The acquisition of lipids by African trypanosomes

Bloodstream forms of African trypanosomes are dependent on their host for fatty acids, choline and other components of membrane lipids. The bulk of their choline requirement is met by their ability to take up lysophospholipids from the host tissue fluids. Trypanosoma brucei has ocyltransferose and phospholipase A(1) activities for the metabolism of exogenous lysophospholipids. The rate of uptake of lysophospholipids can be controlled by changes in the extrocellulor concentration of fatty ocyl-coenzyme A, and this control has potential for chemotherapy.     

Inhibition of yeast sporulation by ethidium bromide

Summary Ethidium bromide blocks ascus formation in the yeast Saccharomyces cerevisiae. This may mean that the presence of the mitochondrial genome is required for sporulation in this organism.     

Mitochondrial membranes and mutagenesis by ethidium bromide

The conversion of wild type (ρ⁺) to cytoplasmic petites (ρ^?) in Saccharomyces cerevisiae, à mutation in mitochondrial DNA, can be brought about with high efficiency by low concentrations of ethidium bromide (EB). The rate and extent of mutagenesis and its expression can be influenced, and even reversed, by a number of genetic lesions, agents or treatments affecting mitochondrial structure and metabolism. Among them are incubation at 45°, exposure to Antimycin A, growth on different carbon sources and the presence or absence of 2 different gene products previously implicated in the repair of UV induced lesions in mitochondrial DNA. Based on these observations a model for EB mutagenesis is advanced which postulates a complex between mitochondrial DNA and the inner membrane as the target susceptible to modification by EB. This model predicts that altered membranes should lead to changes in the susceptibility of cells to the mutagenic action of EB. This prediction has been verified by comparing cells that contain one of 2 structurally quite distinct monounsaturated C₁₈ fatty acids in their mitochondrial phospholipids: greater resistance to mutagenesis and ease of thermal protection is exhibited when cells – and mitochondria – contain oleic (Δ⁹cis, m.p. < 5°) rather than petroselinic (Δ⁶cis, m.p. 28°) acid in their phospholipids. As a corollary, studies on EB mutagenesis and mitochondrial DNA may be used as probes for the mitochondrial inner membrane to reveal some perhaps novel functions.     

Destruction of ethidium bromide in solution by ozonolysis

Ethidium bromide can be rapidly destroyed in aqueous solutions or in isoamyl alcohol by ozonolysis in the presence of H2O2 to give a mixture of organic acids. In a variety of buffers commonly used in recombinant DNA technology destruction of ethidium bromide was more than 99.9%. The yellow reaction mixture after ozonolysis was shown to be nonmutagenic. This method may be used in laboratories for the disposal of ethidium bromide wastes.     

The surface of the African trypanosomes

The African trypanosomes bear on the outside of their cell membrane a single 10-15 nm thick coat of a glycoprotein. This glycoprotein may differ in structure in the predominant populations of parasitemic waves found in relapsing infections. Variant Specific Glycoprotein (VSG) range in MW between 53,000-63,000 d and may have variable amounts of carbohydrate attached at one, two, or several loci. Such differences in carbohydrate content may account in part for their range in molecular size. Approximately 30 C-terminal residues demonstrate isotypy ; i.e. these regions fall into classes having similar amino acid sequence. Modest homology has been demonstrated in two VSGs of T. congolense arising in relapsing infections although comparison of many VSG show little or no obvious homology. More recently, lipid-associated forms of VSG have been described and it is believed that these forms may be transmembrane proteins. Different VSGs appear to have different amounts of the primary sequence which have alpha-helix-forming potential. In some VSG, in excess of 80% of the structure is helical as judged by both Chou-Fasman calculations and by circular dichroism. This raises the possibility that different VSG may have different folding patterns. The arrangement of VSG on the trypanosome surface probably places the basic amino acid-rich carbohydrate-bearing C-terminus of the polypeptide chain close to the membrane. There is some protein-protein association between VSGs for which (in T. evansi) the C-terminal tail is not required. The importance of VSG structure lies not only in the fact that the molecule mediates the phenomenon of antigenic variation but also in the recent observation that VSG may act on the cellular immune system to suppress the humoral immune responses of the host.     

Anisomorphic cell division by African trypanosomes

In the bloodstream of a mammalian host, African trypanosomes are pleomorphic; the shorter, non-proliferative, stumpy forms arise from longer, proliferative, slender forms with differentiation occurring via a range of morphological intermediates. In order to investigate how the onset of morphological change is co-ordinated with exit from the cell cycle we first characterized slender form cell division. Outgrowth of the new flagellum was found to occur at a linear rate, so by using outgrowth of the new flagellum as a temporal marker of the cell cycle we were able determine the order in which single copy organelles (nucleus, kinetoplast and mitochondrion) were segregated. We also found that flagellar length was an effective marker of the slender to stumpy differentiation and were, therefore, able to study both cell division and differentiation. When these differentiating cells were compared to cells undergoing proliferative cell division, they were found to be anisomorphic – showing discernible differences not only in the length of their new flagella but also in the shape and size of the cells and their nuclei.     

A yeast mitochondrial deoxyribonuclease stimulated by ethidium bromide

Several DNase activities, with different substrate and pH requirements, have been identified in yeast mitochondria. One of them is active on double stranded DNA at neutral pH and stimulated by Ethidium Bromide and other DNA intercalating drugs. This activity could be responsible for the yeast mitochondrial DNA degradation induced during mutagenesis by Ethidium Bromide.     

Modulation of innate immunity by African trypanosomes

The experimental studies of Brucei group trypanosomes presented here demonstrate that the balance of host and parasite factors, especially IFN-γ GPI-sVSG respectively, and the timing of cellular exposure to them, dictate the predominant MP and DC activation profiles present at any given time during infection and within specific tissues. The timing of changes in innate immune cell functions following infection consistently support the conclusion that the key events controlling host resistance occur within a short time following initial exposure to the parasite GPI substituents. Once the changes in MP and DC activities are initiated, there appears little that the host can do to reverse these changes and alter the final outcome of these regulatory events. Instead, despite the availability of multiple innate and adaptive immune mechanisms that can control parasites, there is an inability to control trypanosome numbers sufficiently to prevent the emergence and establishment of virulent trypanosomes that eventually kill the host. Overall it appears that trypanosomes have carefully orchestrated the host innate and adaptive immune response so that parasite survival and transmission, and alterations of host immunity, are to its ultimate benefit.     

The reversible inhibition of myoblast fusion by ethidium bromide (EB)

Ethidium bromide (EB) is a reversible inhibitor of myoblast fusion. The range over which EB is both inhibitory and reversible is narrow and is centered around 1 μg/ml. The EB sensitive period for myoblast fusion is very early during the induction of differentiation, at least 15 h prior to cell fusion. Fusion of myoblasts after release from EB inhibition does not require net DNA synthesis, nor is mitochondrial protein synthesis required for myoblast fusion. Rifampicin also causes similar reversible inhibition of fusion with some differences in the release kinetics.     

A synthesis of labeled ethidium bromide

A method is described for the synthesis of labeled ethidium (3,8-diamino-5-ethyl-6-phenyl phenanthridinium) bromide. Based on benzoic acid, the radioactive precursor used, the yield is 15% of a compound that is indistinguishable from authentic ethidium bromide in its absorption spectra (uv, visible, ir), Chromatographie behavior, and mutagenic effectiveness in the induction of respiration-deficient cell lines in baker's yeast.