A method employing aqueous media for isolation of nuclei from rat skeletal muscle is described. The technique involves (a) mincing and then homogenizing in a 0.32 M sucrose-salt solution with a Potter-Elvehjem type homogenizer using a Delrin (an acetal resin) pestle and a carefully controlled, relatively large pestle-to-glass clearance, (b) filtering through fiberglass and stainless steel screens of predetermined mesh size to remove myofibrils and connective tissue, and (c) centrifuging in a 2.15 M sucrose-salt solution containing 0.7 m_u ATP. Electron and phase-contrast microscopic observations show that the nuclei are intact, unencumbered by cytoplasmic tags, and possess well preserved distinct nucleoli, nucleoplasm, and nuclear membranes. Cytoplasmic contamination is minimal and mainly mitochondrial. Chemical assays of the nuclear flacfion show that the DNA/protein and RNA/ DNA ratios are comparable to those obtained in other tissues. These ratios, as well as the low specific activity obtained for cytochrome c oxidase and the virtual absence of myofibrillar ATPase, indicate a high degree of purity with minimal mitochondrial and myofibrillar contamination. The steps comprising the technique and the reasons for their selection are discussed.
During a toxologic study of gold compounds, it was noted that the survivors of midlethal dose of goldthioglucose gained weight excessively. About one-third of the mice became obese (1). Subsequent studies established that goldthioglucose produced lesions in the hypothalamus (2). Other gold compounds of closely related general structure, such as goldthiomalate, failed to produce either the hypothalamic lesions or the obesity (2-4). Goldthioglucose obesity appeared to be of the hypothalamic variety which can be produced both in rats and mice by stereotactic injury to the ventromedial hypothalamic nuclei (S, 6).The apparent specificity of goldthioglucose in producing lesions in the hypothalamus was ascribed to a particular affinity of the gold compound for the ventromedial nucleus, which has been identified as the satiety center. This interpretation supported the glucostatic theory of regulation of food intake which asserted that the cells of the ventromedial nucleus are sensitive to an increasing arterio-venous difference in blood glucose. High blood levels of glucose would load the special receptor cells with glucose and activate discharge of impulses inhibiting further food intake. The presumed selectivity of goldthioglucose for the ventromedial nucleus was ascribed to the special affinity of these specialized nerve cells for the glucose moiety (4).Recently, the specific affinity of the hypothalamic center for goldthioglucose was investigated by quantitative measurement and localization of the gold. This was made possible by activation analysis capable of detecting gold in tissues in micromicrogram quantities and by use of radioautography for localization (7,8). The results clearly indicated that the obese animals had a higher concentration of gold in the hypothalamic area than similarly injected animals that had not become obese.
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