Polyamines and protein kinase I. Induction of ornithine decarboxylase and activation of protein kinase in rat glioma cells

The activities of cAMP-dependent and independent protein kinases were determined after feeding confluent glioma C6-BU-1 cultures. It has been shown that the activity of both enzymes rose considerably after feeding, and that the ratio of ³²P incorporation into histone, in the absence and the presence of cAMP, was maximal 4 hours after feeding. This increase in protein kinase activity was followed by the activation of ornithine decarboxylase and accumulation of putrescine. Spermine, at millimolar concentrations, inhibited protein kinase, apparently by inactivating the catalytic subunit. It is suggested that this inhibition of protein kinase by polyamines is another regulatory mechanism, which controls cellular growth.     

A model for the packing of lipids in bilayer membranes

A number of known structural properties of mixed lipid bilayer membranes and monolayers are accounted for by a model in which lipids pack into bilayers and monolayers like building blocks, each characterized by a surface head group area and characteristic solid angle. In phospholipids above the melting transition the head group area (at a given temperature and degree of hydration) is fairly invariant while the hydrocarbon region may be liquid-like so long as the molecule is not compressed beyond its characteristic solid angle.Phosphotidylcholine and phosphotidylserine are tapered lipids, i.e. their surface head group areas are greater than their non-polar end areas; cholesterol is frayed, i.e. its polar end area is less than its non-polar end area; while phosphotidylethanolamine is almost cylindrical. The “condensing” effect of cholesterol in mixed phospholipid-cholesterol films is seen as a taper-fray accomodation. The lipid distribution in erythrocyte membranes is shown to be conductive to a stable strain-free membrane.     

Salt dissociation of nuclear particles containing DNA-like RNA. Distribution of phosphorylated and nonphosphorylated species.

Electrophoresis of proteins from nuclear particles containing DNA-like RNA gave a pattern with 45 bands. The possibility that some of these proteins arose by contamination with ribosomes, chromatin, or soluble nuclear proteins was examined and eliminated. The fate of the proteins of the particles was studied after partial dissociation with 0.25 and 0.70 M NaCl. The individual proteins were released progressively and in different quantities. A group of easily released species (75 and 95% removed with 0.25 and 0.70 M NaCl) was demonstrated. This group contained 8 species between 29,000 and 39,000 daltons which represented approximately one-half of the total number of molecules. It is suggested that they are bound to repetitive sequences of the RNA. At least 30 and 60% of the other proteins were released at 0.25 and 0.70 M NaCl, respectively. There were no specific proteins tightly bound to the RNA, unless the nature of the remaining species is different from that of the released ones of the same molecular weight. The phosphorylated proteins were more tightly bound to the RNA than the nonphosphorylated species of similar molecular weight. In several instances, the 32-P radioactivity was associated with quantitatively minor bands of proteins.     

Release of euchromatic segments by MgCa-dependent autodigestion of chromatin

Mouse TLT hepatoma chromatin was incubated in the presence of added MgCl₂ and CaCl₂ but in the absence of added exogenous nuclease. Fraction-ation of such autodigested chromatin by glycerol density gradient centrifugation resulted in a heterochromatin-enriched pellet and euchromatin-enriched non-pelletable fractions. This was determined by satellite DNA content analyses and analysis of nascent RNA distribution. Electron microscope examination of the chromatin revealed it to consist of a series of connected spherical particles (nucleosomes) having a diameter of 370 ± 70Å. This native structure of chromatin was not damaged by the autodigestion process.     

Functional role of zinc in poly(A) synthesis catalyzed by nuclear poly(A) polymerase.

Three methods, chromatographic, spectrophotometric and tritium-release assay, were used and compared for the assay of deoxycytidylate methyltransferase. All three methods can be used for assay of this enzyme but the tritium-release assay appears to be the most simple and convenient. With the help of this assay the deoxycytidylate methyltransferase has been isolated and purified from sonically disrupted cells of Xp12-infected Xanthomonas oryzae. Using a procedure that involves fractionation with streptomycin sulfate and ammonium sulfate, filtration through Sephadex G-100 and chromatography on DEAE-cellulose, a 214-fold increase in specific activity was obtained. The enzyme displays a narrow pH optimum at 6.0 Among the buffers tested, 6-morpholinoethane sulfonate with the addition of Mg2 is the best. The enzyme can utilize dCMP as a substrate. The enzyme can also convert tetrahydrofolic acid into dihydrofolic acid. The Km value for dCMP is 31.3 micrometer and the Km value for tetrahydrofolic acid is 71.4 micrometer. There is no absolute requirement of ions for the activity of the enzyme; however, the presence of ions causes stimulating or inhibiting effects on enzyme activity that are dependent on the variety and concentration of ions used.     

Definition of a small core transcriptional circuit regulated by AML1-ETO

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