Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.).

Four new simple sequence repeat (SSR) loci (designated VVMD5, VVMD6, VVMD7, and VVMD8) were characterized in grape and analyzed by silver staining in 77 cultivars of Vitis vinifera. Amplification products ranged in size from 141 to 263 base pairs (bp). The number of alleles observed per locus ranged from 5 to 11 and the number of diploid genotypes per locus ranged from 13 to 27. At each locus at least 75% of the cultivars were heterozygous. Alleles differing in length by only 1 bp could be distinguished by silver staining, and size estimates were within 1 or 2 bp, depending on the locus, of those obtained by fluorescence detection at previously reported loci. Allele frequencies were generally similar in wine grapes and table grapes, with some exceptions. Some alleles were found only in one of the two groups of cultivars. All 77 cultivars were distinguished by the four loci with the exception of four wine grapes considered to be somatic variants of the same cultivar, 'Pinot noir', 'Pinot gris', 'Pinot blanc', and 'Meunier'; two table grapes that are known to be synonymous, 'Keshmesh' and 'Thompson Seedless'; and three table grapes, 'Dattier', 'Rhazaki Arhanon', and 'Markandi', the first two of which have been suggested to be synonymous. Although the high polymorphism at grape SSR loci suggests that very few loci would theoretically be needed to separate all cultivars, the economic and legal significance of grape variety identification requires the increased resolution that can be provided by a larger number of loci. The ease with which SSR markers and data can be shared internationally should encourage their broad use, which will in turn increase the power of these markers for both identification and genetic analysis of grape. Key words : grape, Vitis, microsatellite, simple sequence repeat, DNA typing, identification.

The differential modulation of the enzymes of glutathione metabolism. Indication of overlapping effects of toxicity and repair in mouse liver and kidney after dietary treatment with methyl mercury and sodium selenite.

The effect of methylmercury (MM) and MM plus sodium selenite (SE) on the activity of various GSH-dependent enzymes was studied in the liver and kidney of mice. Ten groups of mice were fed diets containing graded proportions of MM, alone or with graded quantities of SE. GST, GSH-Px, and GSSG-RED were assayed in the cytosolic fraction of liver and kidney homogenates. After treatment with MM, instead of the expected decrease in enzyme activities, an increase was observed in the kidney and a small decrease in the liver with no dose-response relation in either organ. In protected groups, a general pattern of induction was observed in both organs, but again there was little evidence of dose-response relationships. Detailed analysis of the results suggests that the effects observed were not directly caused by MM or SE but are the resultant of complex interactions presumably related to contemporaneous mechanisms of damage and repair.

Disruption of cytoskeleton by methylmercury in cultured CHO cells.

The effect of methylmercury (MM) on three main cytoskeletal components [i.e. microtubules (MT), microfilaments (MF) and intermediate filaments (IF)] and on specific biochemical parameters (i.e. glutathione transferase (GST), glutathione reductase (RED), glutathione peroxidase (GSH-Px), glyoxalase 1 (GLY 1) and total -SH groups (TSH) of the cytosolic fraction) was studied in cultured Chinese hamster ovary (CHO) cells. The experiments were conducted with increasing doses of MM (i.e. 1, 4 and 8 mum), using an exposure time of 16 hr; and with a fixed dose of MM (2 mum), using increasing exposure periods (i.e. 0-24 hr). The morphological changes observed by immunofluorescence seemed to indicate that MF were damaged as much as (if not more than) MT after 16 hr of exposure to 4 mum-MM. At a concentration of 1 mum, MM only affected MF. The time-course experiments revealed that IF as well as MF and MT were severely disorganized after 3 and 6 hr of incubation in the presence of 2 mum-MM. However, an obvious reorganization was observed after 24 hr of exposure. In experiments using increasing MM doses, changes in the enzymatic activities were less noticeable than those observed in the morphology; only a modest decrease in TSH and RED activities (<30%) was recorded at the highest dose of MM used (i.e. 8 mum). In contrast, increasing the time of exposure to MM induced changes in both the cytoskeletal structures and the biochemical parameters: the lowest RED activity and TSH were observed after 3-6 hr exposure; control values were obtained after an exposure period of 24 hr. Ultrastructural observations on cells treated with increasing doses of MM showed changes in plasmamembrane profile, cytoskeleton organization and mitochondrion structure. The results confirm that MM causes non-specific damage to CHO cells and suggest that a functional interaction may exist between GSH-dependent enzymes and cytoskeletal structures.

Glutathione-linked enzymes in normal and tumor cells and their role in resistance against genotoxic agents.

Glutathione is the most abundant low molecular mass thiol in human cells. It is involved in the inactivation of genotoxic electrophilic compounds, and a variety of glutathione-linked enzymes catalyze such detoxication reactions. Within this group, the enzymes occurring in highest intracellular concentrations are the glutathione transferases, which catalyze the detoxication of a broad spectrum of alkylating and oxidizing compounds such as epoxides, reactive alkenes and organic hydroperoxides. Multiple forms of glutathione transferase with distinct substrate specificities exist, and their differential expression in cells contributes to differences in detoxication capacities in tissues. Glyoxalase I catalyzes the inactivation of 2-oxoaldehydes and may also be considered as part of the cellular detoxication system. Characterization of the different enzymes and their differential expression in normal and tumor cells will help to clarify their cellular functions and their significance to human cancer. Clear differences in the occurrence of the various enzyme forms in normal and tumor cells have been demonstrated and variations between different tumors appear to be linked to their degree of resistance to alkylating cytostatic drugs. Modulation of catalytic activities in vitro by administration of enzyme inhibitors may help to overcome this resistance.