Brian D. Hansen

The specificity of purine base and nucleoside uptake in promastigotes of Leishmania braziliensis panamensis

Promastigotes of Leishmania braziliensis panamensis absorbed the purines adenine, hypoxanthine, adenosine and inosine by a combination of diffusion and mediated components. When the uptake rates for these substrates were corrected for diffusion and compared, the purine bases adenine and hypoxanthine were transported at a significantly slower rate than the purine nucleosides adenosine and inosine. Competitive interactions among those purines tested confirmed the presence of mediated and diffusion components and suggested that three transport loci may be operating (Fig. 6). The first transport locus, designated Locus 1, transported inosine, Locus 2, the purine bases hypoxanthine and adenine and Locus 3, adenosine. In addition, adenine and hypoxanthine inhibited the uptake of one another competitively. A comparison of Ki values derived from double reciprocal plots of labelled hypoxanthine and adenine uptake in the presence of the unlabelled substrates as inhibitors suggested that adenine has a greater affinity for the transport locus.

Leishmania mexicana: Purine metabolism in promastigotes, axenic amastigotes, and amastigotes derived from vero cells

Leishmania mexicana mexicana promastigotes, axenic amastigotes, and amastigotes derived from Vero cells were examined for de novo purine synthesis and mechanisms of purine salvage. Both promastigotes and axenic amastigotes were incapable of de novo purine synthesis, as shown by the lack of [14C]formate and [14C]glycine incorporation into purine nucleotide pools. However, the ready incorporation of [14C]hypoxanthine, [14C]adenine, and [14C]guanine suggested that purine salvage pathways were operating. In addition, a significant percentage (greater than or equal to 60%) of the total label from these purine precursors was associated with adenylate nucleotides. Nucleotide pool levels of axenic amastigotes were consistently greater but the specific activities were less than those of promastigotes, suggesting a slower rate of purine metabolism in the axenic amastigote form. Similar results were obtained from amastigotes isolated from infected Vero cells.

Evidence for a membrane adenosine receptor in Leishmania mexicana mexicana (WR 227).

Leishmania spp., a Protozoan Parasite transmitted to the mammalian host by the sand fly (genus Phlebotomus), produces a disease manifested in the cutaneous, mucocutaneous or visceral form. The choice of drugs has been limited primarily to the pentavalent antimonial compounds. Successful treatment within the endemic tropical areas of the world varies due to problems of drug resistance and to strain differences in Leishmania Spp. These difficulties have prompted the search for new effective oral antileishmanial agents and ultimately the development of a vaccine. We are currently studying proteins on the parasite plasma membrane (enzymes, receptors, transporters) which may be functionally vital to the organism and amenable to chemotherapeutic attack or elicit a host immune response. The present study examines evidence for an adenosine receptor on the surface membrane of both the promastigote (insect stage) and the amastigote form (mammalian stage) and the resultant modu-lation of adenylate cyclase activity, intracellular cyclic AMP levels, i the rate of cell growth and parasite transformation.

Regulation of HIV Replication in Monocytes by Interferon

The hallmark of human immunodeficiency virus (HIV) infection is the progressive loss of CD4+ T-cells over a prolonged interval. In the infected individual, two types of cells are infected by HIV: CD4+ T-cells and tissue macrophages. Levels of HIV in blood and tissues are dependent upon and change with the stage of the infection. Acute infection, usually lasting weeks to months after initial exposure to the virus, is characterized by a substantial viremia in which HIV actively replicates within blood leukocytes and high titers of free virus are found in plasma (more than 10 000 infectious virions/ml blood) (Clark et al. 1991; Daar et al. 1991). The chronic subclinical phase of infection is notable for low levels of plasma viremia and of virus-infected cells (less than 100 infectious virions/ml blood) (Ho et al. 1989). The major reservoirs for HIV in blood are CD4+ T-cells which are infected at a frequency of about 0.1 to 1% (Schnittman et al. 1989). HIV-infected CD4+ T-cells have on average only one proviral DNA copy integrated into genomic DNA. Less than 0.1% of these infected cells is transcriptionally active at any given time (Harper et al. 1986; Simmonds et al. 1990). During subclinical infection, the frequency of blood cells that express HIV mRNA and presumably produce infectious virus is only 0.01% (Harper et al. 1986; Clarke et al. 1990; Daar et al. 1991). During subclinical disease when very few cells are producing virus in blood, it is likely that cells in tissue provide most of the actively replicating virus that maintains infection during the long latent interval of 8 to 12 years (Lifson et al. 1988).

EVIDENCE FOR A MEMBRANE ADENOSINE RECEPTOR IN LEISHMANIA MEXICANA MEXICANA (WR224): 79

Plasma membranes from the promastigote and amastigote forms of Leishmania mexicana mexicana were examined for the presence of an adenosine receptor. Specific binding of selected adenosine receptor ligands was tested for modulation of membrane associated adenylate cyclase activity. Time course experiments utilizing amastigote and promastigote membranes demonstrated that total binding of all adenosine receptor ligands equilibrated within 20 min. All incubations of membranes were conducted for 30 min. Specific, nonspecific and total binding of 3H-cyclohexyladenosine (CHA) and 3H-methyl-2-phenylethyl-adenosine (PIA) to promastigote and amastigote membranes over ligand concentrations of 8 to 400 nM were determined. Scatchard plot analysis of the specific binding data indicated single binding sites for both CHA and PIA with Kd values of 75 nM and 200 nM respectively for promastigotes and 55 nM and 175 nM respectively for amastigotes. Membranes were also found to possess adenylate cyclase activity which could be readily inhibited in the presence of increasing concentrations of adenosine, PIA, and CHA (0 to 1 mM). The rate of promastigote replication and transformation to the amastigote form and transformation of the amastigote to the promastigote stage could all be significantly increased in the presence of the adenosine receptor ligands. This receptor mechanism may therefore act to modulate the rate of cell replication and life cycle transformation.