Replacing two conserved amino acids, (Asn-52 to Val and Try-67 to phe), in the Iso-1-cytochrome c gene has little effect on the function of the protein.

Tyr-67 and Asn-52 are among the conserved residues in the amino acid sequence of eukaryotic cytochromes c. These residues, together with Thr-78 are hydrogen bonded to a buried water molecule, (Wat-l66), which is also a conserved structural feature in all naturally occuring eukaryotic cytochromes c whose three dimensional structures have been determined. . Previous studies have suggested that during oxidation-reduction states W at- 166 plays a role in shifting both the side chains and the main chain at the bottom of the protein molecule. Replacement of Tyr-67 in rat cytochrome c with Phe and Asn-52 with He in yeast cytochrome c have been shown to result in more thermally stable proteins than the wild type. The Asn-52 to He mutation also results in exclusion of W at -166 with a substantial re-organization of hydrogen bonding in that region. Other replacement at position 52 using Ala for Asn result in a functional protein. The objective of this project was to replace the two conserved amino acids around the heme environment in the yeast iso-I-cytochrome c with a view of destabilizing Vat-166. The two amino acids (Tyr-67 and Asn-52) were replaced with Phe and Val on the wild type iso-I-cytochrome c gene in a multi-copy pING4 expression plasmid vector which already had a Cys-l 02 to Thr mutation. Yeast cells carrying the triple mutant cytochrome c gene were iv propagated on media with glycerol as the source of carbon. These mutant cells had similar growth pateros as those carrying the wild type gene in the absence of glucose, suggesting that the extra mutations did not have any effect on the expression of a functional cytochrome c in yeast. Cytochromes c were extracted and purified from yeast cells carrying the mutant and wild type genes. The total protein yields were comparable in both cases. The specllum of the oxidized form of the purified mutant protein showed a shift in the 69Snm peak to 70Snm, indicating that the intergrity of the methionine sulfur bonding with the heme. Kinetic assays using polarographic method with purified beef heart cytochrome oxidase showed similarities between the wild type and mutant proteins in the characteristic two activity sites on the oxidase with similar V max l & V m',,2 and Kml around the micro molar range and Km2 around the nano molar range for both proteins, suggesting that the mutations did not affect the conformational structure of the cytochrome c molecule

Epidemiology of vector-borne diseases in cattle from SE Uganda.

Institutions involved in vector-borne diseases research, epidemiological studies as well as vaccine development require reliable and sensitive assays to support the development of vaccine products and new drugs for treatment. These diagnostic assays also aid in identifying disease control target populations, and to monitor infection during trials for assessing the efficacy of preventive or curative drug. Molecular techniques such as the polymerase chain reaction (PCR) amplification have been used in detecting parasites of several species, sub-species and types and are favoured over microscopic examination of blood or the immunological methods because of their superior sensitivity and higher throughput. Two of the most commonly used diagnostic methods, microscopy and molecular techniques for pathogen detection and species characterization, were evaluated for their sensitivity and specificity and subsequently used in screening cattle for parasites in the blood of cattle kept under traditional mixed farming management system. Molecular methods revealed higher VBD prevalence in the cattle from the villages of Tororo and Busia districts of SE Uganda. The prevalence of trypanosome species pathogenic to livestock was found to be higher than previously documented in this area. Based on the data obtained by PCR amplification the effect of prophylactic drug intervention against trypanosomiasis was assessed over a period of six months. While isometamidium chloride treatment of cattle appeared to control trypanosomiasis in areas with low prevalence, the drug had no effect in controlling the disease in high prevalence areas. It would therefore be necessary to combine the use of drug intervention with other methods such as vector control, to reduce the prevalence, in order to realize effective control of trypanosomiasis

The effect of Trypanosoma brucei brucei infection on rabbit plasma iron and zinc concentrations

Changes in plasma iron and zinc concentration were studied in rabbits following a needle challenge with Trypanosoma brucei brucei clone ILTat 2.1. The infection resulted in a decrease of the concentration of both trace elements. Plasma iron concentrations decreased gradually and were decreased maximally to 53.3 percent of pre-infection levels on day 19 post-inoculation. Plasma zinc concentrations, on the other hand, decreased more rapidly and were decreased maximally to 27.4 percent of pre-infection levels on day 3 post-inoculation. The onset of these decreases coincided with the appearance of parasites in the peripheral blood. Furthermore, the magnitude of their decrease correlated closely with the level and duration of the parasitaemia. Other abnormal findings, namely, anaemia and periods of leucocytosis and leukopenia, were also observed. This study therefore demonstrates that depression in plasma iron and zinc concentrations is part of the acute phase response in rabbits infected with this clone of T.b brucei

Kinetics of hematopoietic progenitor cells during a Trypanosoma congolense rechallenge infection in Boran cattle

In a preliminary study, using clonogenic assays, the in vitro kinetics of committed haemopoietic progenitors were monitored during a Trypanosoma congolense rechallenge infection in five trypanosusceptible Boran cattle. Early in the infection (week 2), in the absence of any detectable parasitaemia, a drop in the number of nucleated marrow cells was recorded. This was accompanied by a marked but transient decrease in the levels of the colony-forming units-erythroid (CFU-E) followed by a partial recovery by weeks 3-4 after infection. The burst-forming units-erythroid (BFU-E) and the colony-forming units-granulocyte macrophage (CFU-GM) also significantly decreased between weeks 2 and 4. After a transient rise at weeks 3-5 postinfection, the CFU-GM steadily declined and remained below preinfection levels throughout the infection. The BFU-E remained below preinfection levels until the end of the experiment. The drop in nucleated marrow cells associated with the decreased numbers of CFU-E, BFU-E and CFU-GM was suggestive of a defect at the pluripotential stem cell level early in the infection (week 2). The erythrocyte indices, i.e. mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC), were unchanged until week 10 postinfection. Two animals became severely anaemic; one was euthanised at week 8 and one treated at week 9. The three remaining animals developed chronic anaemia with mean packed cell volume (PCV) fluctuating around 18%-19% between weeks 11 and 14. Low parasitaemia levels were recorded during that period. A CFU-E peak above preinfection levels was noted at week 12 and BFU-E appeared in the peripheral blood culture of two animals between weeks 11 and 14. A progressive rise in MCV associated with a gradual decrease in MCHC also characterised that period. A return to near preinfection levels was recorded for the numbers of all three progenitors three weeks after trypanocidal treatment followed by a full recovery five months after treatment. Although ineffective haemopoiesis has been suggested to contribute to the anaemia of bovine trypanosomiasis, this is the first demonstration of a negative effect on erythroid development in cultures of bone marrow of trypanosome-infected cattle