Biomedical subjects

Richard J S Burchmore

Publications and source records attributed to Richard J S Burchmore.

8 recordsLinked to original sources

PubMed2005

Purine and pyrimidine transport in pathogenic protozoa: from biology to therapy.

Purine salvage is an essential function for all obligate parasitic protozoa studied to date and most are also capable of efficient uptake of preformed pyrimidines. Much progress has been made in the identification and characterisation of protozoan purine and pyrimidine transporters. While the genes encoding protozoan or metazoan pyrimidine transporters have yet to be identified, numerous purine transporters have now been cloned. All protozoan purine transporter-encoding genes characterised to date have been of the Equilibrative Nucleoside Transporter family conserved in a great variety of eukaryote organisms. However, these protozoan transporters have been shown to be sufficiently different from mammalian transporters to mediate selective uptake of therapeutic agents. Recent studies are increasingly addressing the structure and substrate recognition mechanisms of these vital transport proteins.

Animals↗

PubMed2004

The trypanocide diminazene aceturate is accumulated predominantly through the TbAT1 purine transporter: additional insights on diamidine resistance in african trypanosomes.

Resistance to diminazene aceturate (Berenil) is a severe problem in the control of African trypanosomiasis in domestic animals. It has been speculated that resistance may be the result of reduced diminazene uptake by the parasite. We describe here the mechanisms by which [(3)H]diminazene is transported by Trypanosoma brucei brucei bloodstream forms. Diminazene was rapidly accumulated through a single transporter, with a K(m) of 0.45 +/- 0.11 micro M, which was dose dependently inhibited by pentamidine and adenosine. The K(i) values for these inhibitors were consistent with this transporter being the P2/TbAT1 adenosine transporter. Yeast expressing TbAT1 acquired the ability to take up [(3)H]diminazene and [(3)H]pentamidine. TbAT1-null mutants had lost almost all capacity for [(3)H]diminazene transport. However, this cell line still displayed a small but detectable rate of [(3)H]diminazene accumulation, in a nonsaturable manner. We conclude that TbAT1 mediates [(3)H]diminazene transport almost exclusively and that this explains the observed diminazene resistance phenotypes of TbAT1-null mutants and field isolates.

Adenosine↗

PubMed2003

The trypanosomiases.

The trypanosomiases consist of a group of important animal and human diseases caused by parasitic protozoa of the genus Trypanosoma. In sub-Saharan Africa, the final decade of the 20th century witnessed an alarming resurgence in sleeping sickness (human African trypanosomiasis). In South and Central America, Chagas' disease (American trypanosomiasis) remains one of the most prevalent infectious diseases. Arthropod vectors transmit African and American trypanosomiases, and disease containment through insect control programmes is an achievable goal. Chemotherapy is available for both diseases, but existing drugs are far from ideal. The trypanosomes are some of the earliest diverging members of the Eukaryotae and share several biochemical peculiarities that have stimulated research into new drug targets. However, differences in the ways in which trypanosome species interact with their hosts have frustrated efforts to design drugs effective against both species. Growth in recognition of these neglected diseases might result in progress towards control through increased funding for drug development and vector elimination.

Animals↗

PubMed2003

Pentose phosphate metabolism in Leishmania mexicana.

The metabolism of pentose phosphates was studied in Leishmania mexicana promastigotes. Each of the enzymes of the classical pentose phosphate pathway (PPP) has been identified and specific activities measured. Functioning of the PPP was demonstrated in non-growing cells by measuring the evolution of 14CO2 from [1-14C]D-glucose and [6-14C]D-glucose under normal conditions and also under selective stimulation of the PPP by exposure to methylene blue. The proportion of glucose which passes through the PPP increases in the latter condition, thus suggesting a protective role against oxidant stress. The incorporation into nucleic acids of ribose 5-phosphate provided via either glucose or free ribose was also determined. Results indicate that the PPP enables glucose to serve as a source of ribose 5-phosphate in nucleotide biosynthesis. Moreover, free ribose is incorporated efficiently, implying the presence of a ribose uptake system and also of ribokinase. Ribose was shown to be accumulated by a carrier mediated process in L. mexicana promastigotes and ribokinase activity was also measured in these cells.

Animals↗

PubMed2003

Cloning, heterologous expression, and in situ characterization of the first high affinity nucleobase transporter from a protozoan.

While multiple nucleoside transporters, some of which can also transport nucleobases, have been cloned in recent years from many different organisms, no sequence information is available for the high affinity, nucleobase-selective transporters of metazoa, parazoa, or protozoa. We have identified a gene, TbNBT1, from Trypanosoma brucei brucei that encodes a 435-residue protein of the equilibrative nucleoside transporter superfamily. The gene was expressed in both the procyclic and bloodstream forms of the organism. Expression of TbNBT1 in a Saccharomyces cerevisiae strain lacking an endogenous purine transporter allowed growth on adenine as sole purine source and introduced a high affinity transport activity for adenine and hypoxanthine, with Km values of 2.1 +/- 0.6 and 0.66 +/- 0.22 microm, respectively, as well as high affinity for xanthine, guanine, guanosine, and allopurinol and moderate affinity for inosine. A transporter with an indistinguishable kinetic profile was identified in T. b. brucei procyclics and designated H4. RNA interference of TbNBT1 in procyclics reduced cognate mRNA levels by approximately 80% and H4 transport activity by approximately 90%. Expression of TbNBT1 in Xenopus oocytes further confirmed that this gene encodes the first high affinity nucleobase transporter from protozoa or animals to be identified at the molecular level.

Amino Acid Sequence↗

PubMed2003

Genetic characterization of glucose transporter function in Leishmania mexicana.

Both insect and mammalian life cycle stages of Leishmania mexicana take up glucose and express all three isoforms encoded by the LmGT glucose transporter gene family. To evaluate glucose transporter function in intact parasites, a null mutant line has been created by targeted disruption of the LmGT locus that encompasses the LmGT1, LmGT2, and LmGT3 genes. This deltalmgt null mutant exhibited no detectable glucose transport activity. The growth rate of the deltalmgt knockout in the promastigote stage was reduced to a rate comparable with that of WT cells grown in the absence of glucose. deltalmgt cells also exhibited dramatically reduced infectivity to macrophages, demonstrating that expression of LmGT isoforms is essential for viability of amastigotes. Furthermore, WT L. mexicana were not able to grow as axenic culture form amastigotes if glucose was withdrawn from the medium, implying that glucose is an essential nutrient in this life cycle stage. Expression of either LmGT2 or LmGT3, but not of LmGT1, in deltalmgt null mutants significantly restored growth as promastigotes, but only LmGT3 expression substantially rescued amastigote growth in macrophages. Subcellular localization of the three isoforms was investigated in deltalmgt cells expressing individual LmGT isoforms. Using anti-LmGT antiserum and GFP-tagged LmGT fusion proteins, LmGT2 and LmGT3 were localized to the cell body, whereas LmGT1 was localized specifically to the flagellum. These results establish that each glucose transporter isoform has distinct biological functions in the parasite.

Acetyltransferases↗

PubMed2002

Chemotherapy of human African trypanosomiasis.

Human African trypanosomiasis or sleeping sickness is resurgent [1,2]. The disease is caused by subspecies of the parasitic haemoflagellate, Trypanosoma brucei. Infection starts with the bite of an infected tsetse fly (Glossina spp.). Parasites move from the site of infection to the draining lymphatic vessels and blood stream. The parasites proliferate within the bloodstream and later invade other tissues including the central nervous system. Once they have established themselves within the CNS, a progressive breakdown of neurological function accompanies the disease. Coma precedes death during this late phase. Two forms of the disease are recognised, one caused by Trypanosoma brucei rhodesiense, endemic in Eastern and Southern Africa, in which parasites rapidly invade the CNS causing death within weeks if untreated. T. b. gambiense, originally described in West Africa, but also widespread in Central Africa, proliferates more slowly and can take several years before establishing a CNS-involved infection. Many countries are in the midst of epidemics caused by gambiense-type parasites. Four drugs have been licensed to treat the disease [3]; two of them, pentamidine and suramin, are used prior to CNS involvement. The arsenic-based drug, melarsoprol is used once parasites are established in the CNS. The fourth, eflornithine, is effective against late stage disease caused by T. b. gambiense, but is ineffective against T. b. rhodesiense. Another drug, nifurtimox is licensed for South American trypanosomiasis but also been used in trials against melarsoprol-refractory late sage disease. This review focuses on what is known about modes of action of current drugs and discusses targets for future drug development.

Animals↗

PubMedDate not supplied

Detection of arsenical drug resistance in Trypanosoma brucei with a simple fluorescence test.

The resurgence of human African trypanosomiasis (HAT), coupled with an increased incidence of drug resistance, is of concern. We report a quick, simple, and sensitive test for identification of parasites resistant to melarsoprol, the main drug used to treat late stage HAT. Resistant parasites are defective in a plasma membrane transporter responsible for drug uptake. The same transporter carries the fluorescent diamidine DB99 (2,5-bis-(4-amidinophenyl)-3,4-dimethylfuran) into trypanosomes. The two DNA-containing structures in the trypanosome--the nucleus and the kinetoplast--begin to fluoresce within 1 min of introduction of DB99, unless drug resistant.

Animals↗