The hom-thrB operon (homoserine dehydrogenase/homoserine kinase) and the thrC gene (threonine synthase) of Corynebacterium glutamicum ATCC 13,032 and the homFBR (homoserine dehydrogenase resistant to feedback inhibition by threonine) alone as well as homFBR-thrB operon of C. glutamicum DM 368-3 were cloned separately and in combination in the Escherichia coli/C. glutamicum shuttle vector pEK0 and introduced into different corynebacterial strains. All recombinant strains showed 8- to 20-fold higher specific activities of homoserine dehydrogenase, homoserine kinase, and/or threonine synthase compared to the respective host. In wild-type C. glutamicum, amplification of the threonine genes did not result in secretion of threonine. In the lysine producer C. glutamicum DG 52-5 and in the lysine-plus-threonine producer C. glutamicum DM 368-3 overexpression of hom-thrB resulted in a notable shift of carbon flux from lysine to threonine whereas cloning of homFBR-thrB as well as of homFBR in C. glutamicum DM 368-3 led to a complete shift towards threonine or towards threonine and its precursor homoserine, respectively. Overexpression of thrC alone or in combination with that of homFBR and thrB had no effect on threonine or lysine formation in all recombinant strains tested.

T cell receptor signaling results both in T cell proliferation and apoptosis. A key enzyme at the intersection of these downstream pathways is phosphatidylinositol 3-kinase (PI 3-kinase). In a previous report, we showed that the p85␣ subunit of the PI 3-kinase preferentially associated with the CD3-membrane-proximal immunoreceptor tyrosine-based activation motif of the chain

Aptamers represent auspicious ligands for recognition of target molecules on the surface of a specific cell population, such as stem or cancer cells. These ligands are able to capture and enrich desired cells from a cell mixture, and can be used for identification of new biomarkers, development of cell-specific therapeutics, and stem cell therapy. In this study, we investigated the influence of dead cells on single-stranded DNA (ssDNA) binding and established a method to eliminate dead cells from a cell suspension. Flow cytometry analyses demonstrated that all dead cells were stained with fluorescein-labeled ssDNA molecules. The increasing of the proportion of dead cells led to an increased number of cells that were positive for ssDNA staining. Using dead cell removal microbeads, the proportion of dead cells was significantly reduced. The studies demonstrated that dead cells lead to unspecific uptake/binding of ssDNA molecules during cell-Systematic Evolution of Ligands by Exponential enrichment (SELEX) and can cause failure of the selection process. Thus, the elimination of dead cell population before incubation with ssDNA molecules will reduce the loss of target binding sequences and the contamination of the enriched aptamer pool with unspecific ssDNA molecules caused by unspecific binding to dead cells.

Directed mutagenesis has been used to identify a set of amino acids in the Pichia stipitis xylitol dehydrogenase, encoded by the xylitol dehydrogenase gene XYL2, which is involved in specific NAD binding. Within the binding domain, a characteristic beta alpha beta-fold is centered around a glycine motif GXGXXG also containing conserved aspartate and lysine/arginine residues. The mutation D207-->G and the double mutation D207-->G and D210-->G increased the apparent Km for NAD ninefold and decreased the xylitol dehydrogenase activity to 47% and 35%, respectively, as compared to the unaltered enzyme. The introduction of the potential NADP-recognition sequence (GSRPVC) of the alcohol dehydrogenase from Thermoanaerobium brockii into the xylitol dehydrogenase allowed the mutant enzyme to use both NAD and NADP as cofactor with equal apparent Km values. Although this mutant enzyme displayed an unaltered NADP acceptance, the reduction of the NAD specificity in the stably expressed enzyme variant is an important first step towards the long-term goal to reverse the coenzyme specificity from NAD to NADP. The mutagenized XYL2 gene could still mediate growth of Saccharomyces cerevisiae transformants on xylose minimal-medium plates when expressed together with the xylose reductase gene (XYL1).

Directed mutagenesis has been used to identify a set of amino acids in the Pichia stipitis xylitol dehydrogenase, encoded by the xylitol dehydrogenase gene XYL2, which is involved in specific NAD binding. Within the binding domain, a characteristic βαβ‐fold is centered around a glycine motif GXGXXG also containing conserved aspartate and lysine/arginine residues. The mutation D207→G and the double mutation D207→G and D210→G increased the apparent Km for NAD ninefold and decreased the xylitol dehydrogenase activity to 47% and 35%, respectively, as compared to the unaltered enzyme. The introduction of the potential NADP‐recognition sequence (GSRPVC) of the alcohol dehydrogenase from Thermoanaerobium brockii into the xylitol dehydrogenase allowed the mutant enzyme to use both NAD and NADP as cofactor with equal apparent Km values. Although this mutant enzyme displayed an unaltered NADP acceptance, the reduction of the NAD specificity in the stably expressed enzyme variant is an important first step towards the long‐term goal to reverse the coenzyme specificity from NAD to NADP. The mutagenized XYL2 gene could still mediate growth of Sacchammyces cerevisiae trans‐formants on xylose minimal‐medium plates when expressed together with the xylose reductase gene (XYL1).