Partial purification of ferredoxin from Ruminococcus albus and its role in pyruvate metabolism and reduction of nicotinamide adenine dinucleotide by H2.

Extracts of Ruminococcus albus were not able to convert pyruvate to acetyl phosphate, CO2, and H2 after passage through a diethylaminoethyl (DEAE)-cellulose column. Activity was restored by a brown protein fraction eluted from the column with 0.4 M Cl-. The protein was partially purified and shown to have the spectral and biological characteristics of ferredoxin. R. albus ferredoxin, Clostridium pasteurianum ferredoxin, and methyl viologen restored activity for pyruvate decomposition by DEAE-cellulose-treated R. albus extracts. R. albus or C. pasteurianum ferredoxin restored the ability of DEAE-cellulose-treated C. pasteurianum extracts to form H2 and acetyl phosphate from pyruvate. Ferredoxin-free extracts of R. albus reduced nicotinamide adenine dinucleotide (NAD) when supplemented with R. albus or C. pasteurianum ferredoxin or with methyl viologen. These extracts reduced NADP with H2 poorly unless both ferredoxin and NAD were added, which indicates the presence of an NADH:NADP transhydrogenase. Flavin mononucleotide and flavin adenine dinucleotide were rapidly reduced by H2 by ferredoxin-free extracts in the absence of ferredoxin.     

Light scattering and excitation in lobster giant axon Effects of ion substitution

Changes in the light scattering signal from single giant axons of lobster were observed during the propagation of the action potential in order to correlate membrane excitability with possible structural changes reflected in the optical properties of the axolemma. Substitution of guanidine and aminoguanidine for sodium resulted in a decreased action potential amplitude to 69 and 50% of control values, respectively. The amplitude of the light signal was, however, not significantly changed by these substitutions and is, therefore, reported to be independent of the transmembrane potential and current. The venom of the scorpion Leiurus quinquestriatus caused a marked prolongation of the action potential and the light scattering signal without significantly altering their amplitudes. A two-state model of the early (sodium) activation channel is suggested, in which the light scattering signal is correlated with a possible difference in the scattering efficiency between the states of the channel.     

Phosphoenolpyruvate carboxykinase from Fasciola hepatica

Behm C. A. and Bryant C. 1982. Phosphoenolpyruvate carboxykinase from Fasciola hepatica. International Journal for Parasitology12: 271–278. The kinetic properties of a partially purified preparation of phosphoenolpyruvate carboxykinase (PEPCK) from F. hepatica were examined. The pH optimum for the carboxylation reaction is 5.8–6.2. The enzyme is more active with Mn²⁺ than Mg²⁺ and the Mn²⁺ saturation curve was sigmoid. Apparent K_m values for the substrates GDP, IDP, PEP and HCO₃^? were determined and found to be in the same range as those reported for other helminths except that the enzyme is less sensitive to low PEP concentrations. GTP and ATP at 0.5 and 1.0 mM inhibit the enzyme; the GTP inhibition was greater in the presence of Mg²⁺ than Mn²⁺ and was competitive with GDP. It was concluded that the activity of PEPCK from F. hepatica is controlled by the concentration of reactants and the ambient pH, that the accumulation of GTP is a sensitive mechanism for inhibiting the carboxylation reaction and that PEPCK activity in the cytosol is likely to be favoured over that of pyruvate kinase except when pH is high and PEP concentration low.     

The use of cell lethal mutations in the study of Drosophila development

Cell-lethal mutations are currently being used to study the role of cell death in the development of normal and mutant phenotypes, and in highly detailed studies of pattern formation. Histological studies of certain “position-specific” cell-lethals suggest that programmed cell death may be important in the development of normal imaginal discs, and that many mutations may be producing an abnormal phenotype by amplifying this normal process. Somatic mosaic studies using temperature-sensitive cell-lethals have provided much information about the development of pattern duplications, including the number of cells involved, their growth rate, and compartmentalization during duplication. Further work in this area may, when combined with genetic analyses of the loci involved, reveal much about the nature of the processes controlling pattern determination and differentiation.     

Systematic growth studies,cocultivation, and cell hybridization studies of Werner syndrome cultured skin fibroblasts

Summary The growth of 20 independently derived skin fibroblastlike (FL) cell strains from three individuals with Werner syndrome (adult progeria) was compared with the growth of ten FL cell strains from normal individuals. Population growth rates and total replicative life spans of Werner syndrome strains averaged 55% and 27%, respectively, of the growth rates and life spans of non-Werner strains. In the first few passages, four Werner syndrome strains demonstrated population growth rates in the low normal range, but the longest-lived Werner syndrome strain had only 75% of the total replicative potential of the shortest-lived normal strain. Exponential growth rates, cloning efficiencies, and saturation densities of Werner strains were also reduced, whereas cell attachment was normal. Viable cells (identified by dye exclusion) were maintained in post-replicative Werner syndrome and control cultures for periods of at least 10 months; there was no evidence of accelerated post-replicative senescence or cell death of Werner syndrome FL cells. Cocultivation of Werner syndrome and normal strains did not influence population growth rates of either strain. Two proliferating hybrid clones were obtained from fusions of normal and Werner syndrome FL cell strains and these hybrids displayed the reduced growth potential typical of Werner syndrome FL cells. These studies confirm that low growth rate and sharply reduced replicative life span are characteristic of cultured skin FL cells from patients with Werner syndrome, and they suggest that these characteristics are not affected by complementation with non-Werner FL cells.     

Syntrophic Association by Cocultures of the Methanol- and CO(2)-H(2)-Utilizing Species Eubacterium limosum and Pectin-Fermenting Lachnospira multiparus During Growth in a Pectin Medium

Lachnospira multiparus grew very well in an anaerobic 0.2% pectin medium, whereas Eubacterium limosum, which utilizes methanol, H(2)-CO(2), and lactate, did not. Cocultures of the two species grew at a somewhat more rapid growth rate than did L. multiparus alone and almost doubled the amount of growth as measured by optical density. In model experiments with cultures transferred once a day with a 2-day retention time, L. multiparus produced mainly acetate, methanol, ethanol, formate, lactate, CO(2), and H(2) from pectin. The coculture produced one-third more acetate, and butyrate and CO(2) were the only other significant end products. The results are discussed in relationship to microbial metabolic interactions and interspecies hydrogen transfer.     

Thermophilic methane production from cattle waste

Methane production from waste of cattle fed a finishing diet was investigated, using four 3-liter-working volume anaerobic digestors at 60 degrees C. At 55 degrees C a start-up culture, in which waste was the only source of bacteria, was generated within 8 days and readily adapted to 60 degrees C, where efficiency of methanogenesis was greater. Increasing the temperature from 60 to 65 degrees C tended to drastically lower efficiency. When feed concentrations of volatile solids (VS, organic matter) were increased in steps of 2% after holding for 1 months at a given concentration, the maximum concentrations for efficient fermentation were 8.2, 10.0, 11.6, and 11.6% for the retention times (RT) of 3, 6, 9, and 12 days, respectively. The VS destructions for these and lower feed concentrations were 31 to 37, 36 to 40, 47 to 49 and 51 to 53% for the 3-, 6-, 9-, and 12-day RT digestors, respectively, and the corresponding methane production rates were about 0.16, 0.18, 0.20, and 0.22 liters/day per g of VS in the feed. Gas contained 52 to 57% methane. At the above RT and feed concentrations, alkalinity rose to 5,000 to 7,700 mg of CaCo3 per liter (pH to 7.5 to 7.8), NH3 plus NH4+ to 64 to 90 mM, and total volatile acids to 850 to 2,050 mg/liter as acetate. The 3-day RT digestor was quite stable up to 8.2% feed VS and at this feed concentration produced methane at the very high rate of 4.5 liters/day per liter of digestor. Increasing the percentage of feed VS beyond those values indicated above resulted in greatly decreased organic matter destruction and methane production, variable decrease in pH, and increased alkalinity, ammonia, and total volatile acid concentrations, with propionate being the first to accumulate in large amounts. In a second experiment with another lot of waste, the results were similar. These studies indicate that loading rates can be much higher than those previously thought useful for maximizing methanogenesis from cattle waste.     

Nuclear magnetic resonance relaxation in cross-linked lysozyme crystals: an isotope dilution experiment.

Nuclear magnetic resonance (nmr) relaxation times are measured for water protons in cross-linked lysozyme crystals below the freezing event as a function of the mole fraction of protons in the water phase. Proton longitudinal nmr relaxation in these samples is nonexponential and the slow longitudinal relaxation component becomes slower linearly with decreasing proton mole fraction in the water. The data are analyzed using a cross relaxation model that eliminates the necessity of postulating long residence times for water molecules in the domain of the protein. The observed isotope dilution behavior is consistent with the cross relaxation model. The deuterium nmr relaxation is also reported for deuterium oxide in the cross-linked protein crystal sample below the freezing event and the relaxation is shown to be accurately exponential.