Inhibition of the hexokinase/hexose transporter region in the glycosomal membrane of bloodstream Trypanosoma brucei by oligomycin and digitonin

Glycolysis in bloodstream T. brucei is the sole source of energy and remains a favourable chemotherapeutic target. In furtherance of this, an attempt has been made to understand better the contribution of glucose, fructose, mannose and glycerol to the energy charge of these parasites incubated in the presence of oligomycin, salicyhydroxamic acid (SHAM) and digitonin. Their cellular energy charge, when catabolizing glucose was 0.860, and under inhibition by oligomycin (10 microg), SHAM (2 mM) or oligomycin plus SHAM, 0.800, 0.444 and 0.405, respectively. Oligomycin inhibited the rate of catabolism of glucose, mannose and fructose up to 80%. The inhibition could not be alleviated by uncouplers, such as 2,4-dinitrophenol or permeabilization of the membranes by digitonin. Glucose-6-phosphate and other phosphorylated glycolytic intermediates, such as fructose-6-phosphate were catabolized by the permeabilized parasites in the presence of oligomycin, implying that except hexokinase, all the other glycolytic enzymes were active. Glucose oxidation was stimulated by low concentrations of digitonin (up to 4 microg), but at higher concentrations, it was significantly inhibited (up to 90% inhibition at 10 microg). Apparently, the inhibitory effects of oligomycin and digitonin were confined to glucose uptake and hexokinase catalysis. The above observations suggest that the hexose transporter and the enzyme hexokinase might be functionally-linked in the glycosomal membrane and oligomycin inhibits the linkage, by using a mechanism not linked to the energy charge of the cell. Digitonin at concentrations higher than 4 microg disrupted the membrane, rendering the complex in-operative. A hexokinase/hexose transporter complex in the glycosomal membrane is envisaged.     

Photochemistry of dyes and fluorochromes used in biology and medicine: some physicochemical background and current applications

An overview of the basic principles of photochemistry is presented to facilitate discussion of fluorescence, quenching and quantum yields. These topics in turn provide the foundation for an account of fluorescence spectroscopy and its application to microscopy. A brief overview of light microscopy and the application of fluorescence microscopy is given. The influences of molecular features, such as aromatic character and substitution patterns, on color and fluorescence are described. The concept of color fading is considered with particular reference to its effect on microscopic preparations. A survey of representative fluorescent probes is provided, and their sensitivity, application, and limitations are described. The phototoxicity of fluorescent molecules is discussed using biomembranes and DNA as examples of targets of toxicity. Photodynamic therapy, a relatively new clinical application of phototoxicity, is described. Both anticancer and antimicrobial applications are noted, and an assessment is given of the current ideas on the ideal physicochemical properties of the sensitizing agents for such applications.     

Cortisol stress response and histopathological alteration index in Clarias gariepinus exposed to sublethal concentrations of Qua Iboe crude oil and rig wash

The histological effect on and stress response of post juvenile Clarias gariepinus exposed to Qua Iboe crude oil and rig wash were investigated. Fish weighing 60–90 g and measuring 16–18 cm were exposed for 7–28 days to 8.00 ml^?1 Qua Iboe crude oil and 0.0018 ml^–1 rig wash, both being 0.1 of the 96 hr LC₅₀. Blood samples of C. gariepinus were collected every seven days and evaluated for stress by measuring cortisol concentration. The gills and liver were studied and scored for Gill Alteration Index (GAI) and Hepatic Alteration Index (HAI), respectively. There was an increase in cortisol level up to the 7th and 14th day among the group exposed to Qua Iboe crude oil, with a decrease on the 21st and 28th day. The rig wash group increased in cortisol level up to the 7th day and decreased slightly on the 14th day, after which the trend became irregular. The toxic effects of the Qua Iboe crude oil and rig wash were time dependent, as shown by the histopathological alteration index (HAI) of gill and liver. After 28 days of exposure, the gills had irreparable damage due to high frequency of cellular necrosis and degeneration, whereas the liver had from moderate to severe damage due to the high frequency of cellular degeneration and inflammation. Qua Iboe crude oil and rig wash are both toxic to C. gariepinus, therefore their indiscriminate discharge to the environment must be discouraged.     

Pathways of glucose catabolism in procyclic Trypanosoma congolense

Studies of respiration on glucose in procyclic Trypanosoma congolense in the presence of rotenone, antimycin, cyanide, salicylhydroxamic acid and malonate have indicated the presence of NADH dehydrogenase, cytochrome b-c1, cytochrome aa3, trypanosome alternate oxidase and NADH fumarate reductase/succinate dehydrogenase pathway that contributes electrons to coenzyme Q of the respiratory chain. The rotenone sensitive NADH dehydrogenase, the trypanosome alternate oxidase, and cytochrome aa3 accounted for 24.5 +/- 6.5, 36.2 +/- 4.2 and 54.1 +/- 5.5% respectively of the total respiration. Activities of lactate dehydrogenase, NAD(+)-linked malic enzyme and pyruvate kinase were less than 6 nanomoles/min/mg protein suggesting that they play a minor role in energy metabolism of the parasite. Phosphoenolpyruvate carboxykinase, pyruvate dehydrogenase, succinate dehydrogenase, NADP(+)-linked malic enzyme, NADH fumarate reductase, malate dehydrogenase, and alpha-ketoglutarate dehydrogenase and glycerol kinase on the other hand had specific activities greater than 60 nanomoles/min/mg protein. These enzyme activities could account for the production of pyruvate, acetate, succinate and glycerol. The results further show that the amount of glycerol produced was 35-48% of the combined total of pyruvate, acetate and succinate produced. It is apparent that some of the glycerol 3-phosphate produced in glycolysis in the presence of salicylhydroxamic acid is dephosphorylated to form glycerol while the rest is oxidised via cytochrome aa3 to form acetate, succinate and pyruvate.     

Hypomethylation of DNA during differentiation of friend erythroleukemia cells

DNA from mammalian cells has been shown to contain significant amounts of 5-methyl cytosine resulting from enzymatic transfer of methyl groups from s-adenosylmethionine to cytosine residues in the DNA polymer. The function of this modification is not known. We have found that DNA synthesized during chemically induced differentiation of friend erythroleukemia cells is hypomethylated, as measured by its ability to accept methyl groups transferred by homologous DNA methyltransferases in vitro. The extent of hypomethylation detected by this sensitive method is small, a decrease of less than 1.6 percent in 5-methylcytosine content. Hypomethylated DNA can be isolated from friend erythroleukemia cells grown in the presence of dimethyl sulfoxide, butyrate, hexamethylene-bis- acetamide, pentamethylene-bis acetamide, and ethionine. However, hypomethylated DNA is found only under conditions where differentiation is actually induced. DNA isolated from cells of a dimethyl sulfoxide- resistant subclone grown in the presence of that agent is not hypomethylated, although DNA of these cells becomes hypomethylated after growth in the presence of inducers that can trigger their differentiation. We also find that the DNA of friend erythroleukemia cells does not become hypomethylated when the cells are exposed to inducing agents in the presence of substances that inhibit differentiation. These results suggest a close link between genome modification by methylation and differentiation of friend erythroleukemia cells.