[A New Simple Technique for Producing Labeled Monoclonal Antibodies for Antibody Pair Screening in Sandwich-ELISA].

A simple and fast method for obtaining biotin-labeled monoclonal antibodies was developed usingcontent of hybridoma culture supernatant sufficient to select antibody pairs in sandwich ELISA. The method consists in chemical biotinylation of antigen-bound antibodies in a well of ELISA plate. Using as an example target Vaccinia virus A27L protein it was shown that the yield of biotinylated reactant is enough to set comprehensive sandwich ELISA for a moderate size panel of up to 25 monoclonal antibodies with an aim to determine candidate pairs. The technique is a cheap and effective solution since it avoids obtaining preparative amounts of antibodies.

[Inactivation and sensitization of the tumor cells by the gene Bax transfection].

After the transfection of the gene Bax into the cultured tumor cells of human ovary adenocarcinoma SKOV3 and uterus carcinoma HeLa in vitro the high sensitivity of the cells SKOV3 to the protein Bax produced after the gene Bax transfection was found. The sensitivity of the cells HeLa to the gene Bax transfection was much smaller. The hyperexpression of gene Bax and hypersensitivity to doxorubicin were seen in HeLa cells received as a result of the gene Bax transfection and subsequent selection. All cells of the line SKOV3 with the increased expression of the transfected gene Bax died. In the cell line SKOV3 the mutation in a gene Bax was found which has a genotype G7/G9 against a native type of a gene Bax--G8/G8. It was concluded that the found in the exone 3 of the gene Bax mutation G7/G9 in cells SKOV3 results in an inactivation of proapoptotic activity of the protein Bax.

The Ca2+ threshold for the mitochondrial permeability transition and the content of proteins related to Bcl-2 in rat liver and Zajdela hepatoma mitochondria.

Zajdela hepatoma mitochondria were able to accumulate two to five times more Ca2+ than rat liver mitochondria before the permeability transition was induced. Pulses of Ca2+ were given in series to determine the Ca2+ threshold by recording changes in [Ca2+] and membrane potential, the permeability transition causing the release of accumulated Ca2+ and collapse of the membrane potential. Hepatoma mitochondria had lower Ca2+ efflux rates, higher net Ca2+ uptake rates and lower phosphorylation rates than liver mitochondria. Since the differences in regard to induction of the permeability transition might be due to higher expression of the Bcl-2 protein in hepatoma cells than in hepatocytes, the transcription of Bcl-2 and the proteins reacting with a Bcl-2 polyclonal antiserum were estimated by Northern and Western blotting, respectively. Hepatoma cells had two Bcl-2 specific mRNA bands of 7 and 2.4 kb, and substantial amounts of the Bcl-2 protein, whereas in liver cells and mitochondria these were not detected. Both cell lines had a reactive band at 19-20 kDa, and hepatocytes a small band at 31-32 kDa. Bcl-2 antibodies stimulated the permeability transition potently in hepatoma mitochondria.

[Comparative study of Ca/Mg-dependent nucleases from the mammalian thymus]. / Sravnitel'noe issledovanie Ca/Mg-zavisimykh nukleaz timusa mlekopitaiushchikh.

Ca/Mg-dependent nuclease is a possible key enzyme of apoptosis. We isolated and purified nucleases from the human and rat thymus to a homogeneous state and compared some properties of the obtained preparations with those of the earlier isolated Ca/Mg-dependent nuclease from the calf thymus. The activity of the nuclease from the human thymus in the presence of bivalent ions decreases in a sequence:(Ca + Mg) = (Ca + Mn) > Mn, that from the rat thymus: (Ca + Mn) > Mn > (Ca + Mg), and that from the calf thymus: (Ca + Mn) > (Ca + Mg) > Mn. Nuclease are not active in a medium containing only Mg, Ca, Co, and Zn ions. The preparations proved to be unstable during their isolation and storage. If the relative molecular mass of the purifies preparations was according to electrophoresis in 12% DS-Na-polyacrylamide gel 28, 29, and 18.4 and 21 kDa for the calf, human, and rat, respectively, after storage at -20 degrees C for two to six months, the molecular mass of native proteins decreases to 17-14 kDa. Some other properties of the enzymes have been described.