Mediator-assisted simultaneous probing of cytosolic and mitochondrial redox activity in living cells

This work describes an electron transfer mediator-assisted amperometric flow injection method for assessing redox enzyme activity in different subcellular compartments of the phosphoglucose isomerase deletion mutant strain of Saccharomyces cerevisiae, EBY44. The method is demonstrated using the ferricyanide-menadione double mediator system to study the effect of dicoumarol, an inhibitor of cytosolic and mitochondrial oxidoreductases and an uncoupler of the electron transport chain. Evaluation of the role of NAD(P)H-producing pathways in mediating biological effects is facilitated by introducing either fructose or glucose as the carbon source, yielding either NADH or NADPH through the glycolytic or pentose phosphate pathway, respectively. Respiratory noncompetent cells show greater inhibition of cytosolic menadione-reducing enzymes when NADH rather than NADPH is produced. Spectrophotometric in vitro assays show no difference between the cofactors. Respiratory competent cells show cytosolic inhibition only when NADPH is produced, whereas production of NADH reveals uncoupling at low dicoumarol concentrations and inhibition of complexes III and IV at higher concentrations. Spectrophotometric assays only indicate the presence of cytosolic inhibition regardless of the reduced cofactor used. This article shows the applicability of the amperometric method and emphasizes the significance of determining biological effects of chemicals in living cells.     

Accumulation of nitrogen and microbial residues during 2000 years of rice paddy and non‐paddy soil development in the Yangtze River Delta,China

Lowland rice paddy soils may accumulate significant amounts of organic matter. Our aim was to investigate the role of prolonged paddy management on the nitrogen (N) status of the soils, and to elucidate the contribution of bacteria and fungi to long‐term N accumulation processes. For this purpose, we sampled a chronosequence of 0–2000 years of rice cropping with adjacent non‐paddy systems in the Bay of Hangzhou, China. The samples were analyzed for bulk density, total, mineral and microbial N (N_mic), and amino sugars as markers for microbial residues. The results showed that during the first 100 years of land embankment, both paddy and non‐paddy soils accumulated N at a rate of up to 61 and 77 kg ha^?1 per annum, reaching steady‐state conditions after 110–172 years, respectively. Final N stocks in paddy fields exceeded those of the non‐paddies by a factor of 1.3. The contribution of amino sugars to total N increased to a maximum of 34 g N kg^?1 N in both land‐use systems, highlighting a significant accumulation of N in microbial residues of the surface soils. Correspondingly, the ratio of N_mic to microbial residue‐N decreased to a constant value. In the paddy subsoils, we found that bacterial residues particularly contributed to the pool of microbial residue‐N. Nevertheless, the absolute contents of amino sugars in paddy subsoils decreased during the last 1700 years of the chronosequence. We conclude that under paddy cultivation, soil microorganisms may accumulate parts of this N in their residues despite low overall N availability. However, this N accumulation is limited to initial stages of paddy soil development and restricted to the surface horizons, thus challenging its sustainability with future land‐use changes.     

Analysis of Mycoplasma hyorhinis genome by use of restriction endonucleases and by electron microscopy.

The chromosome of Mycoplasma hyorhinis was analyzed by using different restriction endonucleases and electron microscopy. It was found that restriction enzymes BstEII, XhoI, and SacI are the enzymes of choice for analysis and characterization of M. hyorhinis. The bands resulting from digestion of M. hyorhinis DNA with BstEII had apparent molecular weights ranging from 1.2 X 10(6) to 75 X 10(6). The apparent total molecular weight of DNA was calculated from the molecular weights of the individual bands and found to be 251 X 10(6). Electron microscopic contour length measurements of the largest DNA fragments verified the molecular weight values calculated from gel analysis. Electron microscopic contour length measurements of intact DNA of M. hyorhinis revealed a molecular weight of 5.4 +/- 5 X 10(8). The discrepancy between the values of molecular weight of M. hyorhinis DNA as determined by restriction enzyme analysis and contour length measurement is based on the fact that some of the DNA fragments which migrate as an apparent single band in the agarose gel really are double or multiple DNA fragments.     

Characterization of the genome of feline foamy virus and its proteins shows distinct features different from those of primate spumaviruses.

The genome of the feline foamy virus (FeFV) isolate FUV was characterized by molecular cloning and nucleotide sequence analysis of subgenomic proviral DNA. The overall genetic organization of FeFV and protein sequence comparisons of different FeFV genes with their counterparts from other known foamy viruses confirm that FeFV is a complex foamy virus. However, significant differences exist when FeFV is compared with primate foamy viruses. The FeFV Gag protein is smaller than that of the primate spumaviruses, mainly due to additional MA/CA sequences characteristic of the primate viruses only. Gag protein sequence motifs of the NC domain of primate foamy viruses assumed to be involved in genome encapsidation are not conserved in FeFV. FeFV Gag and Pol proteins were detected with monospecific antisera directed against Gag and Pol domains of the human foamy virus and with antisera from naturally infected cats. Proteolytic processing of the FeFV Gag precursor was incomplete, whereas more efficient proteolytic cleavage of the pre125Pro-Pol protein was observed. The active center of the FeFV protease contains a Gln that replaces an invariant Gly residue at this position in other retroviral proteases. Functional studies on FeFV gene expression directed by the promoter of the long terminal repeat showed that FeFV gene expression was strongly activated by the Bell/Tas transactivator protein. The FeFV Bell/Tas transactivator is about one-third smaller than its counterpart of primate spumaviruses. This difference is also reflected by a limited sequence similarity and only a moderate conservation of structural motifs of the different foamy virus transactivators analyzed.     

The Arabidopsis AtNPR1 inversely modulates defense responses against fungal, bacterial, or viral pathogens while conferring hypersensitivity to abiotic stresses in transgenic rice

The nonexpressor of pathogenesis-related (PR) genes (NPR1) protein plays an important role in mediating defense responses activated by pathogens in Arabidopsis. In rice, a disease-resistance pathway similar to the Arabidopsis NPR1-mediated signaling pathway one has been described. Here, we show that constitutive expression of the Arabidopsis NPR1 (AtNPR1) gene in rice confers resistance against fungal and bacterial pathogens. AtNPR1 exerts its protective effects against fungal pathogens by priming the expression of salicylic acid (SA)-responsive endogenous genes, such as the PR1b, TLP (PR5), PR10, and PBZ1. However, expression of AtNPR1 in rice has negative effects on viral infections. The AtNPR1-expressing rice plants showed a higher susceptibility to infection by the Rice yellow mottle virus (RYMV) which correlated well with a misregulation of RYMV-responsive genes, including expression of the SA-regulated RNA-dependent RNA polymerase 1 gene (OsRDR1). Moreover, AtNPR1 negatively regulates the expression of genes playing a role in the plant response to salt and drought stress (rab21, salT, and dip1), which results in a higher sensitivity of AtNPR1 rice to the two types of abiotic stress. These observations suggest that AtNPR1 has both positive and negative regulatory roles in mediating defense responses against biotic and abiotic stresses.     

The function of leak and kuzbanian during growth cone and cell migration

Axonal growth cones require an evolutionary conserved repulsive guidance system to ensure proper crossing of the CNS midline. In Drosophila, the Slit protein is a repulsive signal secreted by the midline glial cells. It binds to the Roundabout receptors, which are expressed on CNS axons in the longitudinal tracts but not in the commissural tracts. Here we present an analysis of the genes leak and kuzbanian and show that both genes are involved in the repulsive guidance system operating at the CNS midline. Mutations in leak, which encodes the Roundabout-2 Slit receptor, were first recovered by Nüsslein-Volhard and co-workers based on defects in the larval cuticle. Analysis of the head phenotype suggests that slit may be able to act as an attractive guidance cue while directing the movements of the dorsal ectodermal cell sheath. kuzbanian also regulates midline crossing of CNS axons. It encodes a metalloprotease of the ADAM family and genetically interacts with slit. Expression of a dominant negative Kuzbanian protein in the CNS midline cells results in an abnormal midline crossing of axons and prevents the clearance of the Roundabout receptor from commissural axons. Our analyses support a model in which Kuzbanian mediates the proteolytic activation of the Slit/Roundabout receptor complex.     

Soil organic carbon stocks in southeast Germany (Bavaria) as affected by land use,soil type and sampling depth

Precise estimations of soil organic carbon (SOC) stocks are of decided importance for the detection of C sequestration or emission potential induced by land use changes. For Germany, a comprehensive, land use–specific SOC data set has not yet been compiled. We evaluated a unique data set of 1460 soil profiles in southeast Germany in order to calculate representative SOC stocks to a depth of 1 m for the main land use types. The results showed that grassland soils stored the highest amount of SOC, with a median value of 11.8 kg m^?2, whereas considerably lower stocks of 9.8 and 9.0 kg m^?2 were found for forest and cropland soils, respectively. However, the differences between extensively used land (grassland, forest) and cropland were much lower compared with results from other studies in central European countries. The depth distribution of SOC showed that despite low SOC concentrations in A horizons of cropland soils, their stocks were not considerably lower compared with other land uses. This was due to a deepening of the topsoil compared with grassland soils. Higher grassland SOC stocks were caused by an accumulation of SOC in the B horizon which was attributable to a high proportion of C‐rich Gleysols within grassland soils. This demonstrates the relevance of pedogenetic SOC inventories instead of solely land use–based approaches. Our study indicated that cultivation‐induced SOC depletion was probably often overestimated since most studies use fixed depth increments. Moreover, the application of modelled parameters in SOC inventories is questioned because a calculation of SOC stocks using different pedotransfer functions revealed considerably biased results. We recommend SOC stocks be determined by horizon for the entire soil profile in order to estimate the impact of land use changes precisely and to evaluate C sequestration potentials more accurately.     

Synthesis of enantiomerically pure perhydro-1,4-diazepine-2,5-dione and 1,4-piperazine-2,5-dione derivatives exhibiting potent activity as apoptosis inhibitors

Apoptosis is the process of programmed cell death and plays a fundamental role in several human diseases. We have previously reported the synthesis of the perhydro-1,4-diazepine-2,5-dione and 1,4-piperazine-2,5-dione derivatives as racemic mixtures. Compounds 1 and 2 showed a potent in vitro and in cellular extracts antiapoptotic activity. In view that the chiral discrimination has been an issue in the development and use of pharmaceutical drugs, the present contribution reports the synthesis of enantiopure peptidomimetics 1 and 2. The biological evaluation of these enantiomers as apoptosis inhibitors is also reported.