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.     

Peptide differential display: a novel approach for phase transition in locusts

Today, the question of the physiological cause of phase transition, the transition from the solitary to the gregarious phase, in locusts remains unanswered. We hereby present a novel approach by which we have attempted to determine whether different phases express or release different peptides in similar physiological conditions. For this purpose, a peptidomic analysis of the corpora cardiaca and hemolymph of crowded and isolated locusts of Schistocerca gregaria was performed using high performance liquid chromatography and matrix-assisted laser desorption ionisation time of flight mass spectrometry. A comparison between the two conditions reveals differences in the number and amount of peptides present in the corpora cardiaca and the hemolymph. Further research will have to identify these phase specific differences and their role in locust phase polymorphism.     

uPA-silica-Particles (SP-uPA): a novel analytical system to investigate uPA-uPAR interaction and to test synthetic uPAR antagonists as potential cancer therapeutics

The urokinase-type plasminogen activation system, including the serine protease uPA (urokinase-type plasminogen activator) and its cell surface receptor (uPAR, CD87), are important key molecules in tumor invasion and metastasis. Besides its proteolytic function, binding of uPA to uPAR on tumor cells exerts various cell responses such as migration, adhesion, proliferation, and differentiation. Hence, the uPA/uPAR system is a potential target for tumor therapy. We have designed a new generation of uPA-derived synthetic cyclic peptides suited to interfere with the binding of uPA to uPAR and present a new technology involving micro silica particles coated with uPA (SP-uPA) and reacting with recombinant soluble uPAR (suPAR), to rapidly assess the antagonistic potential of uPA-peptides by flow cytofluorometry (FACS). For this, we used silica particles of 10 microm in diameter to which HMW-uPA is coupled using the EDC/NHS method. Soluble, recombinant suPAR was added and the interaction of SP-uPA with suPAR verified by reaction with monoclonal antibody HD13.1 directed to uPAR, followed by a cyan dye (cy5)-labeled antibody directed against mouse IgG. Thereby it was possible to test naturally occurring ligands of uPAR (HMW-uPA, ATF) as well as highly effective, synthetic cyclic uPA-derived peptides (cyclo21,29[D-Cys21Cys29]-UPA21-30, cyclo21,29[D-Cys21Nle28Cys29]-uPA21-30, cyclo21,29[D-Cys(21)2-Nal24Cys29]-uPA21-30, and cyclo21,29[D-Cys21Orn23Thi24Thi25Cys29]-uPA21-30. The results obtained with the noncellular SP-uPA/uPAR system are highly comparable to those obtained with a cellular system involving FITC-uPA and the promyeloid cell line U937 as the source of uPAR.     

Asthenozoospermia in mice with targeted deletion of the sperm mitochondrion-associated cysteine-rich protein (Smcp) gene

The sperm mitochondria-associated cysteine-rich protein (SMCP) is a cysteine- and proline-rich structural protein that is closely associated with the keratinous capsules of sperm mitochondria in the mitochondrial sheath surrounding the outer dense fibers and axoneme. To investigate the function of SMCP, we generated mice with a targeted disruption of the gene Smcp by homologous recombination. Homozygous mutant males on a mixed genetic background (C57BL/6J x 129/Sv) are fully fertile, while they are infertile on the 129/Sv background, although spermatogenesis and mating are normal. Homozygous Smcp(-/-) female mice are fertile on both genetic backgrounds. Electron microscopical examination demonstrated normal structures of sperm head, mitochondria, and tail. In vivo experiments with sperm of Smcp(-/-) 129/Sv mice revealed that the migration of spermatozoa from the uterus into the oviduct is reduced. This result is supported by the observation that sperm motility as determined by the computer-assisted semen analysis system (CASA) is significantly affected as compared to wild-type spermatozoa. In vitro fertilization assays showed that Smcp-deficient spermatozoa are able to bind to the oocyte but that the number of fertilized eggs is reduced by more than threefold relative to the wild-type control. However, removal of the zona pellucida resulted in an unaffected sperm-egg fusion which was monitored by the presence of pronuclei and generation of blastocyts. These results indicate that the infertility of the male Smcp(-/-) mice on the 129/Sv background is due to reduced motility of the spermatozoa and decreased capability of the spermatozoa to penetrate oocytes.     

The Streptomyces coelicolor ssgB gene is required for early stages of sporulation

ssgB was identified as a novel early sporulation gene in Streptomyces coelicolor. An ssgB deletion mutant failed to sporulate, over-produced actinorhodin, and its colonies were significantly larger than those of the parental strain, suggesting an important role for the ssgB gene product in the process of growth cessation prior to sporulation-specific cell division. This places ssgB temporally before the paralogous sporulation gene ssgA. Analysis of ssgB mutant hyphae by electron microscopy and by confocal fluorescence microscopy showed that it was defective in the initiation of sporulation, as no sporulation septa could be identified, and DNA segregation had not yet been initiated in the mutant.     

Members of a readily enriched beta-proteobacterial clade are common in surface waters of a humic lake

Humic lakes are systems often characterized by irregular high input of dissolved organic carbon (DOC) from the catchment. We hypothesized that specific bacterial groups which rapidly respond to changes in DOC availability might form large populations in such habitats. Seasonal changes of microbial community composition were studied in two compartments of an artificially divided bog lake with contrasting DOC inputs. These changes were compared to community shifts induced during short-term enrichment experiments. Inocula from the two compartments were diluted 1:10 into water from the more DOC-rich compartment, and inorganic nutrients were added to avoid microbial N and P limitation. The dilutions were incubated for a period of 2 weeks. The microbial assemblages were analyzed by cloning and sequencing of 16S rRNA genes and by fluorescence in situ hybridization with specific oligonucleotide probes. beta-Proteobacteria from a cosmopolitan freshwater lineage related to Polynucleobacter necessarius (beta II) were rapidly enriched in all treatments. In contrast, members of the class Actinobacteria did not respond to the enhanced availability of DOC by an immediate increase in growth rate, and their relative abundances declined during the incubations. In lake water members of the beta II clade seasonally constituted up to 50% of all microbes in the water column. Bacteria from this lineage annually formed a significantly higher fraction of the microbial community in the lake compartment with a higher allochthonous influx than in the other compartment. Actinobacteria represented a second numerically important bacterioplankton group, but without clear differences between the compartments. We suggest that the pelagic microbial community of the studied system harbors two major components with fundamentally different growth strategies.     

Capillary electrophoretic separation and quantification of flavone-O- and C-glycosides in Achillea setacea W. et K

A simple method for the rapid separation and quantification of flavone-O- and C-glycosides in A. setacea W. et K. by capillary zone electrophoresis (CZE) with UV detection is described. Using 25 mM sodium borate with 20% (v/v) of methanol (pH 9.3) as running buffer sufficient separation of the analytes was achieved within 19 min. For the quantitative determination isorhamnetin-3-O-rutinoside was used as internal standard. The method was successfully applied to a rapid characterisation of the flavonoid complex and a precise quantification of the single and total amount of the flavonoids in different samples of A. setacea.     

Isolation,structural characterization,and antiviral activity of positional isomers of monopegylated interferon alpha-2a (PEGASYS)

Interferon alpha-2a plays an essential role in the treatment of chronic hepatitis C, but it is limited in its efficacy by the short in vivo half-life. To improve the half-life and efficacy, interferon alpha-2a is conjugated with a 40-kDa branched polyethylene glycol moiety (PEG-IFN, PEGASYS). From this preparation the positional PEG-IFN isomers were isolated and characterized by different analytical methods and antiviral assay. Two chromatographic steps were used to separate and purify nine isomers. The analytical methods IE-HPLC, RP-HPLC, SE-HPLC, SDS-PAGE, and MALDI-TOF MS indicated that each of these nine isomers is conjugated to the branched polyethylene glycol chain at a specific lysine. No isomer with a modification at the amino terminus was observed. All positional isomers induced viral protection of MDBK cells in the antiviral assay. When comparing the quantitative potency of the individual isomers with the whole mixture of PEG-IFN, significant differences in the specific activities were observed: PEG-Lys(31) and PEG-Lys(134) showed higher activities than the mixture, PEG-Lys(164) was equal to the mixture, whereas the activities of PEG-Lys(49), PEG-Lys(70), PEG-Lys(83), PEG-Lys(112), PEG-Lys(121), and PEG-Lys(131) were lower.     

Alpha-lipoic acid preconditioning reduces ischemia-reperfusion injury of the rat liver via the PI3-kinase/Akt pathway

In liver resection and transplantation ischemia-reperfusion injury (IRI) is one of the main causes of organ dys- or nonfunction. The aim of the present study was to determine whether alpha-lipoic acid (LA) is able to attenuate IRI. Rat livers were perfused with Krebs-Henseleit buffer with or without LA (+/-wortmannin), followed by ischemia (1 h, 37 degrees C) and reperfusion (90 min). Efflux of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) and hepatic ATP content were determined enzymatically. Activation of NF-kappaB and activating protein 1 (AP-1) was examined by EMSA, and protein phosphorylation was examined by Western blot. Caspase-3-like activity served as an indicator for apoptotic processes. Animals treated intravenously with 500 micromol LA were subjected to 90 min of partial no-flow ischemia followed by reperfusion for up to 7 days. Preconditioning with LA significantly reduced LDH and PNP efflux during reperfusion in isolated perfused rat livers. ATP content was significantly increased in LA-treated livers. Postischemic activation of NF-kappaB and AP-1 was significantly reduced in LA-pretreated organs. Preconditioning with LA significantly enhanced Akt phosphorylation. It showed neither effect on endothelial nitric oxide synthase nor on Bad phosphorylation. Importantly, simultaneous administration of wortmannin, an inhibitor of the phosphatidylinositol (PI)3-kinase/Akt pathway, blocked the protective effect of LA on IRI, demonstrating a causal relationship between Akt activation and hepatoprotection by LA. Interestingly, despite activation of Akt, LA did not reduce postischemic apoptotic cell death. The efficacy of LA treatment in vivo was shown by reduced GST plasma levels and improved liver histology of animals pretreated with LA. This study shows for the first time that the PI3-kinase/Akt pathway plays a central protective role in IRI of the rat liver and that LA administration attenuates IRI via this pathway.