[Expression of single-chain Fv antibody for anti-beet necrotic yellow vein virus in Escherichia coli].

The heavy chains variable region gene (VH) of monoclonal antibody against beet necrotic yellow vein virus (BNYVV) was amplified from total DNA extracted from anti-BNYVV hybridoma cells by PCR. Sequencing showed that the VH belongs to mouse subgroup II(A) and contains 360 bp, which code one hundred and twenty amino acids. The VH and VL genes were inserted into a plasmid which contains a linker sequence for constructing scFv gene. The new vector named pTC scFv. The scFv was produced in Escherichia coli and appeared binding activity with BNYVV antigen by ELISA method.

Cell cycle effects on the basal and DNA-damaging-agent-stimulated ADPRT activity in cultured mammalian cells.

ADP-ribosyl transferase (ADPRT) is a DNA-dependent chromatin-associated enzyme which covalently attaches ADP-ribose moieties derived from NAD+ to protein acceptors to form poly(ADP-ribose). ADPRT activity is strongly stimulated by breaks in DNA, and it is suggested that its activity is required for efficient DNA excision repair. In this paper, a cell-cycle-dependent fluctuation of basal ADPRT activity was demonstrated by measuring it in permeabilized FL cells. The cell used was subjected to arginine starvation for 48 h before being released from the block by replacement of deficient medium with complete medium and cells in different proliferating stages were traced by [3H]TdR pulse labelling and obtained at different intervals after block release. The peak basal ADPRT activity appeared 4-6 h after the appearance of the peak of DNA synthesis. After treating the cells with MNNG (10(-4) M), MMS (10(-3)-10(-4) M) and 4NQO (10(-5) M) for 90 min just after release of the block, the ADPRT activity was markedly stimulated. It was further demonstrated that the effects of MNNG/4NQO and cell cycle influence on the level of poly(ADP-ribose) synthesis appear to be additive. While concerning MMS, quite a different pattern of ADPRT stimulation in the cell cycle was demonstrated, i.e., the activity of ADPRT stimulation of 10(-3) M MMS was found to be completely dependent on the basal ADPRT activity. In the cells with the highest basal ADPRT activity 12 h after block release, the MMS-induced ADPRT stimulation could not be observed. It was suggested that more than one pathway might be present in ADPRT stimulation induced by DNA-damaging chemicals, and the cells synchronized in late G1 stage might be the most suitable for demonstrating poly(ADP-ribose) synthesis after DNA damage.

Genotoxic activity of caramel on Salmonella and cultured mammalian cells.

The genetic activity of 2 commercial caramel preparations, manufactured either by heating the malt sugar solution directly (non-ammoniated caramel) or by heating it with ammonia (ammoniated caramel) was studied in the Salmonella mutagenicity test and UDS assay in cultured mammalian cells. The non-ammoniated caramel was found to be mutagenic to S. typhimurium TA100, while the ammoniated one was genetically active in all the tester strains used, namely TA100, TA97 and TA98. It was also demonstrated that non-ammoniated caramel was capable of inducing UDS in cultured human amnion FL cells, but for the ammoniated one, no such activity was observed. Furthermore, based on the results obtained in the DNA synthesis inhibition assay, it was suggested that the DNA synthesis inhibition seen in our experiments with the ammoniated caramel was probably not of DNA damage in origin. These data indicate that the mutagenic fractions formed during ammoniated and non-ammoniated caramelization were quite different.