Reduced pyridine nucleotides in Pseudomonas carboxydovorans are formed by reverse electron transfer linked to proton motive force

In cell suspensions of Pseudomonas carboxydovorans pulsed with lithotrophic substrates (CO or H₂) in the presence of oxygen, formation of reduced pyridine nucleotides and of ATP could be demonstrated using the bioluminescent assay. Experiments employing base-acid transition, an uncoupler and inhibitors of ATPase or electron transport enabled us to propose a model for the formation of NAD(P)H in chemolithotrophically growing P. carboxydovorans.The protonophor FCCP (carbonly-p-trifluormethoxyphenylhydrazon) inhibited both, formation of NAD(P)H and of ATP. In the absence of oxygen, a chemical potential imposed by base-acid transition resulted in the formation of NAD(P)H and ATP when electrogenic substrates (CO or H₂) were present. This suggests proton motive force-driven NAD(P)H formation. The proton motive force was generated by oxidation of substrate, and not by ATP hydrolysis, as obvious from NAD(P)H formation during inhibition of ATP synthesis by oligomycin and N,N-dicyclohexylcarbodiimide.That the CO-born electrons are transferred via the ubiquinone 10-cytochrome b region to NADH dehydrogenase functioning in the reverse direction, was indicated by inhibition of NAD(P)H formation by HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) and rotenone, and by resistance to antimycin A.We conclude that in P. carboxydovorans, growing with CO or H₂, electrons and a proton motive force, generated by respiration, are required to drive an reverse electron transfer for the formation of reduced pyridine nucleotides.Abbreviations CODH carbon monoxide dehydrogenase - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl-p-trifluormethoxyphenylhydrazon - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - pmf proton motive force     

A novel approach to the analysis of the initiation of embryo development in gramineae

An in vivo model system to study the initiation of embryo development is presented. From the so-called Salmon system of wheat (alloplasmic lines with a 1BL-1RS chromosome translocation), three completely isogenic and homozygous lines were produced by selection for uniformity in about 20 selfing/backcross generations as well as between sublines of doubled haploids. The line (aestivum)-Salmon is male fertile and sexual. The lines (caudata)-Salmon and (kotschyi)-Salmon are male sterile and have a parthenogenetic capacity of about 90%. The expression of nuclear-cytoplasmic male sterility is different for the two parthenogenetic lines. The initiation of autonomous embryo development at defined developmental stages of the ovaries and the maximum degree of parthenogenesis are identical in both parthenogenetic lines as proved by the auxin test and progeny analyses. The protein patterns from ovary extracts of the three isogenic lines were identical for more than 200 spots of 2-D polyacrylamide gels, confirming their homogeneity. However, one protein (P 115.1) was found 3 days before and during anthesis only in ovaries of the parthenogenetic lines. It seems to be involved in the initiation of parthenogenesis.     

Interaction of the Neurospora crassa heat shock factor with the heat shock element during heat shock and different developmental stages

The interaction of the heat shock factor (HSF) with the heat shock element (HSE) was determined by a non-radioactive electrophoretic mobility shift assay, in order to analyze HSF regulation in Neurospora crassa. HSF binds to HSE under normal, non-stress conditions and is thus constitutively trimerized. Upon heat shock, the HSF-HSE complex shows a retarded mobility. This was also observed in Saccharomyces cerevisiae, where this mobility shift was shown to be due to HSF phosphorylation [Sorger and Pelham (1988) Cell 54, 855-864]. In N. crassa, HSE-dependent electrophoretic mobility shift is temperature- and time-dependent. Under normal growth conditions, the HSF is located in the cytoplasm as well as in the nucleus. In germinating conidia the HSF shows a retarded mobility typical for heat shock even at normal growth temperatures. No HSF-dependent mobility shift was detectable in aerial hyphae.     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: I. Na+/H+, Cl−/HCO 3 − double exchange and Na+−Cl− symport

Summary Cl^– influx at the luminal border of the epithelium of rabbit gallbladder was measured by 45-sec exposures to³⁶Cl^– and³H-sucrose (as extracellular marker). Its paracellular component was evaluated by the use of 25mm SCN^– which immediately and completely inhibits Cl^– entry into the cell. Cellular influx was equal to 16.7eq cm^–2 hr^–1 and decreased to 8.5eq cm^–2 hr^–1 upon removal of HCO ₃ ^– from the bathing media and by bubbling 100% O₂ for 45 min. When HCO ₃ ^– was present, cellular influx was again about halved by the action of 10^–4 m acetazolamide, 10^–5 to 10^–4 m furosemide, 10^–5 to 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS), 10^–3 m amiloride. The effects of furosemide and SITS were tested at different concentrations of the inhibitor and with different exposure times: they were maximal at the concentrations reported above and nonadditive. In turn, the effects of amiloride and SITS were not additive. Acetazolamide reached its maximal action after an exposure of about 2 min. When exogenous HCO ₃ ^– was absent, the residual cellular influx was insensitive to acetazolamide, furosemide and SITS. When exogenous HCO ₃ ^– was present in the salines, Na⁺ removal from the mucosal side caused a slow decline of cellular Cl^– influx; conversely, it immediately abolished cellular Cl^– influx in the absence of HCO ₃ ^– . In conclusion, about 50% of cellular influx is sensitive to HCO ₃ ^– , inhibitable by SCN^–, acetazolamide, furosemide, SITS and amiloride and furthermore slowly dependent on Na⁺. The residual cellular influx is insensitive to bicarbonate, inhibitable by SCN^–, resistant to acetazolamide, furosemide, SITS and amiloride, and immediately dependent on Na⁺. Thus, about 50% of apical membrane NaCl influx appears to result from a Na⁺/H⁺ and Cl^–/HCO ₃ ^– exchange, whereas the residual influx seems to be due to Na⁺–Cl^– contranport on a single carrier. Whether both components are simultaneously present or the latter represents a cellular homeostatic counterreaction to the inhibition of the former is not clear.     

Genetic population structure as indirect measure of dispersal ability in a Lake Tanganyika cichlid

Diversification and speciation processes are influenced by intrinsic (ecological specialization, dispersal) and extrinsic (habitat structure and instability) factors, but the effect of ecological characteristics on dispersal is difficult to assess. This study uses mitochondrial control region sequences to investigate the population structure and demographic history of the endemic Lake Tanganyika cichlid Neolamprologus caudopunctatus with a preference for the rock-sand interface along two stretches of continuous, rocky shoreline, and across a sandy bay representing a potential dispersal barrier. Populations along uninterrupted habitat were not differentiated; whereas, the sandy bay separated two reciprocally monophyletic clades. The split between the two clades between 170,000 and 260,000 years BP coincides with a period of rising water level following a major lowstand, and indicates that clades remained isolated throughout subsequent lake level fluctuations. Low long-term effective population sizes were inferred from modest genetic diversity estimates, and may be due to recent population expansions starting from small population sizes 45,000–60,000 years BP. Comparisons with available data from specialized rock-dwelling species of the␣same area suggest that habitat structure and lake level fluctuations determine phylogeographic patterns on large scales, while fine-scale population structure and demography are modulated by species-specific ecologies.     

A Jungle in There: Bacteria in Belly Buttons are Highly Diverse,but Predictable

The belly button is one of the habitats closest to us, and yet it remains relatively unexplored. We analyzed bacteria and arachaea from the belly buttons of humans from two different populations sampled within a nation-wide citizen science project. We examined bacterial and archaeal phylotypes present and their diversity using multiplex pyrosequencing of 16S rDNA libraries. We then tested the oligarchy hypothesis borrowed from tropical macroecology, namely that the frequency of phylotypes in one sample of humans predicts its frequency in another independent sample. We also tested the predictions that frequent phylotypes (the oligarchs) tend to be common when present, and tend to be more phylogenetically clustered than rare phylotypes. Once rarefied to four hundred reads per sample, bacterial communities from belly buttons proved to be at least as diverse as communities known from other skin studies (on average 67 bacterial phylotypes per belly button). However, the belly button communities were strongly dominated by a few taxa: only 6 phylotypes occurred on >80% humans. While these frequent bacterial phylotypes (the archaea were all rare) are a tiny part of the total diversity of bacteria in human navels (<0.3% of phylotypes), they constitute a major portion of individual reads (∼1/3), and are predictable among independent samples of humans, in terms of both the occurrence and evolutionary relatedness (more closely related than randomly drawn equal sets of phylotypes). Thus, the hypothesis that “oligarchs” dominate diverse assemblages appears to be supported by human-associated bacteria. Although it remains difficult to predict which species of bacteria might be found on a particular human, predicting which species are most frequent (or rare) seems more straightforward, at least for those species living in belly buttons.     

Transposition of DNA inserted into deletions of the Tn5 kanamycin resistance element

Summary Tn5-trp hybrid transposons have been constructed by insertion of a trpPOED Hind III fragment into an in vivo Tn5 internal deletion mutant or by substitution of trp for the internal Tn5 Hind III fragment. These hybrids are called, respectively, Tn409 and Tn410. Both Tn409 and Tn410 will transpose into in the presence of a complementing Tn5 element. In the absence of a wild Tn5, lysogens carrying R1162::Tn409 and R1162::Tn410 plasmids will yield trp phages at less than six per cent of the complemented frequency. This reduction indicates that Tn409 and Tn410 lack a diffusible transposition function provided by wild Tn5 elements. However, the formation of trp phages without complementation is real. Most of these transducing particles contain Tn409 and Tn410 still linked to the carrier R1162 plasmid. This observation suggests that uncomplemented Tn409 and Tn410 elements mediate the formation of -transposon-plasmid cointegrate structures. Thus, the missing transposition function may be involved in resolving these cointegrate structures to the final ::Tn409 or ::Tn410 product.Abbreviations p.f.u. plaque-forming units - MIC minimal inhibitory concentration - LFT low frequency transducing - HFT high frequency transducing     

SAR studies on dihydropyrimidinone antibiotics

There is an urgent need for the development of novel antimicrobial agents that offer effective treatment against MRSA. Using a new class of dipeptide antibiotic TAN-1057A/B as lead, we designed, synthesized and evaluated analogs of TAN-1057A/B. Several novel dihydropyrimidinone antibiotics demonstrating comparable antibiotic efficacy while possessing favorable selectivity were identified.     

Ueber einen Papuanischen Kasuar im Dresdner Museum

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