Bruce J. Cochrane

Effects of copper and tributyltin on stress protein abundance in the rotifer Brachionus plicatilis

1. Exposure of the rotifer Brachionus plicatilis to elevated temperature resulted in the synthesis of a number of proteins, including a prominent one of 58,000 Da (SP58). 2. This protein is immunologically crossreactive with the 65,000 Da heat shock protein of the moth Heliothis virescens, which is a member of a highly conserved family of mitochondrial proteins. 3. Exposure of rotifers to sublethal doses of CuSO4 leads to a 4-5-fold increase in abundance of SP58, with maximum increase occurring at a dose that is approximately 5% of the LC50 for that compound. 4. A similar response was seen with tributyl tin (TBT). Kinetics of induction were sigmoidal, with induction occurring in the range of 20-30 micrograms/l. 5. No response was observed when rotifers were exposed to aluminum chloride, mercury chloride, pentachlorophenol, sodium arsenite, sodium azide, sodium dodecyl sulfate, or zinc chloride. 6. These results indicate that changes in stress protein abundance may prove useful as a biomarker of exposure to particular toxicants.

The genetics of xenobiotic metabolism in Drosophila. IV: Purification and characterization of the major glutathione s-transferase

Abstract Glutathione S- transferase from Drosophila melanogaster was purified to apparent homogeneity by means of affinity chromatography on a glutathione-agarose column. This single step resulted in a ca 300-fold purification. The enzyme is a dimer, with subunits of 35,000 and 28,500 daltons, and comprises ca 0.3% of the total soluble protein in adults. Monoclonal antibodies to this protein were obtained, and Western blot analysis revealed the existence of one antigenic domain shared by the two subunits. The enzyme was found to possess many of the multiple functions reported for mammalian ones, including heme and bilirubin binding capacities and glutathione peroxidase activity. These results suggest that, in this species, a single enzyme has evolved that possesses many of the properties of the multiple isozymes of higher organisms.

Randomly amplified polymorphism detection (RAPD) reveals high genetic diversity in Thalassia testudinum banks ex König (Turtlegrass)

Abstract Populations of Thalassia testudinum (Banks ex Konig) at the northern and southern limits of the west coast of Florida were compared with a Jamaican population using randomly amplified polymorphism detection (RAPD). With the exception of those from Apalachicola Bay, virtually all samples were distinct genetic individuals. Those putative clone mates that were identified often had other genets dispersed between them. Several distinct differences were observed between the northern and southern populations. The southern populations have higher percentages of the total possible number of bands present, of polymorphic bands, and of bands exclusive to a population, as well as a greater number of RAPD phenotypes and a greater mean number of differences between phenotypes. The biological phenomena that may explain these patterns include increased reproductive success, decreased inbreeding or increased population size in the southern populations. A fourth possibility is unidirectional gene flow from north to south. An analysis of molecular variance (AMOVA) was done indicating that approximately 81% of the variation is within beds, suggesting a homogeneous species. Yet the populations were clearly distinguishable from one another at microgeographic ranges. The level of genetic variation observed is characteristic of species with the same life history traits as Thalassia testudinum and was not predicted based on field observations of the species. This study, in conjunction with other molecular seagrass studies done to date, challenges our understanding of seagrass growth, reproduction, and propagation. There does not appear to be any pattern of reproductive traits, such as dioecy, monoecy, vivipary, or seed banking, that can reliably predict levels of genetic variation in a given seagrass species.

Identification of multiple glutathione S-transferases from Daphnia magna

1. Six anionic glutathione S-transferases (GST) were purified from the crustacean, Daphnia magna, by means of affinity chromatography, that are responsible for ca. 40% of cytosolic GST activity. 2. Electrophoresis in the presence of sodium dodecyl sulfate (SDS) revealed the presence of three proteins, with molecular weights of 27,500, 28,000, and 30,200. 3. Separation under nondenaturing conditions revealed six proteins, all of which exhibited GST activity, with molecular weights ranging from 55,000 to 61,700. 4. Ethacrynic acid is a competitive inhibitor of activity towards CDNB of all six GSTs, binding each with similar affinities. 5. Chlorinated phenols are also competitive inhibitors of the enzyme, with the degree of inhibition being directly correlated with the lipophilicity of the compounds. 6. Analysis of inhibition of separated isoforms reveals that form 4 is most strongly inhibited by these chlorinated phenols, with forms 5 and 6 being inhibited to a lesser degree.

Relationships between the structures of chlorinated phenols, their toxicity, and their ability to induce glutathione S-transferase activity in Daphnia magna

Abstract The effects of six chlorinated phenols on Daphnia magna were assessed with respect to acute toxicity, as well as their abilities to induce activity of the chemical detoxification enzyme glutathione S-transferase. Toxicities were demonstrated to be directly related to lipophilicity, with those compounds having higher numbers of chlorine substitutions being more toxic. All of these compounds induced enzyme activity; however, there was no evident relationship between compound structure and potency as an inducer. It is suggested that in fact all of these compounds act in a similar manner internally to modulate enzyme activity, and that apparent differences in activity are a reflection of differences in bioconcentration of the different compounds.

A rapid method for staining proteins in acrylamide gels

A negative staining procedure for the rapid visualization of proteins in acrylamide gels is described. In the absence of proteins, staining of the gel occurs through the reduction of nitroblue tetrazolium by reduced glutathione. No staining occurs in the presence of proteins. The procedure can be completed in 20 min and is at least as sensitive as Coomassie brilliant blue staining.

Genetics of xenobiotic metabolism in drosophila: I. Genetic and environmental factors affecting glutathione-s-transferase in larvae

The enzyme glutathione-S-transferase, which plays a crucial role in xenobiotic detoxification, was investigated in Drosophila melanogaster. Based upon examination of substrate specificities and pH optima, it was observed that the enzyme in Drosophila is considerably more restricted in its activities than in mammals. The effects of various xenobiotics on activities in third instar larvae were examined. While beta-naphthoflavone and phenobarbital had no effect, pentamethyl benzene (PMB) administration resulted in a 50% increase in enzyme activity. Comparison of lines of known genetic composition indicates that the degree of response to PMB is modulated by genes on chromosome II, and that differences exist with respect to the patterns of response of activities towards the substrates 1-chloro-2, 4-dinitrobenzene and ethacrynic acid. Results obtained suggest the existence of at least two loci on chromosome II that code for glutathione S-transferase isozymes.

The distribution of Basidiobolus and Conidiobolus in soil and litter at sites near Tampa, Florida

In an effort to begin to understand the basic population structure of Basidiobolus, a single, nonlongitudinal field study was conducted to shed light on the number and distribution of the fungus in the soil and litter from several locations near the University of South Florida (USF) in Tampa, Florida. The occurrence of the related fungus Conidiobolus was also documented for comparison with previous studies. An undisturbed cypress stand, an open grassy area on the campus of USF and two pasture lands were chosen for study. Four isolations of Basidiobolus and 48 of Conidiobolus were obtained from 125 soil samples taken from the 5 study plots. Additional sam- ples of soil were taken from the area immediately surrounding the locations of two positive soil collec- tions. One additional isolate of Basidiobolus was re- covered from each of these samplings. RAPD analysis indicates that each Basidiobolus isolate is genetically unique and thus show no evidence of clonal growth in soil. This study suggests the conclusion that in some cases the intestines of reptiles and perhaps oth- er animals may be a more significant reservoir for Basidiobolus than the soil.

Genetic studies in the genus Basidiobolus. II. Phylogenetic relationships inferred from ribosomal DNA analysis.

Abstract We have investigated genetic relationships among isolated of the genus Basidiobolus by examining restriction site polymorphisms in the ribosomal RNA genes. Approximately 10 kb or rDNA was cloned and employed as a probe of genomic DNA from 12 isolates, of human and saprobic origin, digested with nine restriction enzymes. While we found B. microsporus Benjamin to be distinct, 9 human isolates designated B. haptosporus Drechsler, B. meristosporus Drechsler, and B. ranarum Eidam were indistinguishable from one another. In contrast, two unidentified saprobic isolates could be readily differentiated from the human ones. We conclude that all human isolates examined fall within a single monophyletic group, but that significant genetic differentiation has taken place between human and saprobic isolates that is not accounted for in existing taxonomies of the genus.

GENETIC STUDIES IN THE GENUS BASIDIOBOLUS. I. ISOZYME VARIATION AMONG ISOLATES OF HUMAN AND NATURAL POPULATIONS

Genetic variation and its distribution among populations and putative species of 70 isolates in the genus Basidiobolus were analyzed by means of electrophoretic analysis of soluble enzymes. Five enzyme systems were examined and showed large variation. Among laboratory isolates, only one of B. microsporus could be distinguished by means of cluster analysis. Patterns of variation among natural isolates revealed little differentiation with respect to host (toad vs lizard), but very strong patterns of geographical differentiation. Furthermore, natural isolates were quite distinct from laboratory strains tested. These results suggest that the genus Basidiobolus is genetically very heterogeneous, and this fact may contribute to difficulties encountered in defining taxonomic relationships.