Production of a specific antibody against pyruvate kinase type M2 using a synthetic peptide

The pyruvate kinase isozymes M1 and M2 are structurally and immunologically closely related. To obtain an antibody which discriminates between these two forms, a synthetic tetradecapeptide with a sequence specific for pyruvate kinase type M2 from rats was constructed. Antisera from rabbits, immunized with this peptide, reacted specifically with the M2-type holoenzyme of both rat and human origin, and did not cross-react with the M1-type isozyme. This was established by immunoblot analysis, both under dissociating and non-dissociating conditions.     

The effect of quin2 on chemotaxis by polymorphonuclear leukocytes

Exposure of rabbit polymorphonuclear leukocytes to micromolar concentrations of quin2-AM results in high intracellular concentrations of quin2, which lead to inhibition of chemotaxis. The loading efficiency of polymorphonuclear leukocytes, being the percentage of quin2-AM which is taken up by the cells and transformed intracellularly into quin2, is very high, reaches a maximum after 30 min, is independent of the presence of extracellular Ca2+ and is fairly independent of cell concentration. As a consequence, inhibition of chemotaxis is strongly dependent on experimental conditions: with a low cell density (3 X 10(6)/ml) exposure to 20 microM quin2-AM results in complete inhibition of chemotaxis, whereas the same concentration of quin2-AM is nearly without effect when an 8-fold higher cell concentration is used. Inhibition by quin2 is dependent on extracellular Ca2+; inhibition is more pronounced in the absence of extracellular Ca2+ than in its presence. It is suggested that quin2 inhibits chemotaxis by interference with intracellular Ca2+.     

Energy transduction by electron transfer via a pyrrolo-quinoline quinone-dependent glucose dehydrogenase in Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus (var. lwoffi).

The coupling of membrane-bound glucose dehydrogenase (EC 1.1.99.17) to the respiratory chain has been studied in whole cells, cell-free extracts, and membrane vesicles of gram-negative bacteria. Several Escherichia coli strains synthesized glucose dehydrogenase apoenzyme which could be activated by the prosthetic group pyrrolo-quinoline quinone. The synthesis of the glucose dehydrogenase apoenzyme was independent of the presence of glucose in the growth medium. Membrane vesicles of E. coli, grown on glucose or succinate, oxidized glucose to gluconate in the presence of pyrrolo-quinoline quinone. This oxidation led to the generation of a proton motive force which supplied the driving force for uptake of lactose, alanine, and glutamate. Reconstitution of glucose dehydrogenase with limiting amounts of pyrrolo-quinoline quinone allowed manipulation of the rate of electron transfer in membrane vesicles and whole cells. At saturating levels of pyrrolo-quinoline quinone, glucose was the most effective electron donor in E. coli, and glucose oxidation supported secondary transport at even higher rates than oxidation of reduced phenazine methosulfate. Apoenzyme of pyrrolo-quinoline quinone-dependent glucose dehydrogenases with similar properties as the E. coli enzyme were found in Acinetobacter calcoaceticus (var. lwoffi) grown aerobically on acetate and in Pseudomonas aeruginosa grown anaerobically on glucose and nitrate.     

Crystal-induced membrane damage: hydroxyapatite crystal-induced hemolysis of erythrocytes

Microcrystals of hydroxyapatite cause severe membrane damage in human erythrocytes, as is evident from the strong hemolysis that is caused by these crystals. Hemolysis by hydroxyapatite crystals is time and concentration dependent, and is preceded by aggregation of erythrocytes. Polyvinylpyridine-N-oxide, a strong hydrogen acceptor, has no inhibiting effect on hydroxyapatite-induced hemolysis. This suggest that the mechanism of action of these crystals is different from that of urate crystals and silica particles, where hydrogen bonding interaction is supposed to be important. Negatively charged macromolecules, such as dextran sulfate, heparin, and polyglutamic acid, inhibit hydroxyapatite crystal-induced hemolysis, suggesting that positive charges, probably located on the crystals, play an important role in the membrane-damaging effect of these crystals. The structures with which these positive charges interact remain to be determined because removal of negative charges from the erythrocytes by treatment with neuraminidase does not affect crystal-induced hemolysis.     

Transformation of the extremely thermoacidophilic archaeon Sulfolobus solfataricus via a self-spreading vector

Abstract The comparative chromosomal locations of polymeric β-fructosidase SUC genes have been determined by Southern blot hybridization with the SUC2 probe in 91 different strains of Saccharomyces cerevisiae . Most of the strains exhibited a single SUC2 gene, but in some strains two or three SUC genes were found. All Suc⁻ strains carried a silent suc2⁰ sequence. The accumulation of SUC genes was observed in populations derived from sources containing sucrose and seems to be absent in strains from sources promoting the MEL gene.     

Sulfate reduction in methanogenic bioreactors

Abstract: In the anaerobic treatment of sulfate-containing wastewater, sulfate reduction interferes with methanogenesis. Both mutualistic and competitive interactions between sulfate-reducing bacteria and methanogenic bacteria have been observed. Sulfate reducers will compete with methanogens for the common substrates hydrogen, formate and acetate. In general, sulfate reducers have better growth kinetic properties than methanogens, but additional factors which may be of importance in the competition are adherence properties, mixed substrate utilization, affinity for sulfate of sulfate reducers, relative numbers of bacteria, and reactor conditions such as pH, temperature and sulfide concentration. Sulfate reducers also compete with syntrophic methanogenic consortia involved in the degradation of substrates like propionate and butyrate. In the absence of sulfate these methanogenic consortia are very important, but in the presence of sulfate they are thought to be easily outcompeted by sulfate reducers. However, at relatively low sulfate concentrations, syntrophic degradation of propionate and butyrate coupled to HZ removal via sulfate reduction rather than via methanogenesis may become important. A remarkable feature of some sulfate reducers is their ability to grow fermentatively or to grow in syntrophic association with methanogens in the absence of sulfate.