Sulfite Action in Glycolytic Inhibition: In Vivo Real‐Time Observation by Hyperpolarized 13C NMR Spectroscopy

Detecting the molecular targets of xenobiotic substances in vivo poses a considerable analytical challenge. Here, we describe the use of an NMR‐based tracer methodology for the instantaneous in vivo observation of sulfur(IV) action on cellular metabolism. Specifically, we find that glycolytic flux is directed towards sulfite adducts of dihydroxyacetone phosphate and pyruvate as off‐pathway intermediates that obstruct glycolytic flux. In particular, the pyruvate–sulfite association hinders the formation of downstream metabolites. The apparent in vivo association constant of pyruvate and sulfite agrees with the apparent inhibition constant of CO₂ formation, thus supporting the importance of pyruvate interception in disturbing central metabolism and inhibiting NAD regeneration.     

New modules for PCR-based gene targeting in Candida albicans: rapid and efficient gene targeting using 100 bp of flanking homology region

The use of PCR-based techniques for directed gene alterations has become a standard tool in Saccharomyces cerevisiae. In our efforts to increase the speed of functional analysis of Candida albicans genes, we constructed a modular system of plasmid vectors and successfully applied PCR-amplified functional analysis (FA)-cassettes in the transformation of C. albicans. These cassettes facilitate: (a) gene disruptions; (b) tagging of 3'-ends of genes with green fluorescent protein (GFP); and (c) replacements of endogenous promoters to achieve regulated expression. The modules consists of a core of three selectable marker genes, CaURA3, CaHIS1 and CaARG4. Modules for C-terminal GFP-tagging were generated by adding GFP-sequences flanked at the 5'-end by a (Gly-Ala)3-linker and at the 3'-end by the S. cerevisiae URA3-terminator to these selection markers. Promoter exchange modules consist of the respective marker genes followed by the regulatable CaMAL2 or CaMET3 promoters at their 3'-ends. In order to ensure a reliably high rate of homologous gene targeting, the flanking homology regions required a size of 100 bp of gene-specific sequences, which were provided with the oligonucleotide primers. The use of shorter flanking homology regions produced unsatisfactory results with C. albicans strain BWP17. With these new modules only a minimal set of primers is required to achieve the functional analysis of C. albicans genes and, therefore, provides a basic tool to increase the number of functionally characterized C. albicans genes of this human pathogen in the near future.     

CO/sub 2/ laser processing of alumina (Al/sub 2/O/sub 3/) printed circuit board substrates

We report the results of an investigation of the laser-material interaction processes involved in laser drilling of alumina, through the use of an enhanced peak power (2.5 kW) CO/sub 2/ laser and novel temporal pulse formats. Peak power was varied from 30 W to 2 kW for pulses of constant energy to observe the effect produced on scribe depth. High-speed videography of hole formation has been combined with microscopic analysis to investigate the key processes involved in laser processing of alumina. Plasma screening was observed for short, high peak power laser pulses, and optimal scribing was achieved in the weakly plasma absorbing regime. A new processing technique for scribing alumina has been developed, which exploits the fast response of the laser to produce novel temporal pulse shapes, which can be modified to generate cleaner holes. Scribe speeds of up to 280 mm/spl middot/s/sup -1/ were obtained for scribe holes >200 /spl mu/m deep and 150 /spl mu/m apart, with no material plugging the hole, in 0.635-mm-thick 96% alumina.     

Consideration of the BDNF gene in relation to two phenotypes: Hoarding and obesity.

The gene coding for the brain derived neurotrophic factor (BDNF) has emerged as an interesting candidate for multiple brain and brain disorder-related phenomena. The primary aim of the present investigation was to consider the relationship between the BDNF Val66Met variant and two phenotypes: compulsive hoarding as a symptom dimension of obsessive–compulsive disorder (OCD), and body mass index (BMI). We examined the BDNF gene in a large (N = 301) clinical sample of probands with OCD. Participants were classified as hoarding or nonhoarding using a strict, multimeasure grouping approach. Results revealed that the Val/Val genotype was linked with hoarding classification and more severe hoarding behaviors, as well as greater BMI levels. Hoarding status was also associated with greater BMI scores, with individuals in the hoarding group being far more likely to be classified as obese compared with the nonhoarding group. Our findings may provide a distinct avenue through which hoarding and BMI could be linked. These findings are suggestive of a complex gene, body weight, and psychopathology relationship wherein a primitive, survival “thrifty gene” strategy may be conserved and represented in a subgroup of humans manifesting severe hoarding symptoms. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Parameter analysis of the IWA Anaerobic Digestion Model No. 1 for the anaerobic digestion of blackwater with kitchen refuse.

The IWA anaerobic digestion model No.1 (ADM1) had been successfully applied to the lab-scale mesophilic blackwater anaerobic digestion (BWAD) plant for cases of only blackwater (BW) feeding and of BW plus kitchen refuse (KR) feeding. In this paper, the simulation results of BW + KR anaerobic digestion are presented and discussed, followed by the analyses and discussion of the critical and important parameters as well as the performance of ADM1 based on these results. The raw BW can contain up to 30% short chain fatty acids (SCFA) which severely impact the performance of the model. The model proved that the disintegration/hydrolysis rate of BW is around 4.5 d(-1), which is about ten times higher than that of KR (Kdis,KR = 0.5 d(-1)). ADM1 is not sensitive to the distribution ratio among carbohydrates, proteins and lipids. For BWAD the C4 metabolism can be integrated in the uptake of LCFA. The uptake delay phenomenon was observed and cannot be simulated by ADM1, but it is tolerable. No unique KI,NH3,ac is found out for all investigated ammonia concentration ranges. Meanwhile, ADM1 is not sensitive to KLa and kp1 so they can easily be set up.     

p16/INK4a and p15/INK4b gene methylation and absence of p16/INK4a mRNA and protein expression in Burkitt''s lymphoma

So-called sulfur-turf microbial mats, which are macroscopic white filaments or bundles consisting of large sausage-shaped bacteria and elemental sulfur particles, occur in sulfide-containing hot springs in Japan. However, no thermophiles from sulfur-turf mats have yet been isolated as cultivable strains. This study was undertaken to determine the phylogenetic positions of the sausage-shaped bacteria in sulfur-turf mats by direct cloning and sequencing of 16S rRNA genes amplified from the bulk DNAs of the mats. Common clones with 16S rDNA sequences with similarity levels of 94.8 to 99% were isolated from sulfur-turf mat samples from two geographically remote hot springs. Phylogenetic analysis showed that the phylotypes of the common clones formed a major cluster with members of the Aquifex-Hydrogenobacter complex, which represents the most deeply branching lineage of the domain bacteria. Furthermore, the bacteria of the sulfur-turf mat phylotypes formed a clade distinguishable from that of other members of the Aquifex-Hydrogenobacter complex at the order or subclass level. In situ hybridization with clone-specific probes for 16S rRNA revealed that the common phylotype of sulfur-turf mat bacteria is that of the predominant sausage-shaped bacteria.     

Water fluxes and diffuse nitrate pollution at river basin scale: coupling of agro-economic models and hydrological approaches.

An integrated model system has been developed to estimate the impact of nitrogen reduction measures on the nitrogen load in groundwater and in river catchment areas. The focus lies on an area-wide, regionally differentiated, consistent link-up between the indicator "nitrogen balance surplus" and nitrogen charges into surface waters. As a starting point of the analysis actual nitrogen surpluses in the soil were quantified using the agro-economic RAUMIS-model, which considers the most important N-inputs to the soil and N-removals from the soil through crop harvest. The most important pathways for diffuse nitrogen inputs into river systems are modelled with the water balance model GROWA. Additionally, the time-dependent nitrogen degradation along the nitrogen pathways in soil and groundwater are modelled using the WEKU-model. The two selected river basins in Germany cover a variety of landscape units with different hydrological, hydrogeological and socio-economic characteristics. The results indicate a wide range of annual nitrogen surpluses for the rural areas between than 10 kg N ha(-1) x a(-1) and 200 kg N ha(-1) x a(-1) or more, depending on the type and intensity of farming. The level of nitrogen inputs into the surface waters is reduced because of degradation processes during transport in soil and groundwater. Policy impact analyses for a nitrogen tax and a limitation of the livestock density stress the importance of regionally adjusted measures.