PEI-cellulose thin-layer chromatography. Product studies of the creatine kinase and pyruvate kinase reactions

Detailed methods for polyethylenimine-cellulose thin-layer chromatographic determination of the reactants and products of the creatine kinase and pyruvate kinase reactions are presented. The technique has been developed as a highly sensitive method both for estimation of relative initial rates afforded by creatine and creatine analogs in the creatine kinase reaction and also for estimation of trace amounts of inorganic phosphate and ADP contamination in samples of ATP.     

Fatty Acid Composition of Unicellular Strains of Blue-Green Algae

The fatty acids of 34 strains of unicellular blue-green algae provisionally assigned to the genera Synechococcus, Aphanocapsa, Gloeocapsa, Microcystis, and Chlorogloea by Stanier et al. have been chemically characterized. The strains analyzed can be divided into a series of compositional groups based upon the highest degree of unsaturation of the major cellular fatty acids. Twenty strains fall into the group characterized by one trienoic fatty acid isomer (alpha-linolenic acid), and seven strains fall into a group characterized by another trienoic acid isomer (gamma-linolenic acid). These groups in many cases correlate well with groupings based upon other phenotypic characters of the strains, e.g., deoxyribonucleic acid base composition. The assignment of a strain to a compositional group is not altered when the strain is grown under a variety of different culture conditions. All strains contain glycolipids with the properties of mono- and digalactosyldiglycerides.     

Water relations of solute accumulation in Pseudomonas fluorescens

When Pseudomonas fluorescens was grown in a glucose salts medium adjusted with NaCl to a water activity (aw) value of 0.980, the intracellular glutamic acid concentration increased 23-fold and comprised 90% of the total amino acid pool. This increase was not observed when the aw of the medium was reduced to 0.980 with sorbitol. Sorbitol was taken up rapidly over a 30 min period and accumulated intracellularly to a level approximately two-fold greater than the concentration in the growth medium. In continuous culture, the specific rate of glutamic acid production and glucose uptake was greater at 0.980 (NaCl) than at 0.997 aw. The maintenance coefficients for glucose uptake were similar at both aw values but were 2.4-fold greater for glutamic acid production at 0.980 (NaCl) than at 0.997 aw.     

Molecular evolution of mammalian lactate dehydrogenase-A genes and pseudogenes: association of a mouse processed pseudogene with a B1 repetitive sequence

A mouse genomic clone containing a lactate dehydrogenase-A (LDH-A) processed pseudogene and a B1 repetitive element was isolated, and a nucleotide sequence of approximately 3 kb was determined. The pseudogene and B1 element are flanked by perfect 13-bp repeats, and the B1 sequence starts at 14 nucleotides 3' to the presumptive polyadenylation signal of the pseudogene. The nucleotide sequences of the LDH-A genes and processed pseudogenes from mouse, rat, and human were compared, and a phylogenetic tree was constructed. The rate and pattern of nucleotide substitutions in the LDH-A pseudogenes are similar to previously reported results (Li et al. 1984). The average rate of nucleotide substitutions in the LDH-A pseudogenes is 4.3 X 10(- 9)/site/year. The substitutions of C----T and G----A are most frequent, and A----G substitutions are relatively high. The rate of synonymous substitutions in the LDH-A genes is 5.3 X 10(-9), which is not significantly higher than the average rate of 4.7 X 10(-9) for 35 mammalian genes. The rate of nonsynonymous substitutions in the LDH-A genes is 0.20 X 10(-9), which is considerably lower than the average rate of 0.88 X 10(-9) for 35 mammalian genes. Thus, the mammalian LDH-A gene appears to be highly conserved in evolution.      

Evaluation of the overland runoff mode of land wastewater treatment for virus removal.

The removal of enteric and tracer viruses by the overland runoff mode of domestic wastewater treatment was evaluated. Raw and primary and secondary treated wastewaters were sprayed onto grass-covered, 36-m soil plots of fine, sandy loam overlying an impermeable clay subsoil. Tracer bacteriophage f2 was seeded into the applied wastewaters, which were subsequently sampled at several points along the length of the plots. Assay of effluent samples revealed modest tracer virus removals of 30 to 60%. Data from timed experiments indicated that advancement of tracer virus to the bottom of the slopes proceeded at the same rate as wastewater, reaching the plot effluents within 50 to 90 min after application. Indigenous enteric virus levels were reduced by approximately 68 to 85% during migration down the treatment slopes. Soil sampling revealed that, although some f2 virus was found associated with the wastewater-saturated topsoil, little penetration of virus into the soil profile occurred. Laboratory soil adsorption studies revealed that poliovirus I was adsorbed much more readily than f2 virus. Comparison of virus removal characteristics during overland runoff with chemical removal characteristics of wastewater did not reveal any obvious correlations that could be used to predict virus removal.     

The rapid isolation of vacuoles from leaves of crassulacean Acid metabolism plants

A technique is presented for the isolation of vacuoles from Sedum telephium L. leaves. Leaf material is digested enzymically to produce protoplasts rapidly which are partially lysed by gentle osmotic shock and the inclusion of 5 millimolar ethyleneglycol-bis (β-aminoethyl ether)N,N′-tetraacetic acid in the wash medium. Vacuoles are isolated from the partially lysed protoplasts by brief centrifugation on a three-step Ficoll-400 gradient consisting of 5, 10, and 15% (w/v) Ficoll-400. A majority of the vacuoles accumulate at the 5 to 10% Ficoll interface, whereas a smaller proportion sediments at the 10 to 15% Ficoll-400 interface. The total time required for vacuole isolation is 2 to 2.5 hours, beginning from leaf harvest.     

Diurnal Changes in Metabolite Levels and Crassulacean Acid Metabolism in Kalanchoë daigremontiana Leaves

Diurnal changes in levels of selected metabolites associated with glycolysis, the C₃ cycle, C₄-organic acids, and storage carbohydrates were analyzed in active Kalanchoë daigremontiana Crassulacean acid metabolism leaves. Three metabolic transition periods occurred each day. During the first two hours of light, nearly all of the metabolite pools underwent transient changes. Beginning at daylight, stomata opened transiently and closed again within 30 minutes; malate synthesis continued for about 1 hour into the light; C₃ photosynthesis began within 30 minutes; and net quantities of starch and glucan began to accumulate after 2 hours, continuing linearly throughout the rest of the day.     

Fatty acid composition and physiological properties of some filamentous blue-green algae

Summary The fatty acids of 32 axenic strains of filamentous blue-green algae have been analyzed. As an aid to the interpretation of the results, the strains have been assigned to provisional typological groups based upon their morphology and certain physiological characters. The latter are the ability to grow heterotrophically in the dark with glucose as carbon and energy source, the ability to grow in the light at the expense of glucose in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and the ability to synthesize nitrogenase under anaerobic conditions in the light. Each typological group has been given an appropriate generic name.The strains examined for fatty acid composition can be divided into groups according to the major fatty acid of highest degree of unsaturation found in each strain as was done for the unicellular strains examined previously in this laboratory. Four metabolic groups of strains of unicellular and filamentous blue-green algae can be recognized: 1. those in which there is little or no desaturation of oleate; 2. those in which linoleate is desaturated toward the -end of the molecule to give -linolenate; 3. those in which linoleate is desaturated toward the carboxyl end of the molecule to give -linolenate; 4. those in which octadecatetraenoate is synthesized. The nature of the major cellular fatty acids of two of the strains examined is the same whether growth is in the light or in the dark on glucose. All filamentous strains contain glycolipids with the properties of mono- and digalactosyldiglycerides.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea