Induction of chromosome banding by trypsin/EDTA for gene mapping by in situ hybridization

We describe an easy and reproducible procedure that utilizes trypsin/EDTA for the induction of chromosome banding in conjunction with in situ hybridization. The high quality banding resolution required for grain localization is obtained on both elongated and contracted chromosomes derived from synchronized or nonsynchronized human lymphocytes or fibroblasts. This procedure can also be useful for gene localization on chromosomes from cancer cells.     

Redox interconversion of Escherichia coli glutathione reductase. A study with permeabilized and intact cells

Summary The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state.The enzyme from toulene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 M NADPH, while 700 M NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)⁺. The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact E. coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.     

Incorporation of [3H]thymidine into DNA and of [35S]sulfate into sulfatides of oligodendroglial cells during development: Effect of malnutrition

Incorporation of [³H]thymidine into DNA and of [³⁵S]sulfate into sulfatides of oligodendroglial cells isolated from brain slices incubated with the radioactive precursor was studied in normal and malnourished rats at different ages. The pattern and the values of incorporation of [³H]thymidine into DNA were similar in both groups of animals. The maximum value of incorporation was observed at 7 days of age decreasing rapidly thereafter and leveling off between 18–21 days. In both groups of animals labeling of sulfatides attained a maximum at 18 days of age, showing similar values of incorporation up to that age. However, at 21 days of age; the values corresponding to malnourished rats were found to be 40% lower in comparison to controls. The results suggest that (a) proliferation of oligodendroglial cells stops at similar ages in normal and malnourished rats, (b) expression of sulfatide synthesis by oligodendroglial cells is similar in both groups of animals up to 18 days, and (c) the starved rats seem to be unable to maintain normal synthesis of these galactolipids throughout the entire period of active myelinogenesis.     

Neonatal malnutrition in the rat affects the delivery of sulfatides from microsomes and their entry into myelin

Brain slices from 18 day old normal and malnourished rats were incubated in the presence of [³⁵S]sulfate to explore its incorporation into sulfatides of a total brain homogenate and the appearance of labeled sulfatides in different subcellular fractions. While the incorporation of label into sulfatides of the total homogenate was similar in both groups of animals, in subcellular fractions separated on a linear sucrose density gradient, labeling of sulfatides in malnourished animals was relatively higher in the region corresponding to the microsomal fraction. Time course incorporation and pulse-chase experiments were carried out to explore the kinetics of labeling of microsomal and myelin sulfatides. In pulse-chase experiments, normal controls showed a decrease in the specific radioactivity of sulfatides in the microsomal fraction after the chase, which was not observed in malnourished animals, while the appearance of labeled sulfatides in the myelin fraction of the latter group of animals was found to be lower than in normals. These results suggest that in neonatal malnutrition there is a defect in the transport of de novo synthesized sulfatides towards myelin or/and a problem in the assembly of these lipids into the myelin membrane.     

Effect of rhodium (III) complex on experimental carcinogenesis in the livers of rats treated with thioacetamide

Enzyme activities and protein content were determined in the cytosolic and mitochondrial fractions of liver homogenates obtained from Rh(III) complex-, thioacetamide- and thioacetamide + Rh(III) complex-treated rats. The Rh(III) complex administered to nonthioacetamide-treated rats produced no significant changes either in the enzymatic activities assayed or in the protein concentration. The Rh(III) complex administered to thioacetamide-treated rats produced significant restoration of the following altered values: cytosolic and mitochondrial aspartate aminotransferase, glutamate dehydrogenase, NADP-isocitrate dehydrogenase, and protein concentration. However, a further increase was produced in the activities of glucose-6-phosphate dehydrogenase and malic enzyme. These increases can be interpreted in terms of an enhancement of the NADPH-dependent detoxifying processes and of nucleic acid synthesis and repair.     

A NEW GENUS OF PROTOSTELIDS SHOWING AFFINITIES WITH CERATIOMYXA

A new genus and species of the Protosteliida (Mycetozoa), Ceratiomyxella tahitiensis, was isolated from dead plant material—var. tahitiensis from Tahiti and var. neotropicalis from Brazil and Colombia. The sporocarps have deciduous spores borne singly on slender hollow stalks; zoocysts with anteriorly flagellate planonts are produced. The trophic stage is comprised of uninucleate to plurinucleate amoeboid cells and reticulate plasmodia; the uninucleate cells become flagellate in water. The prespore cells and spores are plurinucleate. Sexuality has not been demonstrated. Var. tahitiensis has globose spores and produces its zoocysts just after spore germination, whereas var. neotropicalis has subglobose spores and forms zoocysts later in the life cycle. The species is thought to show phylogenetic relationships with Ceratiomyxa, which was recently transferred to the Protosteliida by Olive.