Genetic engineering of brewing yeast to reduce the content of ethanol in beer

The GPD1 gene encoding the glycerol-3-phosphate dehydrogenase was overexpressed in an industrial lager brewing yeast (Saccharomyces cerevisiae ssp. carlsbergensis) to reduce the content of ethanol in beer. The amount of glycerol produced by the GPD1-overexpressing yeast in fermentation experiments simulating brewing conditions was increased 5.6 times and ethanol was decreased by 18% when compared to the wild-type. Overexpression of GPD1 does not affect the consumption of wort sugars. Only minor changes in the concentration of higher alcohols, esters and fatty acids could be observed in beer produced by the GPD1-overexpressing brewing yeast. However, the concentrations of several other by-products, particularly acetoin, diacetyl and acetaldehyde, were considerably increased.     

Receptor Binding Sites and Antigenic Epitopes on the Fiber Knob of Human Adenovirus Serotype 3

The adenovirus fiber knob causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s), the interaction of labeled cell membrane proteins to synthetic peptides covering the adenovirus type 3 (Ad3) fiber knob was studied. Peptide P6 (amino acids [aa] 187 to 200), to a lesser extent P14 (aa 281 to 294), and probably P11 (aa 244 to 256) interacted specifically with cell membrane proteins, indicating that these peptides present cell receptor binding sites. Peptides P6, P11, and P14 span the D, G, and I β-strands of the R-sheet, respectively. The other reactive peptides, P2 (aa 142 to 156), P3 (aa 153 to 167), and P16 (aa 300 to 319), probably do not present real receptor binding sites. The binding to these six peptides was inhibited by Ad3 virion and was independent of divalent cations. We have also screened the antigenic epitopes on the knob with recombinant Ad3 fiber, recombinant Ad3 fiber knob, and Ad3 virion-specific antisera by enzyme-linked immunosorbent assay. The main antigenic epitopes were presented by P3, P6, P12 (aa 254 to 269), P14, and especially the C-terminal P16. Peptides P14 and P16 of the Ad3 fiber knob were able to inhibit Ad3 infection of cells.     

Mutations of the mitochondrial holocytochrome c-type synthase in X-linked dominant microphthalmia with linear skin defects syndrome

The microphthalmia with linear skin defects syndrome (MLS, or MIDAS) is an X-linked dominant male-lethal disorder almost invariably associated with segmental monosomy of the Xp22 region. In two female patients, from two families, with MLS and a normal karyotype, we identified heterozygous de novo point mutations--a missense mutation (p.R217C) and a nonsense mutation (p.R197X)--in the HCCS gene. HCCS encodes the mitochondrial holocytochrome c-type synthase that functions as heme lyase by covalently adding the prosthetic heme group to both apocytochrome c and c(1). We investigated a third family, displaying phenotypic variability, in which the mother and two of her daughters carry an 8.6-kb submicroscopic deletion encompassing part of the HCCS gene. Functional analysis demonstrates that both mutant proteins (R217C and Delta 197-268) were unable to complement a Saccharomyces cerevisiae mutant deficient for the HCCS orthologue Cyc3p, in contrast to wild-type HCCS. Moreover, ectopically expressed HCCS wild-type and the R217C mutant protein are targeted to mitochondria in CHO-K1 cells, whereas the C-terminal-truncated Delta 197-268 mutant failed to be sorted to mitochondria. Cytochrome c, the final product of holocytochrome c-type synthase activity, is implicated in both oxidative phosphorylation (OXPHOS) and apoptosis. We hypothesize that the inability of HCCS-deficient cells to undergo cytochrome c-mediated apoptosis may push cell death toward necrosis that gives rise to severe deterioration of the affected tissues. In summary, we suggest that disturbance of both OXPHOS and the balance between apoptosis and necrosis, as well as the X-inactivation pattern, may contribute to the variable phenotype observed in patients with MLS.     

HPLC-based bioactivity profiling of plant extracts: a kinetic assay for the identification of monoamine oxidase-A inhibitors using human recombinant monoamine oxidase-A

An assay for the HPLC-based search for monoamine oxidase-A (MAO-A) inhibitors in plant extracts was established. It combines human recombinant MAO-A, expressed as GST-fusion protein in yeast, with a kinetic measurement of the conversion of kynuramine to 4-hydroxyquinoline. Substrate selectivity and kinetic parameters of the GST-fusion protein were comparable to the wild-type enzyme. The applicability of the assay to HPLC-based activity profiling was tested with plant extracts spiked with small amounts of known MAO inhibitors.     

Microbial stabilization of riverine sediments by extracellular polymeric substances

Sediment stability is a critical component for the understanding of cohesive sediment dynamics. Traditionally, physico-chemical sediment conditions have been regarded as most important drivers of sediment stability. However, over the last decade, the stabilization of sediment by biological activity, particularly the influence of highly hydrated matrices of extracellular polymeric substances (EPS) has been given increasing attention. However, most studies have focused on the sediment/water interface and, usually, of marine systems. The present study exploits current knowledge of EPS dynamics from marine systems and applies it to freshwater habitats, also considering a wide range of biological and physico-chemical variables. Natural sediments were taken from a freshwater site with high levels of heavy metal pollution (Lauffen reservoir, River Neckar, Germany). Vertical profiles from the flocculent surface layer to depth of 50 cm within the sediment were investigated, monthly, over the course of year. Tubificidae and Chironomidae larvae constituted the majority of the macrofauna. Despite the turbidity of the water column, a highly diverse and abundant microphytobenthic community of diatoms (11-82 microg g(-1) DW) was found at the sediment surface closely associated with high numbers of bacteria (10(9) cells g(-1) DW). The concentrations of all EPS moieties were remarkably high (0.1-0.5, 1.7-3.8, 0.9-5.2 mg g(-1) DW, for colloidal and bound carbohydrates and proteins, respectively) and levels were comparable to those determined in intertidal studies. The microalgal and bacterial biomass both showed strong correlations with the colloidal and bound EPS carbohydrate fractions. The data suggested that the present macrofauna as well as the metabolic activities of microalgae and bacteria interact with sedimentological factors to influence the properties of the sediment by binding fine-grained sediment, changing water content and enhancing the organic content through secretion products. The colloidal and bound EPS moieties showed strong correlation with the critical shear stress for erosion over sediment depth. It is suggested that the cohesive strength of the sediment was controlled by a high number of active adsorption sites and higher charge densities in fine grained sediments. The EPS network may significantly enhance this by embedding particles and permeating the void space but also in offering additional ionic binding sites and cross-linkages.