Dielectric cytometry with three-dimensional cellular modeling

We have developed what we believe is an efficient method to determine the electric parameters (the specific membrane capacitance C(m) and the cytoplasm conductivity kappa(i)) of cells from their dielectric dispersion. First, a limited number of dispersion curves are numerically calculated for a three-dimensional cell model by changing C(m) and kappa(i), and their amplitudes Deltaepsilon and relaxation times tau are determined by assuming a Cole-Cole function. Second, regression formulas are obtained from the values of Deltaepsilon and tau and then used for the determination of C(m) and kappa(i) from the experimental Deltaepsilon and tau. This method was applied to the dielectric dispersion measured for rabbit erythrocytes (discocytes and echinocytes) and human erythrocytes (normocytes), and provided reasonable C(m) and kappa(i) of the erythrocytes and excellent agreement between the theoretical and experimental dispersion curves.     

Asymmetric synthesis of arylselenoalcohols by means of the reduction of organoseleno acetophenones by whole fungal cells

A series of novel organoseleno acetophenones (3a–f) have been synthesized. The microbial reduction of the seleno ketones (3) has been evaluated using whole cells of Rhizopus oryzae CCT 4964, Aspergillus terreus CCT 3320, A. terreus CCT 4083 and Emericella nidulans CCT 3119. These microorganisms showed Prelog and anti-Prelog stereoselectivity, leading to the arylselenoalcohols in moderate to high enantiomeric excesses. The organoselenium compounds were compatible with the biocatalytic conditions employed.     

Evidence for the presence of DNA-binding proteins involved in regulation of the gene expression of indole-3-pyruvic acid decarboxylase, a key enzyme in indole-3-acetic acid biosynthesis in Azospirillum lipoferum FS.

We isolated the ipdc gene coding for indole-3-pyruvic acid decarboxylase (IPDC), a key enzyme in the indole-3-pyruvic acid pathway for indole-3-acetic acid biosynthesis, in the plant growth-promoting rhizobacterium Azospirillum lipoferum FS. Gel mobility-shift assay showed the presence of two DNA-binding proteins that might be involved in regulation of the ipdc gene expression.     

Bacterial and spontaneous dehalogenation of organic compounds

Only 3 of more than 500 soil enrichments contained organisms able to use 1,9-dichlorononane as a sole carbon source. One isolate, a strain of Pseudomonas, grew on the compound and released much of the halogen as chloride. Resting cells dehalogenated 1,9-dichlorononane aerobically but not anaerobically. Pseudomonas sp. grew on and resting cells dehalogenated 1,6-dichlorohexane, 1,5-dichloroheptane, 2-bromoheptanoate, and 1-chloro-, 1-bromo-, and 1-iodoheptane, but the bacterium cometabolized but did not grow on 3-chloropropionate. p-Methylbenzyl alcohol, chloride, and p-methylbenzoate were formed when resting cells were incubated with alpha-chloro-p-xylene; the first two products were also formed in the absence of the bacteria. Similarly, o- and m-methylbenzyl alcohols were generated from the corresponding chlorinated xylenes in the presence or absence of Pseudomonas sp. The formation of m- and p-chlorobenzoic acid from m- and p-chlorobenzyl chloride proceeded only in the presence of the cells, but p-chlorobenzyl alcohol was generated from p-chlorobenzyl chloride even in the absence of the bacterium. These results are discussed in terms of possible mechanisms of dehalogenation.     

TAK1 kinase switches cell fate from apoptosis to necrosis following TNF stimulation

TNF activates three distinct intracellular signaling cascades leading to cell survival, caspase-8–mediated apoptosis, or receptor interacting protein kinase 3 (RIPK3)–dependent necrosis, also called necroptosis. Depending on the cellular context, one of these pathways is activated upon TNF challenge. When caspase-8 is activated, it drives the apoptosis cascade and blocks RIPK3-dependent necrosis. Here we report the biological event switching to activate necrosis over apoptosis. TAK1 kinase is normally transiently activated upon TNF stimulation. We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation. In addition, we also demonstrated that activation of RIPK1 and RIPK3 promoted TAK1 activation, suggesting a positive feedforward loop of RIPK1, RIPK3, and TAK1. Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro. Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis. TAK1 acts as a switch between apoptosis and necrosis.     

Replication of G4 progeny double-stranded DNA in dna mutants of Escherichia coli.

Host functions required for replication of progeny double-stranded DNA of bacteriophage G4 were examined by using metabolic inhibitors and Escherichia coli dna mutants. In dna+ bacteria, synthesis of the progeny replicative form (RF) was relatively resistant to 30 microgram/ml of chloramphenicol, but considerably sensitive to 200 microgram/ml of rifampicin. The RF replication was severely inhibited by 50 microgram/ml of mitomycin C, 50 microgram/ml of nalidixic acid, or 200 microgram/ml of novobiocin. At 41 degrees C, synthesis of G4 progeny RF was distinctly affected in a dnaC(D) mutant and in a dnaG host. The progeny RF replication was prevented at 42 degrees C in a dnaE strain as well as in a dnaB mutant. In a dnaZ strain, the synthetic rate of the progeny RF was markedly reduced at 42 degrees C. At 43 degrees C, the rate of G4 progeny RF synthesis was reduced even in dna+ or dnaA bacteria, but significant amounts of the progeny RF were still synthesized in these hosts at the high temperature. In addition to five dna gene products, host rep function was essential for the RF replication.     

Coexistence of different pathways in the metabolism of n-propylbenzene by Pseudomonas sp.

Pseudomonas desmolytica S449B1 and Pseudomonas convexa S107B1 grown on n-propylbenzene oxidized n-propylbenzene to beta-phenylpropionic acid and benzoic acid by initial oxidation of the n-propyl side chain and the following beta-oxidation, respectively. The same strains also oxidized n-propylbenzene to 3-n-propylcatechol by initial oxidation of positions 2 and 3 of the aromatic nucleus. A ring fission product, 2-hydroxy-6-oxononanoic acid, was also isolated from the culture broth. Together with the results of oxygen uptake experiments, the data obtained suggested not only the existence of a reductive step to form 2-hydroxy-6-oxononanoic acid, but also the coexistence of two different pathways in the metabolism of n-propylbenzene by the strains used.     

Phospholipid-Derived Fatty Acids and Quinones as Markers for Bacterial Biomass and Community Structure in Marine Sediments

Phospholipid-derived fatty acids (PLFA) and respiratory quinones (RQ) are microbial compounds that have been utilized as biomarkers to quantify bacterial biomass and to characterize microbial community structure in sediments, waters, and soils. While PLFAs have been widely used as quantitative bacterial biomarkers in marine sediments, applications of quinone analysis in marine sediments are very limited. In this study, we investigated the relation between both groups of bacterial biomarkers in a broad range of marine sediments from the intertidal zone to the deep sea. We found a good log-log correlation between concentrations of bacterial PLFA and RQ over several orders of magnitude. This relationship is probably due to metabolic variation in quinone concentrations in bacterial cells in different environments, whereas PLFA concentrations are relatively stable under different conditions. We also found a good agreement in the community structure classifications based on the bacterial PLFAs and RQs. These results strengthen the application of both compounds as quantitative bacterial biomarkers. Moreover, the bacterial PLFA- and RQ profiles revealed a comparable dissimilarity pattern of the sampled sediments, but with a higher level of dissimilarity for the RQs. This means that the quinone method has a higher resolution for resolving differences in bacterial community composition. Combining PLFA and quinone analysis as a complementary method is a good strategy to yield higher resolving power in bacterial community structure.