Effect of nickel on oxygen free radical metabolism

The effect of nickel on superoxide dismutase activity (SOD), as well as on rate of hydroxydopamine oxidation, was studied in vitro since lipid peroxidation has been implicated in cell damage by nickel, whose toxicity and carcinogenicity are well established. Nickel strongly inhibits SOD activity. The degree of inhibition is directly proportion to the nickel concentration (tested range 0.066 to 0.33 microgram/mL in the reaction mixture); to the substrate concentration (tested range 0.4 x 10 4M to 1.1 x 10 4M 6-hydroxydopamine); and to reaction mixture. Autoxidation of 6-hydroxydopamine was increased by nickel concentrations higher than 15 micrograms/mL. The combination of excessive oxygen free radical production and inhibition of their elimination by inhibition of SOD activity may contribute to the nickel toxicity that has been reported in industrial accidents, as well as to the high incidence of cancer occurring in nickel workers. It may also contribute to many complications in uremic patients, in whom increased serum nickel levels were reported to be in a similar range to those inhibiting SOD.     

Flow cytometry sorting of viable bacteria and yeasts according to beta-galactosidase activity.

We describe a novel method for quantitative measurement of beta-galactosidase (beta-gal) levels in bacteria and yeasts by using flow cytometry, a method which allows viable microbial cells to be sorted on the basis of the expressed activity and to be recultivated. The method is based on encapsulating single cells in agarose microbeads 20 to 30 microns in diameter and analyzing the beta-gal activity of the colonies that develop (containing several hundred cells) by using the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside (FDG). Three strains of Escherichia coli, containing different levels of beta-gal, served as a model system. A high degree of correlation was found between the average fluorescence measured per bead and the level of the enzyme in extracts of the respective strain. Although the use of FDG necessitates cell permeabilization, conditions were found under which a small part of each colony remained viable, yet most of the enzyme was exposed to the substrate. This allowed sorting of microcolonies and plating with close to 100% efficiency. The potential of the technique was demonstrated by selecting beta-gal-positive cells from an artificial mixture of beta-gal-positive and beta-gal-negative E. coli strains.     

Fluorescence detection of enzymatically formed hydrogen peroxide in aqueous solution and in reversed micelles

A sensitive enzymatic assay for oxidase reactions both in aqueous solution and in hexadecyltrimethylammoniumbromide (CTAB) reversed micelles has been developed. The assay is based on the fluorescence detection of dichlorofluorescein, which is formed by hydrogen peroxide oxidation of the nonfluorescent precursor dichlorofluorescin. Hydrogen peroxide as product of the reaction catalyzed by glucose oxidase served to select the reaction conditions. The reaction rate is distinctly enhanced in CTAB reversed micelles as compared to the rate in aqueous solution. This effect, combined with the high sensitivity owing to the strong fluorescence of dichlorofluorescein, makes the assay attractive for the detection of low enzyme, substrate, or peroxide concentrations.     

Alterations of auxin perception in rolB-transformed tobacco protoplasts. Time course of rolB mRNA expression and increase in auxin sensitivity reveal multiple control by auxin.

Expression and physiological effects of the root-inducing rolB gene of Agrobacterium rhizogenes T-DNA were studied simultaneously in tobacco (Nicotiana tabacum) mesophyll protoplasts. The kinetic study of the expression of rolB mRNA following exogenous auxin application showed that auxin transiently stimulated rolB expression, with mRNA levels starting to accumulate 6 to 9 h after auxin was supplied and increasing 300-fold after 12 to 18 h. The parallel study of the auxin sensitivity of rolB-transformed protoplasts, as assayed by their electrical response to the hormone, showed that the auxin treatment generated an increase in sensitivity by a factor of up to 100,000, whereas in untransformed protoplasts the same auxin treatment induced an increase in auxin sensitivity that never exceeded 30- to 50-fold. This reflects a strong cooperative effect of auxin and rolB in transformed protoplasts. Surprisingly, the maximal increase in sensitivity was observed several hours before the maximal accumulation of rolB mRNA, suggesting that the dramatic control of auxin sensitivity by auxin in rolB-transformed protoplasts requires only low levels of rolB expression. Antibodies directed against ZmER-abp1, the major auxin-binding protein from maize, differentially altered the auxin sensitivity of the electrical response of rolB-transformed and normal protoplasts. This suggests that alterations of the auxin reception-transduction pathway at the plasma membrane of rolB-transformed protoplasts may account for their increased auxin sensitivity.     

Linkage of bovine erythrocyte antigen loci B, C, L, S, Z, R' and T' and the serum protein loci post-transferrin 2 (PTF 2), vitamin D binding protein (GC) and albumin (ALB) to DNA microsatellite markers

Seven bovine erythrocyte antigen loci and three serum protein loci were tentatively assigned to chromosomes or synteny groups by linkage analysis to previously assigned microsatellite DNA markers. The erythrocyte antigen locus EAB was mapped to synteny group U27; EAC to chromosome 18, synteny group U9; EAL to chromosome 3, synteny group U6; EAS to chromosome 21, synteny group U4; EAZ to chromosome 10, synteny group U5; EAR' to chromosome 16, synteny group U1; and EAT' to chromosome 19, synteny group U21. The vitamin D binding protein (GC) and albumin (ALB) loci were assigned to chromosome 6, synteny group U15 and post-transferrin 2 (PTF 2) to chromosome 19, synteny group U21.     

Conservation of two predator species for biological control ofChrysophtharta bimaculata (Col.: Chrysomelidae) in tasmanian forests

Chrysophtharta bimaculata (Olivier) (Coleoptera: Chrysomelidae) is a major defoliator of regeneration eucalypt trees in Tasmania causing a significant reduction in height and diameter increment of trees which reduces wood volume per hectare. A study to conserve and enhance the efficiency of coccinellid species chieflyCleobora mellyi (Mulsant) (Coleoptera: Coccinellidae), and the cantharid,Chauliognathus pulchellus (Macleay) (Coleoptera: Cantharidae), for the biological control ofC. bimaculata was conducted in young regeneration forests in southern Tasmania from 1991–92. Cantharid adults and coccinellid adults and larvae feed onC. bimaculata eggs and, to a lesser extent, young larvae. The study found that coccinellids were more active throughout the egg and early (1^st and 2^nd) stage ofC. bimaculata. The cantharid, however was active only during the egg stage of the prey and then disappeared from the plantation. The coccinellids were therefore the most useful predators, but their population declined when the prey reached the 3^rd and 4^th stages. As shortage of food may account for this decline, supplementary food was provided in the form of sucrose sprays or sugar granules at a feeding station. This resulted in the retention of both predators and particularly the coccinellids and enhanced their efficacy.