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. Pentamidine, dibromopropamidine and CGP-4O-215 induced the appearance of the novel polyamine in the trophozoites after only 8 h of incubation with the drugs while induction by berenil occurred after 32 h. It is suggested that diamidine drugs either induce encystment or alter the pathway of polyamine metabolism in Acanthamoeba. If inducing encystment is the main mode of reaction, then drugs that induce early encystment will be less effective in treatment than those that delay it.

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. The quantity of dye bound to the trophozoites correlated (r = 0.99) with the number of organisms per well. When inhibition of the growth of Acanthamoeba was the only parameter used to measure the effectiveness of these drugs, saperconazole was found to be most active with IC50 (concentration of drug that inhibited by 50%, the growth of A. polyphaga trophozoites incubated at 28 degrees C for 48 h) of 0.95 microM. The IC50s of the other drugs ranged from > or = 1500 microM for micronazole, the least active, to 3.0 microM for berenil. However, when efficacy was assessed by combining inhibition of growth with the uptake of [14C-8]adenosine by the drug treated organism, the diamidines, particularly berenil and pentamidine were considered most potent.

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 +/- 0.45 pmol/ml/mg protein/min and 1.02 +/- 0.06 microM, respectively. The IC50s for the enantiomers were similar for (+)-R-AG and (-)-R-ChAG (0.86 +/- 0.06 and 0.89 +/- 0.15 microM, respectively. (+)S-ChAG was most potent with IC50 of 0.075 +/- 0.003 microM. The IC50s for (-)-S-AG, (+)-R-RG, and (-)-S-RG were in the same range (23.15 +/- 2.74, 24.58 +/- 2.46, and 24.43 +/- 2.20 microM, respectively.

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. In contrast, a strain of Leishmania donovani promastigotes clonally selected to grow in defined medium was only slightly (approximately 10%) inhibited at the logarithmic but not at the stationary phase. These results suggest that the differences in expression of adenosine in the parasites previously reported, may be related to uptake by a channel-like pathway in the promastigotes.

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. polyphaga 435/89 of active de novo purine biosynthesis in addition to a purine salvage pathway may be a general characteristics of free-living protozoan organisms since parasitic protozoans are devoid of de novo purine biosynthesis.

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. However, when 3H-adenosine was used as permeant, considerable differences in the inhibition profiles were noted among strains. Thus, both inosine transporter-selective nucleosides such as inosine and guanosine and adenosine transporter-selective nucleosides such as 2'-deoxyadenosine and tubercidin showed variable activity as inhibitors of 3H-adenosine transport in different strains. These observations indicated that an adenosine transporter was variably expressed in different strains, and that inhibition profiles for adenosine transport indicated cellular entry via both the inosine and adenosine transporters. The existence of different types of adenosine transporters as an alternative explanation could not be ruled out. The apparent uniform expression of an inosine transporter among different species and strains of Leishmania suggests that inosine derivatives may be useful as anti-leishmanial drugs.

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 3.9 +/- 0.2 of Syrian golden hamsters infected with Leishmania major. There was a statistical difference (p < 0.01) in the size of the foot pad by the fifth day between the infected groups that received treatment and the controls, as well as between the groups that were treated with combined drugs and FoA only. The initial reduction in size of the foot pad noted in the group that received only FoA was transient. The effect of FoA or FoA combined with NBMPR on the in vitro cultured promastigotes was similar, indicating that the transport inhibitor might be manipulating the availability of FoA in the host's macrophages where the leishmania amastigotes are resident. The results further indicate the need to explore the usefulness of combining cytotoxic nucleoside analogues with host protecting nucleoside transport inhibitors in the treatment of protozoan parasitic infections.

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. With the exception of formycin B, all nucleosides were phosphorylated intracellularly to varying degree, adenosine being almost fully phosphorylated. During the time course of routine experiments (30 s) formycin B was concentrated twofold over external medium levels (1 microM) without any drop-off in the transport rate. On the basis of metabolic studies it was estimated that uridine and tubercidin were also transported against a concentration gradient. Inosine, guanosine, 2'-deoxyadenosine, tubercidin, formycin B, and the pyrimidines uridine, thymidine, and cytidine (all 100 microM) inhibited transport of adenosine and inosine about 50-100%, while 3'-deoxyinosine showed weak inhibitory action. Transport of thymidine was strongly inhibited by nucleosides except by 3'-deoxyinosine. The Na(+)-dependent, active, and concentration transport system appears to be a feature of many immune-type cells, and its presence offers particular conceptual possibilities for the therapy of infections located in these cells.

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. This treatment also led to rounding of cells and thus permitted convenient and reproducible counting by either hemocytometer or electronic particle counter. The detaching fluid did not impair viability even after exposure up to 1 h.

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. The inhibition profile with [3H]inosine as permeant was essentially identical in S1 and S2 promastigotes, indicating that the same inosine transporter was present in both strains. However, with [3H] adenosine as permeant, significant differences were noted between the two strains. Thus, only adenosine, 2'-deoxyadenosine, tubercidin, uridine, and thymidine were strongly inhibitory in S1 promastigotes, while essentially all nucleosides tested were effective in S2 promastigotes. This indicates that adenosine transport in S2 promastigotes seems to involve a transporter differing from that described for S1 promastigotes.

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. The overall conclusion from this and previous studies is that the evaluation of relative potencies in screening of potential nucleoside transport inhibitors is best carried out at the level actual nucleoside transport studies in intact cells, since [3H(G)]NBMPR binding studies yield discrepant data.

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 +/- 0.4 and 0.44 +/- 0.05 nM, and maximal binding (Bmax) of 3.7 +/- 0.4 and 1.04 +/- 0.01 pmol/mg, respectively. The binding of [G-3H]NBMPR was inhibited in a concentration dependent manner by unlabelled NBMPR, dilazep and a new group of chemically related nucleoside transport inhibitors, mioflazine, soluflazine and R57974, the latter being the most potent derivative. R57974 displaced bound [G-3H]NBMPR as effectively as unlabelled NBMPR suggesting a common binding site. The assay procedure used appears useful for the rapid screening of the effectiveness of nucleoside transport inhibitors which will be of value for the selection of inhibitors suitable for combination with cytotoxic nucleosides in the treatment of selected cancers or parasitic diseases.

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. NBTI-P dephosphorylation progressed with similar rates in the presence or absence of NBTI in the incubation medium; ii. adenosine-5'-monophosphate inhibited the dephosphorylation of NBTI-P but did not affect the binding of NBTI. The inhibition was competitive and dependent on concentration; iii. the effect of the NBTI nonisotopic medium on the binding of free G-3H-NBTI and G-3H-NBTI derived from G-3H-NBTI-P was different, and an instantaneous isotopic dilution was observed with G-3H-NBTI, but not with G-3H-NBTI-P, as substrate.

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 0.38 +/- 0.06 mumole min-1 ml-1 packed cells respectively). Efflux of the nucleoside from erythrocytes of normal and infected mice were inhibited to the same extent by a specific nucleoside transport inhibitor, nitrobenzylthioinosine. Enumeration of nitrobenzylthioinosine binding sites on the erythrocytes showed that both have similar numbers of sites (6.2-6.6 X 10(3) sites/erythrocyte). It is concluded that the membrane permeability of the erythrocytes from infected mice was affected by the trypanosome in such a way as to enhance the purine nucleoside transport capacity. This may result in an increased supply of vital purine bases and nucleosides to trypanosomes which depend on their hosts for these nutrients.

Adenosine cycle in African trypanosomes.

African trypanosomes can convert adenosine to adenosine monophosphate. However, in Trypanosoma brucei, as in T. vivax and T. congolense, most of the adenosine is broken down to adenine before conversion to the nucleotide by adenine phosphoribosyltransferase. Trypanosoma brucei and T. vivax use the purine nucleoside hydrolase for adenosine cleavage while T. congolense uses purine nucleoside phosphorylase for the nucleoside cleavage. Trypanosoma vivax also deaminates adenine to hypoxanthine before its conversion to adenosine monophosphate by way of inosine monophosphate. All African trypanosomes lack adenosine deaminase. This finding particularly demonstrates that the effectiveness of the therapy of African trypanosomiasis with adenosine analogue drugs will depend upon the strain of trypanosome which causes the infection.