Reconstitution of the Na+K+ pump of Ehrlich ascites tumor and enhancement of efficiency by quercetin

Plasma membranes from Ehrlich ascites tumor cells were solubilized by octylglucoside in the presence of phospholipids. The Na+K+-ATPase was purified from this extract by adsorption and elution from thio-Seph-arose 4B. The enzyme (specific activity, 7 mumoles of ATP hydrolyzed min-1 mg of protein -1) was reconstituted into liposomes by the octyglucoside dilution procedure. An ATP-dependent Na+ influx with low efficiency was observed. On addition of appropriate amounts of quercetin, the Na+ flux/ATP hydrolysis ratio was increased from 0.4 to 1.4.     

Genetics of NAD metabolism in Salmonella typhimurium and cloning of the nadA and pnuC loci.

The nadA and pnuC loci of S. typhimurium were cloned and found to reside within a 2.2-kilobase region. Two-dimensional O'Farrell gel electrophoresis of the proteins produced after chloramphenicol amplification and subsequent release from chloramphenicol inhibition revealed NadA and PnuC to be 43,000- and 25,000-molecular-weight proteins, respectively. The data indicated that nadA and pnuC represent two distinct genes.     

Somatic instability of carotenoid biosynthesis in the tomato ghost mutant and its effect on plastid development

The tomato (Lycopersicon esculentum (L.) Mill.) ghost plant is a mutant of the San Marzano cultivar affected in carotenoid biosynthesis. ghost plants exhibit a variable pattern of pigment biosynthesis during development. Cotyledons are green but true leaves are white. Green sectors, which appear to be clonal in origin, are frequently observed in the white tissue. Because of the lack of photosynthesis ghost plants have a very low viability in soil. We have developed a strategy for propagating ghost plants that employs organ culture to generate variegated green-white plants which, supported by the photosynthetic green areas, develop in soil to almost wild-type size. These plants were used to analyze the pigment content of the different tissues observed during development and plastid ultrastructure. Cotyledons and green leaves contain both colored carotenoids and chlorophyll but only the colorless carotenoid phytoene accumulates in white leaves. the plastids in the white tissue of ghost leaves lack internal membrane structures but normal chloroplasts can be observed in the green areas. The chromoplasts of white fruits are also impaired in their ability to form thylakoid membranes.     

Intracellular cyclic AMP produces effects opposite to those of cyclic GMP and calcium on shape and motility of neuroblastoma cells.

We have directly evaluated the effects of various intracellular second messengers including cyclic nucleotides, calcium ion, and inositol polyphosphates on shape and motility of differentiating mouse neuroblastoma cells. The messengers were microinjected into cells and the responses of the soma, neurite, and growth cone were monitored using time-lapse video microscopy. Each messenger altered cell shape and motility in a characteristic manner. Cyclic AMP promoted lamellipodial expansion, neurite outgrowth, and motility. The other injected messengers opposed motility. Cyclic GMP caused motile structures to freeze and to retract permanently, while the inhibitory effects of calcium injection were concentration-dependent. Small calcium injections affected specifically actin-containing motile structures which froze and retracted temporarily. Intermediate calcium injections caused a strong contraction at the site of injection in all cells. With large injections, cells retracted long neurites, rounded up, and frequently began vigorous blebbing that continued to cell death. Injections of the inositol polyphosphates IP3(1,4,5) and IP4(1,4,5,6) mimicked the effects of small calcium injections, as did electrical stimulation that elicited action potentials. The results suggest that in mouse neuroblastoma cells, intracellular cAMP elevation increases cytoskeletal organization and promotes neurite extension perhaps through an enhancement of cell-substratum adhesion. On the other hand, a rise of intracellular cGMP or intracellular calcium interferes directly with the function and organization of the actin-microfilament system. The integrated action of these second messenger systems may, therefore, operate in vivo to allow substances released from neighboring cells to regulate neuronal architecture.     

Conversion of 15-hydroxyeicosatetraenoic acid to 11-hydroxyhexadecatrienoic acid by endothelial cells

Cultured endothelial cells take up 15-hydroxyeicosatetraenoic acid (15-HETE), a lipoxygenase product formed from arachidonic acid, and incorporate it into cellular phospholipids and glycerides. Uptake can occur from either the apical or basolateral surface. A substantial amount of the 15-HETE incorporated into phospholipids is present in the inositol phosphoglycerides. 15-HETE is converted into several metabolic products that accumulate in teh extracellular fluid; this conversion does not require stimulation by agonists. The main product has been identified as 11-hydroxyhexadecatrienoic acid [16:3(11-OH)], a metabolite of 15-HETE that has not been described previously. Formation of 16:3(11-OH) decreases when 4-pentenoic acid is present, suggesting that it is produced by beta-oxidation. The endothelial cells can take up 16:3(11-OH) only 25% as effectively as 15-HETE, and 16:3(11-OH) is almost entirely excluded from the inositol phosphoglycerides. These results suggest that the endothelial cells can incorporate 15-HETE when it is released into their environment. Through partial oxidation, the endothelium can process 15-HETE to a novel metabolite that is less effectively taken up and, in particular, is excluded from the inositol phosphoglycerides.