Expression of recombinant cytochromes c from various species in Saccharomyces cerevisiae: post-translational modifications.

A complete protocol for the expression of recombinant cytochrome c genes from yeast, Drosophila melanogaster, and rat in a yeast strain, GM-3C-2, which does not express its own cytochromes c is described. The construction of the expression vectors, transformation and large-scale growth of the yeast, and preparation and purification of the recombinant cytochromes c are described. It was found that, contrary to the way yeast modifies its own cytochromes c, the recombinant proteins were partially acetylated at their N-terminus, except for the drosophila protein, which remained entirely unblocked. Furthermore, the yeast and rat proteins were close to fully trimethylated at lysine 72, while the drosophila protein could be separated chromatographically into forms containing tri-, di-, mono-, and unmethylated lysine 72 showing corresponding resonances in the NMR spectrum. These observations emphasize that, in employing expression procedures to obtain native or mutant forms of cytochrome c, it is essential to identify the variety and extent of post-translational modifications and to separate the preparation into pure monomolecular species. Otherwise, it may become impossible to distinguish between the influence of a site-directed mutation and unexamined post-translational modifications.     

The genetic organization of integrated hepatitis B virus DNA in the human hepatoma cell line PLC/PRF/5.

Hepatitis B virus (HBV) DNA is often found integrated in the genome of infected human liver cells and is supposed to be related to the development of primary liver carcinoma (PLC). Four clones of HBV DNA-containing sequences derived from DNA of the human PLC-derived cell line PLC/PRF/5 are discussed. The viral sequences show no intricate rearrangements excepting for a duplication and an inversion in one case, and a deletion in another. In all cases integration of the viral DNA was seen to be in a region which is single-stranded in the unintegrated HBV DNA. Sequence homologies between human and viral DNA flanking the integration sites have been detected. That may have a functional role in integration. Nucleotide sequence analyses of regions encompassing the viral-human junctions reveal open reading frames which consist of viral and/or human information. The possible expression of chimeric or cellular proteins may play a role in tumour development, and offers directions for further investigations.     

Mosaic double aneuploidy: Down syndrome and XYY

Chromosomal abnormalities are seen in nearly 1% of live born infants. We report a 5-year-old boy with the clinical features of Down syndrome, which is the most common human aneuploidy. Cytogenetic analysis showed a mosaicism for a double aneuploidy, Down syndrome and XYY. The karyotype was 47, XY,+21[19]/48, XYY,+21[6]. ish XYY (DXZ1 × 1, DYZ1 × 2). Mosaic double aneuploidies are very rare and features of only one of the aneuploidies may predominate in childhood. Cytogenetic analysis is recommended even if the typical features of a recognized aneuploidy are present so that any associated abnormality may be detected. This will enable early intervention to provide the adequate supportive care and management.     

Early presynaptic changes during plasticity in cultured hippocampal neurons

Long-lasting increase in synaptic strength is thought to underlie learning. An explosion of data has characterized changes in postsynaptic (pstS) AMPA receptor cycling during potentiation. However, changes occurring within the presynaptic (prS) terminal remain largely unknown. We show that appearance of new release sites during potentiation between cultured hippocampal neurons is due to (a) conversion of nonrecycling sites to recycling sites, (b) formation of new releasing sites from areas containing diffuse staining for the prS marker Vesicle-Associated Membrane Protein-2 and (c) budding of new recycling sites from previously existing recycling sites. In addition, potentiation is accompanied by a release probability increase in pre-existing boutons depending upon their individual probability. These prS changes precede and regulate fluorescence increase for pstS GFP-tagged-AMPA-receptor subunit GluR1. These results suggest that potentiation involves early changes in the prS terminal including remodeling and release probability increase of pre-existing synapses.     

RNA interference for CFTR attenuates lung fluid absorption at birth in rats

Background

Small interfering RNA (siRNA) against αENaC (α-subunit of the epithelial Na channel) and CFTR (cystic fibrosis transmembrane conductance regulator) was used to explore ENaC and CTFR function in newborn rat lungs.

Methods

Twenty-four hours after trans-thoracic intrapulmonary (ttip) injection of siRNA-generating plasmid DNA (pSi-0, pSi-4, or pSi-C₂), we measured CFTR and ENaC expression, extravascular lung water, and mortality.

Results

αENaC and CFTR mRNA and protein decreased by ~80% and ~85%, respectively, following αENaC and CFTR silencing. Extravascular lung water and mortality increased after αENaC and CFTR-silencing. In pSi-C₂-transfected isolated DLE cells there were attenuated CFTR mRNA and protein. In pSi-4-transfected DLE cells αENaC mRNA and protein were both reduced. Interestingly, CFTR-silencing also reduced αENaC mRNA and protein. αENaC silencing, on the other hand, only slightly reduced CFTR mRNA and protein.

Conclusion

Thus, ENaC and CFTR are both involved in the fluid secretion to absorption conversion around at birth.     

Adhesion of acidic lipid vesicles by 21.5 kDa (recombinant) and 18.5 kDa isoforms of myelin basic protein

Myelin basic protein (MBP) is thought to be responsible for adhesion of the intracellular surfaces of compact myelin to give the major dense line. The 17 and 21.5 kDa isoforms containing exon II have been reported by others to localize to the cytoplasm and nucleus of murine oligodendrocytes and HeLa cells while the 14 and 18.5 kDa isoforms lacking exon II are confined to the plasma membrane. However, we show that the exon II(-) 18.5 kDa form and a recombinant exon II(+) 21.5 kDa isoform both caused similar aggregation of acidic lipid vesicles, indicating that they should have similar abilities to bind to the intracellular lipid surface of the plasma membrane and to cause adhesion of those surfaces to each other. The circular dichroism spectra of the two isoforms indicated that both had a similar secondary structure. Thus, both isoforms should be able to bind to and cause adhesion of the cytosolic surfaces of compact myelin. The fact that they do not could be due to differences in post-translational modification in vivo, trafficking through the cell and/or subcellular location of synthesis, but it is not due to differences in their lipid binding.