AndréJ. Bester

The synthesis of myofibrillar and soluble proteins in cell-free systems and in intact cultured muscle cells from newborn polymyopathic hamsters

Abstract Myofibrillar proteins from both hearts and skeletal muscles of control and myopathic hamsters, at different ages, were extracted and analysed electrophoretically. Significant quantitative differences were found, more or less uniformly affecting all the myofibrillar components, even of embryonic hearts. In an attempt to determine whether the observed differences become established as the result of defective biosynthesis or for some other reason, reconstituted cell-free systems were used to study the in vitro synthesis of some of the specific myofibrillar protein components. A generally defective biosynthesis of myosin, (both heavy and light chains) and actin was demonstrated, while gel-electrophoretic analysis of soluble proteins synthesized in vitro showed that the profile of radioactivity in systems derived from myopathic animals was also generally lower than in those from controls. In order to demonstrate that the defect in protein synthesis was present in intact beating heart cells, myopathic and control heart cells were treated with radioactive amino acids, and the soluble and myofibrillar proteins subsequently extracted and fractionated. The biosyntheses of both types of proteins were clearly impaired in the case of cells derived from myopathic animals.

Effect of propranolol on the metabolism and function of the rat heart

Abstract The effect of propranolol, a beta-adrenergic blocking agent, was studied on the metabolism and function of the isolated, perfused rat heart. Propranolol (20 μ m ) depressed the oxygen consumption, coronary flow rate, palmitate uptake and oxidation as well as the high energy phosphate contents of the perfused heart. Propranolol significantly reduced heart rate while the time to peak height of developed tension period was increased. The effect of propranolol was also studied on the metabolism of heart muscle slices and the sarcosomal oxidative phosphorylation process. With pyruvate or glucose as substrate, propranolol stimulated the respiration and metabolism of heart slices, while it depressed oxidative phosphorylation. In order to eliminate the effect of the reduction in heart rate on substrate metabolism in the perfused heart, additional studies were performed in which the effect of propranolol was studied in hearts driven at a controlled heart rate, by a pacemaker. The results showed that the chronotropic effect of propranolol had a significant effect on the reduction in substrate metabolism.

Protein synthesis in myocardial ischaemia and infarction

Abstract Protein synthesis, as indicated by the in vitro and in vivo incorporation of isotopic amino acids into myocardial proteins, was determined at various time intervals after ligation of the left coronary artery. The results show that the in vitro incorporation of l -[4,5- 3 H]leucine and l -[U- 14 C]lysine into the soluble protein and actomyosin fractions of heart muscle slices was normal up to 6 h after the onset of ischaemia. The rate of amino acid incorporation was significantly higher 12 h after occlusion of the left coronary artery and remained elevated for at least 2 months. After 3 months, the rate of amino acid incorporation into both protein fractions returned to normal. Protein synthesis was elevated in both the uninfarcted and infarcted areas of the myocardium. Studies on the in vivo incorporation of l -[4,5- 3 H]leucine into total myocardial proteins also showed that protein synthesis was normal up to 6 h after occlusion of the left coronary artery.

Autoantibodies from patients with systemic lupus erythematosus can affect DNA and RNA synthesis in cultured cells

Sera reacting positively for anti-DNA antibodies from systemic lupus erythematosus (SLE) patients were tested for their effect on DNA and RNA synthesis in permeabilized cultured cells and isolated nuclei. The immunoglobulin fraction obtained by ammonium sulfate precipitation of serum was shown to exert considerable influence on DNA and RNA synthesis in cultured cells and nuclei. A component of this antibody population is anti-DNA. These antibodies exert different effects on DNA template activity which is a function of their conformational specificity. Intracellular penetration of autoantibodies as noted in SLE may be one of the reasons for clinical manifestations of disease in these patients.

Naturally occurring anti-DNA antibodies from systemic lupus erythematosus patients may be used to study gene expression in vitro

Anti-B-DNA and anti-Z-DNA antibodies were prepared from the serum of systemic lupus erythematosus (SLE) patients by affinity chromatography. The anti-Z-DNA antibodies were shown to exhibit site-specific binding preferences in pBR322 negatively supercoiled (plasmid) DNA, as assayed by restriction-enzyme cleavage. The anti-B-DNA antibodies were found to stimulate in vitro transcription of pBR322, whereas little effect was observed on combination with anti-Z-DNA antibodies. The results support the proposal that the formation of Z-DNA is a down-regulatory mechanism and that the B to Z conformational change may be a flip-flop control for gene expression.

The subunit structure of heart muscle chalones.

Abstract Recently, chalones have been isolated from adult bovine hearts and purified to homogeneity (2). A combination of equilibrium centrifugation in 6.0 M Guanidinium chloride and SDS polyacrylamide gelelectrophoresis of the chalones both under reduced conditions suggest the involvement of four indentical subunits with a molecular weight of about 150 000 each. Electron microscopy of negatively stained preparations showed a globular structure. From the amino acid analysis it is evident that the chalones has a net negative charge and a predominant hydrophobic amino acid composition. Isoelectric focusing shows a pI of 5.1. The sugars detected were, in molar proportions, D-Mannose (3.0), D-Galactose (2.5), D-Glucose (1.0) and D-Glucosamine (1.7).

Nucleic acid synthesis in myocardial ischaemia and infarction

The rate of in vivo and in vitro labelling of DNA and RNA in left and right ventricles were studied at various time intervals after right coronary artery ligation. The RNA concentration of both left and right ventricles were significantly higher than the controls 7 days after ligation and remained elevated for at least 14 days. The DNA concentration remained significantly higher for one month. In vitro as well as in vivo labelling of myocardial RNA, were unaffected by coronary artery ligation. In vitro labelling of DNA was unchanged during ischaemia and infarction, while in vivo DNA labelling was significantly increased.