Advanced protein crystallization using water-soluble ionic liquids as crystallization additives.

The application of five water-soluble, halogen-free, alkylammonium-based ionic liquids (ILs) as additives for advanced crystallization of lysozyme was investigated. Their biocompatibility was determined by long-term measurement of the overall mean relative enzyme activities. These were maximally reduced by about 10-15% when up to 200 g IL l(-1) was added.

Effect of cyclins and Cdks on the cyclin B1 promoter activation.

To study the effect of several cyclins on cyclin B1 promoter activation, we co-transfected cyclin A. cyclin E and cyclin D1 expressing plasmids with a cyclin B1 promoter construction driving a luciferase reporter gene into NIH 3T3 cells. All three cyclins produced activation of the reporter gene, however, co-transfection of cyclin A with a Cdk2 dominant negative mutant blocks activation by cyclin A.

Adenovirus E1A activates cyclin A gene transcription in the absence of growth factors through interaction with p107.

Using the infection of quiescent human fibroblasts with adenovirus type 5 and various deletion mutants, we show that E1A can stimulate transcription of the cyclin A gene in the absence of exogenous growth factors. Required for this activity is conserved region 2 (CR2), while both the N-terminal part of E1A and CR1 are dispensable. This indicates that activation of cyclin A gene expression requires the binding of E1A to p107, while binding to either pRB or p300 is not involved in transcriptional activation.

Identification of domains required for transcriptional activation and protein dimerization in the human papillomavirus type-16 E7 protein.

To analyse the potential of the E7 oncogene of HPV-16 to activate transcription, we constructed hybrid proteins containing various portions of the HPV-16 E7 protein fused to the DNA binding region of the bacterial LexA repressor. We found that full length HPV-16 E7 is capable to mediate activation of two different reporter genes, which carry LexA binding sites in their promoters. In contrast, E7 from HPV-11, a low-risk type papillomavirus, was unable to activate transcription, when analysed in the same assay.

Modulation of cyclin gene expression by adenovirus E1A in a cell line with E1A-dependent conditional proliferation.

To investigate how adenovirus E1A controls cell proliferation, we have fused E1A to the hormone-binding domain of the human estrogen receptor (ER) and introduced the E1A-ER chimeric gene together with an activated ras gene into primary rat embryo fibroblasts. Cell lines derived from this transfection proliferate in an estrogen-dependent manner. Estrogen-dependent activation of E1A-ER led to a rapid induction of both cyclin E and cyclin A gene expression.

Binding of the human E2F transcription factor to the retinoblastoma protein but not to cyclin A is abolished in HPV-16-immortalized cells.

The adenovirus E1A, SV40 large T and papillomavirus E7 proteins immortalize primary cells by virtue of their ability to bind the retinoblastoma gene product (pRB) and other cellular proteins, including cyclin A and the prRB-related protein, p107. It has been demonstrated that these viral oncogene products will prevent the inhibition of positive growth regulators by pRB, one of them being the E2F transcription factor. Here we show that the interactions of pRB and cyclin A with E2F are present also in normal keratinocytes and in primary human fibroblasts.