Identification by flow cytometry of two distinct rhodamine-123-stained mitochondrial populations in rat liver

Isolated rat liver mitochondria were split into three fractions of increasing density when applied to a Percoll gradient. NADH-ubiquinone oxidoreductase, succinate dehydrogenase and cytochrome-c oxidase but not F1-ATPase activities increased with density as well as respiratory rate in state 3 and the respiratory control index. Flow cytometry of mitochondrial density fractions stained with rhodamine-123 revealed the occurrence in each density fraction of two distinct mitochondrial populations with different fluorescence intensity. The high fluorescence population was minor and its proportion decreased with density. The extent of high fluorescence population staining depended on the deenergized state of the mitochondria suggesting that this population represents an immature form of the mitochondria which may develop into a fully functional organelle by the incorporation of structural and/or functional proteins.     

Nitrogen metabolism in tumor bearing mice

In experiments with whole animals infested with a highly malignant strain of Ehrlich ascites tumor cells, serial concentrations of amino acids were determined for host plasma, ascitic fluid, and tumor cells, throughout tumor development. Concentration gradients of glutamine, asparagine, valine, leucine, isoleucine, phenylalanine, tyrosine, histidine, tryptophan, arginine, serine, methionine, and taurine from the host plasma toward the ascitic liquid were established; while on the other hand, concentration gradients from the ascitic liquid toward the plasma were established for glutamate, aspartate, glycine, alanine, proline, and threonine. With the exception of aspartate the concentrations of these amino acids were highest inside the cells. Arginine was the only amino acid not detected in tumor cells. In vitro incubations of tumor cells in the presence of glutamine and/or glucose, as the energy and nitrogen sources, confirmed the amino acid fluxes previously deduced from the observed relative concentrations of amino acids in plasma, ascitic liquid, and tumor cells, suggesting that glutamate, alanine, aspartate, glycine, and serine can be produced by tumors. These findings support that changes in amino acid patterns occurring in the host system are related to tumor development.     

Rapid postnatal changes in F1-ATPase proteins and in the uncoupling protein in brown adipose tissue mitochondria of the newborn rat

Changes in F1-ATPase and UCP protein contents and in the activity of respiratory complexes I, II and IV of brown adipose tissue mitochondria are reported during the first 0-6 hours of life in the rat. Mitochondrial UCP/F1-ATPase protein ratio is used to define the onset of thermogenic differentiation of brown adipose tissue mitochondria. It is concluded that mitochondrial differentiation occurs soon after birth and that the process is accelerated by hypothermic conditions.     

Purification of phosphate-dependent glutaminase from isolated mitochondria of Ehrlich ascites-tumour cells.

Phosphate-dependent glutaminase was purified to homogeneity from isolated mitochondria of Ehrlich ascites-tumour cells. The enzyme had an Mr of 135,000 as judged by chromatography on Sephacryl S-300. SDS/polyacrylamide-gel electrophoresis displayed two protein bands, with Mr values of 64,000 and 56,000. Two major immunoreactive peptides of Mr values of 65,000 and 57,000 were found by immunoblot analysis using anti-(rat kidney glutaminase) antibodies. The concentration-dependences for both glutamine and phosphate were sigmoidal, with S0.5 values of 7.6 mM and 48 mM, and Hill coefficients of 1.5 and 1.6, respectively. The glutaminase pH optimum was 9. The activation energy of the enzymic reaction was 58 kJ/mol. The enzyme showed a high specificity towards glutamine. A possible explanation for the different kinetic behaviour found for purified enzyme and for isolated mitochondria [Kovacević (1974) Cancer Res. 34, 3403-3407] should be that a conformational change occurs when the enzyme is extracted from the mitochondrial inner membrane.     

Processing of SP1 precursor in a cell-free system from poly(A+) mRNA of human placenta

Cell-free translation of polyadenylated mRNA from human term placenta in a wheat germ extract, after immunoprecipitation with antibodies directed against purified pregnant serum SP₁, yielded a single polypeptide of 31 kDa. Addition of dog pancreatic microsomal vesicles to the translation system resulted in the appearance of two polypeptides, one of them of 46 kDa and the other of 28 kDa. Both polypeptides were protected from limited proteolysis and when the assay was performed with lytic detergent concentrations in addition to proteases, this protection was abolished indicating that the polypeptides were segregated into the microsomal vesicles. The cleavage of a signal peptide of 3 kDa from the 31 kDa primary translation product gives rise to 28 kDa and accounts for the slight increase in electrophoretic mobility. The treatment of the immunoprecipitated products with Endoglycosidase H and -mannosidase, suggested that only the 46 kDa polypeptide is a glycoprotein.From the results obtained we conclude that SP₁ is synthesized and processed to a glycoprotein of 46 kDa which would be a protomeric form of the oligomers reported in pregnant serum by other authors.Abbreviations PMSF phenylmethyl sulfonylfluoride - TCA trichloroacetic acid - DTT dithiotreitol     

Stretch-activated ion channels modulate the resting membrane potential during early embryogenesis

By using the patch-clamp technique, stretch-activated ionic channels were found in the membrane of cleaving freshwater fish embryos at the early stages of embryogenesis (2-256 cells). The application of negative pressure to the pipette increased the frequency of activation and the duration of bursts. This type of channel has a preferential K+ selectivity. When bathed on both membrane surfaces with 140 mM KCl the channel conductance was 71 pS. The kinetic behaviour did not depend markedly on either membrane potential (in the range from -70 to +70 mV) or calcium concentration on the cytoplasmic side of the membrane. On continuous recording, the probability of the channel being open was found to change periodically over a 5- to 20-fold range for different cells. These variations correlated with changes in resting potential and membrane conductance during the cell cycle. These results suggest that the oscillation of resting potential within the cell cycle is associated with the operation of stretch-activated ion channels.     

A quantitative study of enterotoxin production by sheep milk staphylococci

Of 124 staphylococcal strains isolated from sheep milk, 78 produced enterotoxin A, B, C, or D when evaluated by an enzyme-linked immunosorbent assay. Enterotoxins A and D, elaborated by 44 and 43 strains, respectively, showed the highest incidence. Enterotoxin production by coagulase-negative strains (one Staphylococcus cohnii, three S. epidermidis, five S. haemolyticus, and four S. xylosus) was detected. Linear and logarithmic-logarithmic regressions of optical density on enterotoxin concentration yielded the best-fitting equations for enterotoxin quantitation. A significantly higher incidence of enterotoxin producers and significantly higher levels of enterotoxins produced were recorded for coagulase-positive, thermostable nuclease-positive, hemolysis-positive, or mannitol-positive strains. Mannitol utilization was the best test for discriminating between enterotoxigenic and nonenterotoxigenic staphylococci.     

Simultaneous utilization of glucose and xylose byCandida curvata D in continuous culture

Summary Of ten, mainly oleaginous, yeasts examined for the ability to use glucose and xylose simultaneously, only one,Candida curvata D, was found which could do so. This yeast was examined further in a single-stage chemostat wherein it produced similar biomass yields, lipid contents and fatty acids on glucose plus xylose mixed in varying proportions. This oleaginous yeast would therefore be capable of growing on hydrolysed wood and straw wastes as a potential source of single cell oil.     

Nutrient dynamics within amazonian forests

Summary Relationships between fine root growth, rates of litter decomposition and nutrient release were analysed in a mixed forest on Tierra Firme, a Tall Amazon Caatinga and a Low Bana on podsolized sands near San Carlos de Rio Negro. Fine root growth in the upper soil layers (root mat+10 cm upper soil) was considerably higher in the Tierra Firme forest (1117 g m^-2 yr^-1) than in tall Cattinga (120) and Bana (235). Fine root growth on top of the root mat was stimulated significantly by added N in Tall Caatinga and Low Bana forests, by P in Tierra Firme and Bana forests, and by Ca only in the Tierra Firme forest. Rate of fine root growth in Tierra Firme forest on fresh litter is strongly correlated with the Mg and Ca content of litter. Rate of litter decomposition was inversely related to % lignin and the lignin/N ratio of litter. Litter contact with the dense root mat of the Tierra Firme increased rates of disappearance for biomass, Ca and Mg as compared with litter permanently separated or lifted weekly from the root mat to avoid root attachment. Nitrogen concentration of decomposing litter increased in all forests, net N released being observed only in Caryocar glabrum and Aspidosperma megalocarpum of the Tierra Firme forest after one year of exposure. Results emphasize the differences in limiting nutrients in amazonian forest ecosystems on contrasting soil types: Tierra Firme forests are particularly limited by Ca and Mg, while Caatinga and Bana forests are limited mainly by N availability.     

Centrifugation of 2-cell mouse ova: cytoplasm stratification and recovery

Summary Two-cell mouse ova, which were centrifuged for l h at 70 000–90 000xg, showed a precise stratification of the cytoplasm and an elongation of the nucleus. The ova were fixed at different times and observed by light and electron microscopy using cytochemical methods and detergent extractions. Within 40 min after centrifugation the normal-looking morphology was recovered except for the persisting lipid caps at the centripetal poles of the blastomeres. Cleavage, compaction and blastulation were not prevented by centrifugation. Treatments with colcemid or cytochalasin D delayed but did not impair recovery. These results suggest that a resilient cytoskeletal structure may be involved in this kind of embryonic regulation.