Phosphorus-31 nuclear magnetic resonance of phosphoenzymes of sodium- and potassium-activated and of calcium-activated adenosinetriphosphatase

Prior studies identified phosphoenzyme intermediates in the turnover of sodium- and potassium-activated adenosinetriphosphatase [(Na,K)ATPase] from several sources and of the calcium-activated adenosinetriphosphatase [(Ca)-ATPase] of skeletal muscle sarcoplasmic reticulum. In both cases, the transphosphorylation is to a beta-aspartyl carboxyl group at the active site. We now report observation of a K+-sensitive phosphorylated intermediate of purified (Na,-K)ATPase from the salt gland of the duck using high-field 31P nuclear magnetic resonance. Addition of ATP to a suspension of this enzyme in the presence of Mg2+ and Na+ produced a resonance at about +17 ppm relative to 85% phosphoric acid. Addition of inorganic phosphate and Mg2+ to (Na,K)ATPase also produced a resonance at about +17 ppm which was enhanced in the presence of a saturating concentration of the inhibitor, ouabain; again, addition of K+ made this resonance disappear. These findings are consistent with earlier kinetic characterization of an acid-stable (Na,K)ATPase phosphoenzyme intermediate by 32P-labeled phosphate incorporation into a denatured precipitate of the enzyme. We attribute the +17-ppm resonance to formation of an acyl phosphate at an aspartyl residue of the catalytic site of (Na,K)ATPase. This is supported by our finding of a similar resonance at +17 ppm after phosphorylation of another membrane-bound cation transport enzyme, sarcoplasmic reticulum (Ca)ATPase, as well as by a similar resonance at about +17 ppm after phosphorylation of the model dipeptide L-seryl-L-aspartate.     

Extinction of differentiated glial functions in somatic ce-l hybrids is incomplete.

Rat glioma cells of clone C₆ were hybridized in vitro with mouse L cells of clone A9 or with freshly isolated mouse macrophages, and the hybrids were assayed for glial cell functions. C₆ cells expressed high levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP; EC 3.1.4.37), β-hydroxybutyrate dehydrogenase (HBDH; EC 1.1.1.30), glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8), and inducibility of GPDH by hydrocortisone (HC). A9 cells and macrophages had very low activities of these functions. Hybrids between C₆ and A9 or between C₆ and macrophages had greatly reduced activities of these functions, but the hybrids expressed significantly higher activities than the non-glial parent. This incomplete extinction was not due to fusion of two glioma cells with one L cell or macrophage. The difference in GPDH activity in the hybrids as compared with the non-glial parent was due to incomplete shut-off of GPDH of the glial parent, and not to an increase in GPDH production by the non-glial genome.     

Extinction of differentiated glial functions in somatic cell hybrids is incomplete

Rat glioma cells of clone C₆ were hybridized in vitro with mouse L cells of clone A9 or with freshly isolated mouse macrophages, and the hybrids were assayed for glial cell functions. C₆ cells expressed high levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP; EC 3.1.4.37), β-hydroxybutyrate dehydrogenase (HBDH; EC 1.1.1.30), glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8), and inducibility of GPDH by hydrocortisone (HC). A9 cells and macrophages had very low activities of these functions. Hybrids between C₆ and A9 or between C₆ and macrophages had greatly reduced activities of these functions, but the hybrids expressed significantly higher activities than the non-glial parent. This incomplete extinction was not due to fusion of two glioma cells with one L cell or macrophage. The difference in GPDH activity in the hybrids as compared with the non-glial parent was due to incomplete shut-off of GPDH of the glial parent, and not to an increase in GPDH production by the non-glial genome.     

Lipid-protein interactions in model membranes from bovine brain white matter. An ESR spin label and electron microscopy study.

Lipid-protein model membranes, prepared from bovine brain white matter and containing all the lipids and Folch-Lees proteolipids, have been studied in macroscopically oriented multibilayers. To examine the lipid environment the membranes were spin labeled with the cholestane spin label (3'-spiro(2'=(N-oxyl-4',4'-dimethyl-oxazolidine))5alpha-cholestane) and a fatty acid spin label (4',4'-dimethyloxazolidine-N-oxyl derivative of 5-ketostearic acid). The ESR spectra exhibit two components arising from fairly well oriented and completely unoriented lipids. Up to a temperature of 55 degrees C the amount of oriented lipids is almost constant, being about 35%. At higher temperatures this percentage drops rapidly to zero. It is shown that the presence of unoriented lipids arises mainly from disrupted areas in the lipid bilayer structure. This is confirmed by electron miccroscopy and from an analysis of the temperature dependence of the order parameters of the spin labels. The presence of locally disrupted lipid parts in the bilayer is discussed in relation to the interaction of the brain white matter lipids with Folch-Lees protein.     

Inhibition of bacteriophage PBS2 replication in Bacillus subtilis by phleomycin.

Phleomycin is an effective inhibitor of the replication of Bacillus subtilis bacteriophage PBS2, whose DNA contains uracil instead of thymine. Phleomycin does not affect the induction of the known phage enzymes involved in deoxyribonucleotide metabolism. But phage DNA synthesis is severely inhibited by phleomycin, and late virion protein synthesis is eliminated. These effects appear to result from a phleomycin-induced degradation of the parental phage DNA. Similar inhibitory and degradative effects on DNA are seen in phleomyinc-treated, uninfected cells. This system is unaffected by the related antibiotic, bleomycin.     

Rhythmic oscillations in amygdala excitability during kindling

Unilateral amygdala electrodes were implanted in male Sprague-Dawley rats stimulated once daily with a 200 μamp pulse of 500 millisecond duration to produce kindling. Forty-six percent (12 of 26) of the animals that eventually developed after-discharges demonstrated rhythmic oscillations in after-discharge duration. The presence or absence of generalized bilateral clonic seizures also showed rhythmic oscillations in close association with after-discharge duration. It is suggested that during kindling some animals, independent of electrode placement, develop rhythmic oscillations in excitability of the amygdala. This model may represent a means of experimentally eliciting or uncovering neuronal substrates which show regular alterations in excitability and may be relevant to the oscillations in mood and behavior observed in the affective disorders.     

Rap1 Can Bypass the FAK-Src-Paxillin Cascade to Induce Cell Spreading and Focal Adhesion Formation

We developed new image analysis tools to analyse quantitatively the extracellular-matrix-dependent cell spreading process imaged by live-cell epifluorescence microscopy. Using these tools, we investigated cell spreading induced by activation of the small GTPase, Rap1. After replating and initial adhesion, unstimulated cells exhibited extensive protrusion and retraction as their spread area increased, and displayed an angular shape that was remodelled over time. In contrast, activation of endogenous Rap1, via 007-mediated stimulation of Epac1, induced protrusion along the entire cell periphery, resulting in a rounder spread surface, an accelerated spreading rate and an increased spread area compared to control cells. Whereas basal, anisotropic, spreading was completely dependent on Src activity, Rap1-induced spreading was refractory to Src inhibition. Under Src inhibited conditions, the characteristic Src-induced tyrosine phosphorylations of FAK and paxillin did not occur, but Rap1 could induce the formation of actomyosin-connected adhesions, which contained vinculin at levels comparable to that found in unperturbed focal adhesions. From these results, we conclude that Rap1 can induce cell adhesion and stimulate an accelerated rate of cell spreading through mechanisms that bypass the canonical FAK-Src-Paxillin signalling cascade.     

The chromosomes of the Filariae

An understanding of the nature of the chromosomes of the filariae is expected to greatly assist the future interpretation of genome data. Filarial development is not eutelic, and there does not seem to be a fixed number of cell divisions in the way that there is in Caenorhabditis. It is not clear whether the chromosomes of the filariae have localized centromeres or whether they are holocentric. Sex determination is by a chromosomal "balance" X0 system in most filariae, but in some Onchocercidae there has been a chromosomal fusion to create a neo-XY system. It is presumed that the molecular basis of sex determination in filariae is similar to Caenorhabditis. The ancestral karyotype of the filariae is probably 5A+X0, but in some Onchocercidae this has been reduced to 4A+XY, and in O. volvulus and O. gibsoni it has been further reduced to 3A+XY. Onchocerca volvulus and O. gibsoni both have supernumary (B-) chromosomes and in O. volvulus there is a single active nucleolus organising region near the middle of the long autosome.