Molecular cloning, expression and characterization of ribokinase of Leishmania major.

Ribokinase (EC 2.1.7.

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.

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.

In vitro effect of diamidines on intracellular polyamines of Acanthamoeba polyphaga.

Treatment with the drugs berenil, dibromopropamidine, pentamidine and CGP-4O-215 were found to alter levels of intracellular polyamines of Acanthamoeba polyphagia trophozoites in organisms. While the polyamine, spermidine, consistently remained higher in the control organisms incubated at 8, 16 and 32 h respectively, the level fluctuated significantly from below 5% to 40% in drug-treated organisms. A novel polyamine (polyamine F), yet to be identified, representing 80% of the total polyamine extracts and seen in control organisms after 48 h of incubation (stationary phase), was seen much earlier in the drug treated organisms.

In vitro assessment of susceptibility of Acanthamoeba polyphaga to drugs using combined methods of dye-binding assay and uptake of radiolabelled adenosine.

A technique based on binding of eosin dye to cell was applied to quantitate Acanthamoeba trophozoites in culture. Using this technique in combination with the uptake of radiolabelled adenosine, we assessed the activities of triazole (saperconazole), imidazole (ketoconazole, miconazole) and diamidine (berenil, pentamidine, dibromopropamidine) compounds against A. polyphaga trophozoites.

In vitro inhibition of aromatase by the enantiomers of aminoglutethimide and analogs.

The in vitro aromatase activity in microsomal fractions from rat ovary and its inhibition by enantiomers of aminoglutethimide (AG), rogletimide (RG), and cyclohexylaminoglutethimide (ChAG) were studied by analysing the [3H]H2O released when [1 beta-3H]androstenedione was converted to estrone. Maximum velocity (Vmax) and the Michaelis-Menten constant (Km) of the microsomal aromatase enzyme were 17.40 +/- 0.

Expression of a channel-like pathway for adenosine transport in Leishmania donovani promastigotes.

L. donovani promastigotes (MHOM/ET/67/HA3) transport adenosine by a route that is sensitive to inhibition by a nonspecific channel blocker, propranolol. At the logarithmic and stationary phases of growth, the transport of 1 microM-3H-adenosine was significantly inhibited (40-50%) by 100 microM-propranolol.

De novo purine biosynthesis in a free living protozoan parasite, Acanthamoeba polyphaga [correction of polypha].

A free living protozoon, Acanthamoeba polyphaga 435/89, was shown to utilize radiolabelled formate and glycine as precursors for purine biosynthesis and to grow in medium devoid of any source of preformed purine compounds. The organism also incorporated into its nucleic acids radiolabel derived from adenosine and guanosine indicating presence of purine salvage pathway. Thus, the presence in A.

Nucleoside transporters in Leishmania major: diversity in adenosine transporter expression or function in different strains.

Cytotoxic nucleoside derivatives may become useful in the treatment of parasitic infections. As part of our drug development studies, the effect of a number of nucleosides (100 microM) on the cellular transport of 3H-adenosine and 3H-inosine (each at 1 microM) in promastigotes from four Leishmania major strains was investigated. When 3H-inosine was used as permeant, all strains exhibited essentially the same inhibition profile, with unlabeled inosine, guanosine, formycin B, and 3'-deoxyinosine being strongly inhibitory, and adenosine-related compounds such as 2'-deoxyadenosine and tubercidin being inactive.

Sodium-dependent nucleoside transport in mouse lymphocytes, human monocytes, and hamster macrophages and peritoneal exudate cells.

Mouse splenocytes and hamster peritoneal exudate cells (PEC), including macrophages, were shown to contain a predominantly Na(+)-dependent and inhibitor (6-[(4-nitrobenzyl)-mercapto]purine ribonucleoside, NBMPR)-resistant transport system for adenosine and other nucleosides. Adenosine (1 microM) was transported about equally in mouse thymocytes and human monocytes from peripheral blood by a Na(+)-dependent system and the NBMPR-sensitive facilitated diffusion system. Hamster PEC also transported inosine, tubercidin, formycin B, uridine, and thymidine in a NBMPR-insensitive manner.

Experimental chemotherapy of leishmaniasis with adenosine analogue Formycin A, in combination with inhibitor of nucleoside transport, nitrobenzylthioinosinate.

A single dose of the adenosine analogue Formycin A (FoA) (20 mg/kg), combined with nitrobenzyl mercaptopurine ribonucleoside 5'-monophosphate (NBMPR-P) (10 mg/kg), a prodrug of nitrobenzylthioinosine (NBMPR), was effective in reducing the size of the foot pad lesions from 7.4 +/- 0.2 to 3.

New method for detachment and quantitation of Acanthamoeba trophozoites in culture.

Trophozoites of two Acanthamoeba polyphaga strains (RYD and 435) were readily detached from plastic culture flasks or multiwell plates by 5 min exposure to a detaching fluid containing 0.005% of Triton X-100 and 0.05% ammonium sulphate.

Leishmania donovani: characteristics of adenosine and inosine transporters in promastigotes of two different strains.

The nucleoside transport characteristics of two strains of Leishmania donovani promastigotes were studied. Strain S1, growing in fully defined medium, and strain S2 (MHOM/ET/67/HA3) both transported adenosine and inosine, but only strain S1 transported uridine and thymidine. Competition studies in the presence of 100 microM of unlabeled adenosine, inosine, guanosine, 2'-deoxyadenosine, tubercidin, formycin B, 3'-deoxyinosine as well as uridine, thymidine and cytidine, with either 1 microM [3H]adenosine or [3H]inosine as permeant, were carried out.

Competition of nucleoside transport inhibitors with binding of 6-[(4-nitrobenzyl)-mercapto]purine ribonucleoside to intact erythrocytes and ghost membranes from different species.

The potency of nucleoside transport inhibitors, including 6-[(4-nitrobenzyl)-mercapto]purine ribonucleoside (NBMPR), dilazep, mioflazine and its derivatives soluflazine and R57974 as inhibitors of the binding of [3H(G)]NBMPR to intact erythrocytes and respective ghost membranes from human, mouse and hamster was determined. There was no close agreement between the IC50 profiles for the different inhibitors when comparing values obtained for intact cells and membranes from each species, and there was no consistent profile of differences when considering individual drugs and comparing their actions in the three species. Present data also were compared with potency values obtained previously with the same drugs directly in nucleoside transport inhibition studies with erythrocytes from the same species as well as with [3H(G)]NBMPR binding studies in isolated liver and lung membranes from hamster.

Binding of [G-3H]6-(4-nitrobenzylmercapto)purine ribonucleoside to isolated membranes. Inhibitory effect of mioflazine and its derivatives.

The binding of [G-3H]-6-(4-nitrobenzylmercapto)purine ribonucleoside [( G-3H]NBMPR) was investigated using a centrifugation assay with membrane preparations from hamster tissues including liver, lung, kidney and heart. Only liver and lung membranes showed high specific binding, with dissociation constants (Kd) values of 2.4 +/- 0.

A simple procedure for the isolation of schistosomules for biochemical studies or culture.

Incubation of cercariae of Schistosoma mansoni in a 1:1 mixture of Eagle's Minimal Essential Medium and rat serum at 42 degrees C for only 5 minutes, plus subsequent vortexing, resulted in at least 98% conversion of cercariae to schistosomules. Subsequent centrifugation before or after settling for 30 minutes in an ice bath, resulted in schistosomule preparations with about 20% or 10% contamination with tail segments, respectively.

Nitrobenzylthioinosinate binding sites in HeLa cells: relationship with ecto-5'-nucleotidase.

Nitrobenzylthioinosine (NBTI), a substrate for the ecto-5'-nucleotidase of HeLa cells, was used to probe the relationship between ecto-5'-nucleotidase dephosphorylation site and the dephosphorylated NBTI binding site. It may be assumed that the dephosphorylation site of the enzyme is separate and functions independently of the NBTI binding site. Evidences supporting these conclusions were based on the following observations: i.

Efflux of 3H-thymidine by erythrocytes from mice infected with Trypanosoma brucei brucei.

Erythrocytes from mice infected with Trypanosoma brucei brucei showed a higher rate of efflux of labelled thymidine than did control erythrocytes from uninfected mice (0.56 +/- 0.10 and 0.

Adenosine cycle in African trypanosomes.

African trypanosomes can convert adenosine to adenosine monophosphate. However, in Trypanosoma brucei, as in T. vivax and T.

Dephosphorylation of nitrobenzylthioinosine 5'-monophosphate by ecto 5'-nucleotidase of HeLa cells.

HeLa cells as well as human and mouse erythrocytes possess membrane sites which bind the inhibitor of nucleoside transport, nitrobenzylthioinosine (NBMPR), reversibly but tightly (KD, 10(-9)-10(-10) M). Site-specific binding of the ligand correlates with inhibition of nucleoside transport. The present study showed that the 5'-phosphate of NBMPR, NBMPR-P, was not transport inhibitory.

Comparative aspects of purine metabolism in some African trypanosomes.

Some enzymes of purine salvage were detected in the cell-free preparations from bloodstream forms of African trypanosomes: Trypanosoma vivax; T. brucei and T. congolense.