Protein diffusion in crowded electrolyte solutions

We report on a combined cold neutron backscattering and spin-echo study of the short-range and long-range nanosecond diffusion of the model globular protein bovine serum albumin (BSA) in aqueous solution as a function of protein concentration and NaCl salt concentration. Complementary small angle X-ray scattering data are used to obtain information on the correlations of the proteins in solution. Particular emphasis is put on the effect of crowding, i.e. conditions under which the proteins cannot be considered as objects independent of each other. We thus address the question at which concentration this crowding starts to influence the static and in particular also the dynamical behaviour. We also briefly discuss qualitatively which charge effects, i.e. effects due to the interplay of charged molecules in an electrolyte solution, may be anticipated. Both the issue of crowding as well as that of charge effects are particularly relevant for proteins and their function under physiological conditions, where the protein volume fraction can be up to approximately 40% and salt ions are ubiquitous. The interpretation of the data is put in the context of existing studies on related systems and of existing theoretical models.     

Dibutyryl-cyclic AMP inhibits cholesterol esterification in J 774 monocyte-like cells

The effect of dibutyryl-cyclic AMP (dbcAMP) and theophylline was investigated on oleic acid incorporation into cholesteryl esters and triacylglycerols in the mouse monocyte-macrophage cell line J 774. 24h pretreatment of macrophages with dbcAMP decreased cholesteryl ester formation in a dose-dependent manner (about 4 fold reduction for dbcAMP 10(-4)M + theophylline 10(-3)M), while oleic acid incorporation into triacylglycerols was markedly (2 to 3 fold) enhanced. The catabolism of acetylated LDL was only slightly affected (about 15-20% reduction with dbcAMP 5 X 10(-4)M + theophylline 10(-3)M). Acyl Coenzyme A: cholesterol-O-acyl-transferase activity, measured in vitro on cell homogenates, was reduced in dbcAMP-treated cells, whereas diacylglycerol acyltransferase activity was increased. These results suggest that cyclic AMP can modulate cholesteryl ester and triacylglycerol formation in macrophages, and that these metabolisms are inversely regulated. Agents which increase cyclic AMP intracellular level could be of interest for reducing cholesteryl ester accumulation in macrophages.     

Introduction of the Escherichia coli gdhA gene into Rhizobium phaseoli: effect on nitrogen fixation.

Rhizobium phaseoli lacks glutamate dehydrogenase (GDH) and assimilates ammonium by the glutamine synthetase-glutamate synthase pathway. A strain of R. phaseoli harboring the Escherichia coli GDH structural gene (gdhA) was constructed. GDH activity was expressed in R. phaseoli in the free-living state and in symbiosis. Nodules with bacteroids that expressed GDH activity had severe impairment of nitrogen fixation. Also, R. phaseoli cells that lost GDH activity and assimilated ammonium by the glutamine synthetase-glutamate synthase pathway preferentially nodulated Phaseolus vulgaris.     

Ammonium assimilation in Rhizobium phaseoli by the glutamine synthetase-glutamate synthase pathway.

Evidence from in vitro and in vivo studies showed that in Rhizobium phaseoli ammonium is assimilated by the glutamine synthetase (GS)-glutamate synthase NADPH pathway. No glutamate dehydrogenase activity was detected. R. phaseoli has two GS enzymes, as do other rhizobia. The two GS activities are regulated on the basis of the requirement for low (GSI) or high (GSII) ammonium assimilation. When the 2-oxoglutarate/glutamine ratio decreases, GSI is adenylylated. When GSI is inactivated, GSII is induced. However, induction of GSII activity varied depending on the rate of change of this ratio. GSII was inactivated after the addition of high ammonium concentrations, when the 2-oxoglutarate/glutamine ratio decreased rapidly. Ammonium inactivation resulted in alteration of the catalytic and physical properties of GSII. GSII inactivation was not relieved by shifting of the cultures to glutamate. After GSII inactivation, ammonium was excreted into the medium. Glutamate synthase activity was inhibited by some organic acids and repressed when cells were grown with glutamate as the nitrogen source.     

Nitrogen Source Regulates Glutamate Dehydrogenase NADP Synthesis in Neurospora crassa

Neurospora crassa glutamate dehydrogenase-NADP (EC 1.3.1.3) has a higher activity when mycelium is grown on ammonium or nitrate as nitrogen source than when grown on glutamate or glutamine. Quantitative immunoelectrophoresis established that, under all conditions, enzyme activity corresponded to enzyme concentration. Isotope incorporation studies demonstrated that the nitrogen source exerts its regulation at the level of de novo enzyme synthesis.     

Long-acting contraceptive agents: Norethisterone esters of arylcarboxylic acids

The synthesis of esters of norethisterone (17α-ethynyl-17β-hydroxy-estr-4-en-3-one) with acids containing a benzene ring is described, two methods of esterification being compared in terms of yield and convenience. The activities of these esters as long-acting contraceptive agents have been evaluated.     

Long-acting contraceptive agents: norethisterone esters of monoalkenyl and monoalkynyl acids

The synthesis of nine new esters of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) is described, with the esterifying acids bearing an acetylenic or olefinic function in a chain of eight or nine carbon atoms, for evaluation as long-acting contraceptive agents.