Stimulatory effect of soluble supernatant on hydroxylase activity of rat testis microsomes.

Addition of soluble supernatant to testis microsomes results in 42% increase in steroid 17,20-lyase activity and a 65% increase in 17alpha-hydroxylase activity. This stimulatory activity could be partially purified by salt fractionation. The activating factor(s) was not removed by dialysis nor did it appear to be lipid. It was destroyed by trypsin. Differential effects of heat were observed with the hydroxylase and lyase activators. The activation did not affect Km but only increased Vmax. The supernatant could be added to each enzyme to the point of maturation. No binding of steroids by the supernatant could be detected. Corpus luteum and placental supernatant did not stimulate enzymic activity, but supernatant from an adrenal adenoma was active.     

DNA-Mediated gene transfection into primary cultures of adult mouse keratinocytes

Summary An efficient and reproducible technique for the transfection of primary cultures of adult mouse keratinocytes has been developed. The procedure involves culturing the primary adult mouse epidermal cells at 32° C in an enriched media until they reach 70 to 95% confluency, followed by transfection with exogenous DNA in a low potassium environment. Using chloramphenicol acetyl transferase (CAT) transient gene expression assays and various strong viral promoter/CAT constructs, the transfection procedure was optimized for media formulation, plasmid DNA concentration, carrier DNA concentration, incubation temperature, incubation period, and cell density. Optimized parameters include the use of 6 μg plasmid DNA and 10 μg pUC19 carrier DNA per 60-mm tissue culture dish. Since primary keratinocytes undergo a well-characterized pattern of differentiation in vitro in response to extracellular calcium concentrations, this transfection procedure should provide a useful model in which to study both tissue- and differentiation-specific gene expression.     

Isolation and separation of nucleic acids from Streptomyces hydrogenans (author's transl)]

Different methods for homogenization of cells of Streptomyces hydrogenans, for extraction of nucleic acids and for fractionation of the RNA and DNA obtained were critically examined. The only way to prepare high molecular weight rapidly labelled RNA and polysomes was to grind freeze-dried cells together with kieselguhr with a mortar and pestle. The best results for extraction of nucleic acids from the cell homogenate were obtained in the presence of diethyl pyrocarbonate (diethyl oxydiformate), yielding nucleic acids of considerable purity in a minimal amount of time. The best resolution of extracted nucleic acids was achieved by electrophoresis in 2% agarose acrylamide gels. This technique proved that during the cell homogenization and extraction procedure the bulk of nucliec acids was not degraded to low molecular weight material. An improved device for the registration of the profile of the absorption after gel electrophoresis is described.     

Thyroxine Treatment and Chicken (Gallus gallus) Embryo Duodenum in vitro

To study direct action of thyroxine (T₄) on duodenal development in chicken embryos, duodena from stage 42 White Leghorn (strain Hyline 934F) Gallus gallus embryos were cultured, cut into quarter segments and slit open on grids. L-T₄ was added to McCoy's 5A medium to concentrations of 14 ng/ml or 14 ng/100 ml. To control for possible physicochemical effects, D-T₄ was added to other media in the same concentrations, and saline vehicle was added to control media. Bovine serum albumin (BSA) was added to a duplicate set of cultures (0.2% w/vol) as a nutritive supplement. After 48 h alkaline phosphatase (E.C. 3.1.3.1) specific activity was significantly elevated (p < 0.05) in segments cultured with 14 ng L-T₄/100 ml (non-BSA) compared to saline control cultures. With 14 ng L-T₄/ml (BSA) phosphatase activity had increased significantly compared to segments treated with 14 ng L-T₄/100 ml (BSA), while those cultured with D-T₄ (BSA) had significantly lower activities than all other BSA-treated groups including controls. Total protein content was significantly elevated in all D-T₄ treated segments, both non-BSA and BSA-treated, compared to protein values of all other treatment groups. Following 48 h of culture the fine structure of epithelial mucosal cells usually showed condensed cytoplasm, while goblet cells were well developed, structural features appearing in all segments regardless of treatment. The responses after treatment further support a direct role for T₄ in duodenal development.