Latency of Botrytis cinerea Pers.: Fr. and Biochemical Studies During Growth and Ripening of Two Grape Berry Cultivars, Respectively Susceptible and Resistant to Grey Mould

Latency and development of Botrytis cinerea were assessed under field conditions and after artificial inoculation of two grape varieties, Gamay (susceptible) and Gamaret (resistant). When the percentage of latent Botrytis was the same for both varieties, severity of visible grey mould remained very low in Gamaret berries, while Gamay clusters were destroyed by the disease to a high percentage. Some biochemical parameters were measured in berries, such as constitutive and induced anti‐fungal compounds, polymeric proanthocyanidins and lipid peroxidation products as markers of senescence. Differences were observed in polymeric proanthocyanidins (PPRA) of Gamaret compared with those of Gamay. Concentration and mean degree of polymerization (mDP) of PPRA were always higher in the berries of the resistant variety. The inhibitory effect of Gamaret PPRA on enzyme activity remained until harvest whereas Gamay PPRA lost their inhibitory activity at the beginning of véraison. Based on these results, resistance to B. cinerea seems to be linked to the maintainance of the fungus in its latent form in berries, mainly due to the ability of Gamaret PPRA to inhibit macerating fungal enzyme activities.     

Isolation of high molecular weight DNA from wholeEuglena cells

Summary A method for isolating high quality DNA from wholeEuglena cells is described. The procedure consists in: the weakening of the cell pellicle in glycerol avoiding the mechanical disruption of cells and shearing damage in DNA molecules; the decondensation ofEuglena compact chromatin directly inside the cells; the complete dissociation of cells and nucleoproteins in sarkosyl detergent; the optional digestion of proteins and RNA with DNase-free enzymes and the final purification of DNA by isopycnic banding in CsCl gradients. Degradation of DNA is prevented all along the extraction procedure by glycerol, antioxydants, EDTA and sarkosyl detergent. Using the enzymatic digestion step, DNA containing few single-stranded nicks is obtained with a yield approaching 100%. DNA with no single-stranded nick could be obtained with a 35% yield when the enzymatic digestion step was omitted. In both cases, the double-stranded DNA has an average molecular weight equal or greater than 6×10⁷. It is free of contaminants and could be easily digested with restriction enzymes. After digestion with Eco RI and size-fractionation in agarose gel this DNA has permitted specific hybridization of the rDNA sequences with a radioactive rRNA probe.Abbreviations Kbp kilobasepairs - Kb kilobases     

Phleomycin resistance as a dominant selectable marker for plant cell transformation

Tobacco cells are sensitive to bleomycin and phleomycin. The Tn5 and the Streptoalloteichus hindustanus (Sh) bleomycin resistance (Ble) genes conferring resistance to these antibiotics have each been inserted into two plant expression vectors. They are flanked by the nopaline synthase (nos) or the cauliflower mosaic virus (CaMV) 35S promoters on one side, and by the nos polyadenylation signal on the other. These four chimaeric genes were introduced into the binary transformation vector pGA 492, which were thereafter mobilized into Agrobacterium tumefaciens strain LBA 4404. The resulting strains were used to transform Nicotiana tabacum cv. Xanthi nc using the leaf disc transformation procedure. In all cases, phleomycin- and bleomycin-resistant tobacco plants were regenerated from transformed cells under selective conditions; however the highest frequency of rooted plants was obtained when transformation was carried out with the Sh Ble gene under the control of the 35S promoter. Phleomycin resistance was stably transmitted to sexual offspring as a dominant nuclear trait as confirmed by Southern blotting.     

Reactivity of HDL subfractions towards lecithin-cholesterol acyltransferase. Modulation by their content in free cholesterol

(1) Human HDL2 (d 1.070-1.125) and HDL3 (d 1.125-1.21) labelled with unesterified [14C]cholesterol, were incubated with a source of lecithin-cholesterol acyltransferase. For optimal activity, the reaction required the addition of albumin in excess, at least 3-times greater than the concentration of HDL-free cholesterol. Under such conditions, the reaction appeared saturable. HDL3 was found the most efficient substrate and the Vmax values expressed for 1.5 IU LCAT/ml and with an albumin/free cholesterol ratio of 3, were 8.3 nmol free cholesterol esterified/ml per h and 4.1 nmol/ml per h for HDL3 and HDL2, respectively. (2) HDL3 were modified in the presence of VLDL by inducing triacylglycerol lipolysis with a semipurified lipoprotein lipase from bovine milk. The newly formed HDL had gained free cholesterol and phospholipids, so that about 50% of these modified HDL, referred to as light-LIP-HDL3, were reisolated in the HDL2 density range. Light-LIP-HDL3 were enriched mostly in free cholesterol (+ 160%) and in phospholipid (+ 40%). Their reactivity towards LCAT was half-reduced compared to parent HDL3, which correlated well with a decrease in their phospholipid/free cholesterol molar ratio. Moreover, HDL3 artificially enriched in free cholesterol and exhibiting a comparable PL/FC behaved like lipolysis-modified HDL in their reactivity towards LCAT. (3) HDL3 were also modified by co-incubation with VLDL (post-VLDL-HDL3), or with VLDL and a source of lipid transfer protein (CET-HDL3). The latter treatment greatly affected the lipid composition of the core particle (-25% esterified cholesterol, +190% TG). In both cases, the moderate decreasing LCAT reactivity observed could be related to the phospholipid/free cholesterol ratio. Thus, like in artificial substrates, the lipid composition of the HDL surface may control the rate of LCAT-mediated cholesterol esterification.     

Topological orientation of mitochondrial GDPmannose: dolichyl-monophosphate mannosyltransferase in the outer membrane

Previous studies have shown the existence of an autonomous mitochondrial GDPmannose: dolichylmonophosphate mannosyltransferase, located in mitochondiral outer membrane of liver cells. As nothing is known about glycosylation sites in mitochondria, we have investigated the topological orientation of this enzyme in intact mitochondria, using controlled proteolysis with trypsin. Mitochondria were purified sequentially by mild ultrasonic treatment and sucrose density gradient. Purity and homogeneity of mitochondrial fraction were assessed by electron microscopy and specific marker enzymes measures. Our data provide evidence for a mitochondrial GDPmannose: dolichylmonophosphate mannosyltransferase facing the cytoplasmic side of the outer membrane. However, the exposure of this enzyme to the water phase has been shown to be dependent on the ionic strength of the environment.     

Evolutionary divergence of enterobacterial genes coding for the glucose phosphotransferase system components

Abstract The DNA relatedness of 64 enterobacterial species to Escherichia coli genes pts I, pts H, crr , pts G, and pts LPM, was determined by quantitative filter hybridization. DNA relatedness was expressed relative to E. coli K-12 DNA. Enterobacterial DNAs were 0 to 100% related to E. coli genes and the level of relatedness (except for crr data) reflected the known taxonomic (phylogenetic) position of species with respect to E. coli . When pts I relatedness data were plotted against pts H data, correlation was excellent. In pts G versus pts LPM plots, the data points (species) were scattered along the diagonal with a large gap separating E. coli strains (80–100% relatedness to both probes) from the 63 other species (1 to 40% relatedness to E. coli genes). Serratia (9 species), Buttiauxella agrestis , and Klebsiella planticola gave higher relatedness values with crr probe than with the other probes tested.     

SIMS microscopy in the biomedical field.

We attempted to indicate the requirements for biomedical applications of SIMS microscopy. Sample preparation methodology should preserve both the structural and the chemical integrity of the tissue. Furthermore, it is often necessary to correlate ionic and light microscope images. This implies a common methodological approach to sample preparation for both microscopes. The use of low or high mass resolution depends on the elements studied and their concentrations. To improve the acquisition and processing of images, digital imaging systems have to be designed and require both ionic and optical image superimposition. However, the images do not accurately reflect element concentration; a relative quantitative approach is possible by measuring secondary ion beam intensity. Using an internal reference element (carbon) and standard curves the results are expressed in micrograms/mg of tissue. Despite their limited lateral resolution (0.5 microns) the actual SIMS microscopes are very suitable for the resolution of biomedical problems posed by action modes and drug localization in human pathology. SIMS microscopy should provide a new tool for metabolic radiotherapy by facilitating dose evaluation. The advent of high lateral resolution SIMS imaging (less than 0.1 microns) should open up new fields in biomedical investigation.     

The expression of the sIgD isotype in wild-derived mice

IgD and IgM are concomitantly expressed on the surface of most mouse B lymphocytes and both molecules serve as receptor for antigen. In this communication we report that in contrast to IgM, which is expressed in a constant manner on the surface of spleen B lymphocytes of different laboratory and wild-derived mice, IgD expression is variable among the spleen cells of wild-derived mice. SPE, SEI, and SFM mice belonging to the Mus 3 subgroup show a fluorescence profile characterized by a marked diminution in the population of B lymphocytes expressing the IgD isotype; in addition, these cells have a low sIgD density on their membranes. These findings were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the iodinated membrane proteins. Polyclonal in vitro activation with lipopolysaccharide increases the frequency of surface IgD (sIgD)-bearing spleen cells and sIgD density in the SPE strain but decreases both the frequency and the density of IgD bearing cells in the BALB/c strain. This result suggests that delta gene expression is regulated differently in SPE and BALB/c mice. In addition, genetic analysis of sIgD expression in (BALB/c X SPE)F1 hybrids suggests that the proportion of sIgD-bearing cells and sIgD density are independently regulated.