Identification of human GC-box-binding zinc finger protein, a new Krüppel-like zinc finger protein, by the yeast one-hybrid screening with a GC-rich target sequence.

A new human zinc finger DNA-binding protein was identified by using a yeast one-hybrid selection system. Two versions of the cDNA, encoding the same protein, were detected that differ for a 584 bp extension at the 5' region. Sequence analysis showed that the longer clone is a full length version containing part of the 5' untranslated region. The smaller version was fused in frame with the yeast GAL4 activation domain whereas the 5' region of the longer clone displayed a stop codon interrupting the fusion with the GAL4 domain. Nevertheless, this clone activated the yeast HIS3 reporter gene with the same efficiency as the smaller version. Sequence comparison of the derived protein with the database showed that it belongs to a family of zinc finger DNA-binding proteins which regulate the expression of genes involved in cell proliferation. Expression of the protein in an in vitro system, DNA-binding studies and genetic experiments identify this factor as a new zinc finger DNA-binding protein which binds GC-rich sequences and contains a domain probably functioning as a transcriptional activator. The new human protein identified in this study was therefore named GC-box-binding zinc finger protein).

Tunable stability of monodisperse secondary O/W nano-emulsions.

Stable and biodegradable oil in water (O/W) nano-emulsions can have a huge impact on a wide range of bio-applications, from food to cosmetics and pharmaceuticals. Emulsions, however, are immiscible systems unstable over time; polymer coatings are known to be helpful, but an effective procedure to stabilize monodisperse and biodegradable O/W nano-emulsions is yet to be designed. Here, we coat biodegradable O/W nano-emulsions with a molecular layer of biodegradable polyelectrolytes such as polysaccharides--like chitosan--and polypeptides--like polylysine--and effectively re-disperse and densify the polymer coating at high pressure, thus obtaining monodisperse and stable systems. In particular, focusing on chitosan, our tests show that it is possible to obtain unprecedented ultra-stable O/W secondary nano-emulsions (diameter sizes tunable from ∼ 80 to 160 nm and polydispersion indices below 0.1) by combining this process with high concentrations of polymers. Depending on the polymer concentration, it is possible to control the level of coating that results in a tunable stability ranging from a few weeks to several months. The above range of concentrations has been investigated using a fluorescence-based approach with new insights into the coating evolution.

The melanogenic system of the liver pigmented macrophages of Rana esculenta L.--tyrosinase activity.

The enzyme system responsible for Amphibian Kupffer Cell (KC) melanogenesis has not been entirely elucidated. This research demonstrates that the KC melanosomes of Rana esculenta L. possess a tyrosine-hydroxylase (TH) activity, showing that a tyrosinase is the enzyme involved in the melanogenesis. The TH reaction depends on catalytic Dopa as a cofactor and is not affected by catalase or H2O2, showing that it is catalysed by the tyrosinase and not by the peroxidase present in the melanosomes. The TH reaction is activated by Cu2+ ions but not by other tyrosinase activators such as limited proteolysis, protein ageing, and Sodium Dodecyl Sulphate (SDS). SDS inhibited the KC TH activity even below the critical micelle concentration. All these results suggest that the KC-tyrosinase differs in structure from other known tyrosinases. Using anti-KC-tyrosinase antobodies, we observed that the sites of the tyrosinase location within the cell are the same as those described in the melanocytes. In the immunoblots, the anti-KC-tyrosinase antibodies also recognised two protein bands, at the higher molecular weight ranges, in the protein electrophoretic pattern. Moreover, the tyrosinase activity was limited to the highest molecular weight band of about 260 kDa, suggesting that the enzyme activity could depend on a molecular aggregate. The melanin produced in the liver was found to be a 5,6-dihydroxyindole-rich eumelanin similar to the Sepia melanin.

A FastA based compilation of higher plant mitochondrial tRNA genes.

A new version of the compilation of higher plant mitochondrial tRNA genes (http://www.ebi.ac.uk/service ) has been obtained by means of the FastA program for similarity searching in nucleotide sequence Databases. This approach improves the previous collection, which was based on literature data analysis. The current compilation contains 158 sequences with an increase of 43 units. In this paper, some interesting features of the new entries are briefly presented.

Regulation of the expression of the sea urchin mitochondrial D-loop binding protein during early development.

The Paracentrotus lividus mitochondrial D-loop binding protein (mtDBP) is a DNA-binding protein which is involved in the regulation of sea urchin mtDNA transcription. Immunoblots of Heparin Sepharose-bound proteins at selected early developmental stages, as well as electrophoretic mobility shift assay, show that mtDBP is present in the egg at a concentration of about 1 x 10(6) molecules/egg. Its level increases after fertilization of about twofold, remaining substantially unchanged between 16-h blastula stage and early pluteus stage and declines thereafter. The content of mtDBP mRNA, determined by RNase protection experiments, increases about sevenfold at the 16-h blastula stage compared to the egg. A considerable decrease occurs at the 40-h pluteus stage, which precedes that of the protein. These results suggest that the expression of mtDBP is regulated at transcriptional level up to blastula stage, while other factors, in addition to the level of the RNA, may control the content of this protein in the following stages of embryogenesis.