mitochondrial genes were found for glutamyl transfer RNA, they were not homologous in DNA sequence and were located at two separate loci.The data indicate that the petite mitochondrial genome is the result of a primary deletion followed by successive additional deletions. Thus an unequivocal gene arrangement cannot be readily established by deletion mapping with petite mutants alone. Nevertheless, we have derived a tentative circular map of the yeast mitochondrial genome from the data; the map indicates that all but one of the transfer RNA genes are found between the C and P markers without forming a tight cluster. The following arrangement is suggested:-P-pro-val-ile-(phe, ala, tyr, asp)-glu2-(lys-leu)-his-C-E-OI-glu1-OII-P-.