Karsten Rothbarth

One single mRNA encodes the centrosomal protein CCD41 and the endothelial cell protein C receptor (EPCR)

The cDNA encoding the centrosomal protein CCD41 is identical with the cDNA for the endothelial cell protein C receptor. This finding is not due to an artefact, e.g. caused by selection of false positive clones. The segment of the CCD41 cDNA encoding the protein originally termed CCD41 and deletion mutants of it were fused with the nucleotide sequence encoding the enhanced green fluorescent protein (EGFP). Transfection and expression of the full length construct produces a fusion protein mainly located in cell membranes reflecting the receptor‐type protein. Deletion mutants, e.g. those where the signal sequence is deleted, result in fusion proteins which are exclusively incorporated into a small perinuclear structure which is the site of the centrosome. This result suggests that post‐translational modification, namely deletion of the signal sequence, is decisive for the centrosomal location of the resulting centrosomal protein while the unprocessed protein is incorporated into cell membranes.

A multifunctional protein: involvement of the alpha-1 serum protease inhibitor in SDS and high salt-stable DNA-protein complexes.

Occasionally new and intriguing roles arise for proteins with well established functions. The alpha‐1 serum protease inhibitor (α‐1 PI) represents another example. Sequence identities exist in the α‐1 PI and in a nuclear 52‐kDa glycoprotein which is involved in resistant DNA‐polypeptide complexes. The results of Western blots support the identity of the two proteins and immunocytochemical studies indicate the nuclear location of the α‐1 PI. Consistently, e.g. Ehrlich ascites tumor cells express the α‐1 PI, and the fusion protein between the α‐1 PI and the green fluorescent protein from Aequorea victoria shows intracellular accumulation and partly nuclear location.

Proteasome-mediated degradation antagonizes critical levels of the apoptosis-inducing C1D protein

The C1D gene is expressed in a broad spectrum of mammalian cells and tissues but its product induces apoptotic cell death when exceeding a critical level. Critical levels are achieved in a fraction of cells by transient transfection with EGFP-tagged C1D expression constructs. However, transfected cells expressing sub-critical levels of C1D(EGFP) escape apoptotic cell death by activation of a proteasome-mediated rescue mechanism. Inhibition of the proteasome-dependent degradation of the C1D(EGFP) protein results in a parallel increase of the intracellular C1D level and in the fraction of apoptotic cells.

Cell density dependent DNA replication in Ehrlich ascites tumour cells

DNA replication of Ehrlich ascites tumour cells was investigated in suspensions with different cell densities by incorporation in vitro of tritiated thymidine and alkaline sucrose gradient analysis of the newly formed DNA. It is demonstrated that the incorporation of [3H]thymidine and chain growth of newly made DNA decreases with increasing cell density. The inhibition of DNA synthesis observed at high cell densities can be prevented if diffusible substances are removed by incubating the cells in dialysis tubes. This indicates that the changes in DNA synthesis are caused by diffusible inhibitors released from the tumour cells.