HYPK has the ability of reducing rate of 4-Hydroxytamoxifen concentration aggregate formation and subsequent toxicity caused by mutant Huntingtin. Further investigation revealed that HYPK is involved in diverse cellular processes and required for normal functioning of cells. In this study we observed that hyperthermia increases HYPK expression in human and mouse cells in culture. Expression of exogenous Heat Shock Factor 1 (HSF1), upon heat treatment could
induce HYPK expression, whereas HSF1 knockdown reduced endogenous as well as heat-induced HYPK expression. Putative HSF1-binding site present in the promoter of human HYPK gene was identified and validated by reporter assay. Chromatin immunoprecipitation revealed in vivo interaction of HSF1 and RNA polymerase II with HYPK promoter sequence. Additionally, acetylation of histone H4, a known epigenetic buy Nutlin-3 marker of inducible HSF1 binding, was observed in response to heat shock in HYPK gene promoter. Overexpression of HYPK inhibited cells from lethal heat-induced death whereas knockdown of HYPK made the cells susceptible to lethal heat shock-induced death. Apart from
elevated temperature, HYPK was also upregulated by hypoxia and proteasome inhibition, two other forms of cellular stress. We concluded that chaperone-like protein HYPK is induced by cellular stress and under transcriptional regulation of HSF1.”
“The generation of transgenic cell lines is acquired by facilitating the uptake and integration of DNA. Unfortunately, most of the systems generating VX770 stable expression systems are cost and time-consuming and transient expression is optimized to generate milligram amounts of the recombinant protein. Therefore we improved and compared two transfection systems, one based on cationic liposomes consisting of DOTAP/DOPE and the second one on polyethylenimine (PEI). Both systems have been used as chemically defined transfection systems in combination with serum-free cultivated host cell line. At first we had determined the toxicity and ideal ratio of DNA to PEI followed by determination of the optimal transfection
conditions in order to achieve maximum transfection efficiency. We then directly compared DOTAP/DOPE and PEI in transient transfection experiments using enhanced green fluorescence protein (EGFP) and a human monoclonal antibody, mAb 2F5, as a model protein. The results which were achieved in case of EGFP were more than 15% transfectants at a viability of 85%. Despite the fact that expression of the mAb was found negligible we used both techniques to generate stable mAb 2F5 expressing cell lines that underwent several cycles of screening and amplification with methotrexate, and resulted in cell lines with similar volumetric production titers. These experiments serve to demonstrate the potential of stable cell lines even in case where the transient systems did not show satisfying results.