Engineering yeast alcohol dehydrogenase. Replacing Trp54 by Leu broadens substrate specificity

Analysis of a crystal structure of alcohol dehydrogenase (Adh) from horse liver suggests that Trp54 in the homologous yeast alcohol dehydrogenase prevents the yeast enzyme from efficiently catalysing the oxidation of long-chain primary alcohols with branching at the 4 position (e.g. 4-methyl-1-pentanol, cinnamyl alcohol). This residue has been altered to Leu by site-directed mutagenesis. The alteration yields an enzyme that serves as an effective catalyst for both longer straight-chain primary alcohols and branched chain alcohol

Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule

The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that has been identified as a marker of cancer-initiating cells. EpCAM is highly expressed on most carcinomas, and transient silencing of EpCAM expression leads to reduced oncogenic potential. To silence the EpCAM gene in a persistent manner via targeted DNA methylation, a low activity mutant (C141S) of the CpG-specific DNA methyltransferase M.SssI was coupled to a triple-helix-forming oligonucleotide (TFO−C141S) specifically designed for the EpCAM gene. Reporter plasmids encoding the green fluorescent protein under control of different EpCAM promoter fragments were treated with the TFO−C141S conjugate to determine the specificity of targeted DNA methylation in the context of a functional EpCAM promoter. Treatment of the plasmids with TFO−C141S resulted in efficient and specific methylation of the targeted CpG located directly downstream of the triple helix forming site (TFS). No background DNA methylation was observed neither in a 700 bp region of the EpCAM promoter nor in a 400 bp region of the reporter gene downstream of the TFS. Methylation of the target CpG did not have a detectable effect on promoter activity. This study shows that the combination of a specific TFO and a reduced activity methyltransferase variant can be used to target DNA methylation to predetermined sites with high specificity, allowing determination of crucial CpGs for promoter activity

Erforschung von biokompatiblen und korrosionsbestandigen Beschichtungen, die auf Titanlegierung Vt6 durch Elektroerosivemetallbearbeitung aufgetragen werden

В статье рассмотрены фазовый и химико-элементный составы покрытия для ортопедии и дентальной медицины, нанесенного на титановый сплав ВТ6методом электроискровой обработки металлов с целью предотвращения попадания с поверхности сплава в организм человека химических элементов, оказывающих токсическое воздействие