Burt Goldberg

Production of superoxide anion during the oxidation of hemoglobin by menadione

Menadione in the presence of oxyhemoglobin will accelerate the formation of methemoglobin and result in the generation of superoxide anion. Menadione appears to oxidize slowly ferrohemoglobin to ferrihemoglobin, while forming menadione semiquinone in the process. Menadione semiquinone is known to react with molecular oxygen to yield superoxide anion. The superoxide anion appears to be the source of hydrogen peroxide which accounts for most of the observed methemoglobin formation when hemoglobin is reacted with menadione.

Resistance to dl-α-difluoromethylornithine by clinical isolates of Trypanosoma brucei rhodesiense: Role of S-adenosylmethionine

The ornithine decarboxylase (ODC) inhibitor DL-alpha-difluoromethylornithine (DFMO) has emerged as a new treatment for West African sleeping sickness but is less effective against East African sleeping sickness. We examined uncloned clinical isolates of Trypanosoma brucei rhodesiense, agent of the disease in East Africa, which were refractory to DFMO in laboratory infections, for characteristics that would explain their resistance. None of the isolates were from patients treated with DFMO. Two isolates took up [3H]DFMO at 50-70% lower rates than drug-sensitive strains but ODC activities, Ki values for DFMO, spermidine and spermine uptake rates, polyamine content and inhibition of polyamine metabolism by DFMO were statistically (P < 0.05) similar between sensitive and refractory isolates. One cloned strain, continuously passaged in vivo under DFMO pressure and included for comparison, had > 85% lower ODC activity and up to 14-fold higher putrescine uptake rates than sensitive controls. A statistically important trend was the metabolism of S-adenosylmethionine (AdoMet): activities of AdoMet synthetase and AdoMet decarboxylase were 2- to 5-fold and 3- to 40-fold lower in resistant strains, respectively, while intracellular AdoMet pools (AdoMet + decarboxylated AdoMet) that were > 60-fold elevated in sensitive strains during DFMO treatment, increased only 9-fold in refractory isolates. The extreme elevation of the AdoMet pool in sensitive isolates from 0.7 to 44 nmol/mg protein and an intracellular pool concentration of approximately 5 mM may lead to an imbalance in methylation of proteins or other cell constituents as a consequence of DFMO action. These studies indicate that the metabolism of AdoMet is altered significantly in DFMO refractory isolates and suggest that differences in AdoMet metabolism may be responsible for increased tolerance to DFMO.

S-adenosylmethionine synthetase in bloodstream Trypanosoma brucei.

S-adenosylmethionine synthetase was studied from bloodstream forms of Trypanosoma brucei brucei, the agent of African sleeping sickness. Two isoforms of the enzyme were evident from Eadie Hofstee and Hanes-Woolf plots of varying ATP or methionine concentrations. In the range 10-250 microM the Km for methionine was 20 microM, and this changed to 200 microM for the range 0.5-5.0 mM. In the range 10-250 microM the Km for ATP was 53 microM, and this changed to 1.75 mM for the range 0.5-5.0 mM. The trypanosome enzyme had a molecular weight of 145 kDa determined by agarose gel filtration. Methionine analogs including selenomethionine, L-2-amino-4-methoxy-cis but-3-enoic acid and ethionine acted as competitive inhibitors of methionine and as weak substrates when tested in the absence of methionine with [14C]ATP. The enzyme was not inducible in procyclic trypomastigotes in vitro, and the enzyme half-life was > 6 h. T. b. brucei AdoMet synthetase was inhibited by AdoMet (Ki 240 microM). The relative insensitivity of the trypanosome enzyme to control by product inhibition indicates it is markedly different from mammalian isoforms of the enzyme which are highly sensitive to AdoMet. Since trypanosomes treated with the ornithine decarboxylase antagonist DL-alpha-difluoromethylornithine accumulate AdoMet and dcAdoMet (final concentration approximately 5 mM), this enzyme may be the critical drug target linking inhibition of polyamine synthesis to disruption of AdoMet metabolism.

Subcellular localization of the enzymes of the arginine dihydrolase pathway in Trichomonas vaginalis and Tritrichomonas foetus

The enzymes of the arginine dihydrolase pathway were demonstrated in Tritrichomonas foetus and their subcellular localization determined for both T. foetus and Trichomonas vaginalis. Ornithine carbamyltransferase (anabolic and catabolic activities), ornithine decarboxylase and carbamate kinase activity were localized predominately (56–80%) in the non sedimentable fraction of both species. A large proportion (35–40%) of the arginine deiminase was, however, recovered in the large granular fraction, and this distribution was unchanged by increasing the ionic strength of the buffer. Upon density gradient centrifugation the particles containing arginine deiminase activity had an isopycnic density of 1.09 g/ml in percoll, and separated from hydrogenosomes (1.18 g/ml) and lysosomes (1.12 g/ml). Arginine deiminase was also the only enzyme of the dihydrolase pathway which demonstrated latency upon treatment of the 1.09 g/ml fraction with non‐ionic detergents. The results demonstrate the presence of the arginine dihydrolase pathway in T. foetus and indicate that at least a portion of the arginine deiminase in trichomonads is membrane associated.

Differential sensitivity of Trypanosoma brucei rhodesiense isolates to in vitro lysis by arsenicals

Clinical isolates of Trypanosoma brucei rhodesiense, which were resistant to arsenical drugs in murine infections, were examined for resistance in vitro. A rapid lysis assay was developed which was able to predict in vivo sensitivity to melarsoprol (Mel B, Arsobal) and melarsen oxide. The assay was based on the finding that long slender bloodforms of drug-sensitive isolates would lyse in the presence of arsenicals upon incubation in heat-inactivated fetal bovine serum. On the basis of plots of decrease in the absorbance of trypanosome suspensions vs time of incubation with drug, L50 values, reflecting the drug concentration necessary for lysis of 50% of the cells within 30 min. were calculated for five strains. These values ranged from less than 30 microM for arsenical-sensitive strains to greater than 75 microM in proven arsenic refractory isolates. Calcium was essential for lysis, and the presence of the Ca2+ chelator EGTA (10 mM) in serum delayed lysis of sensitive strains. Ca2+ channel antagonists (Verapamil, Diltiazem), however, did not enhance lysis of refractory isolates when used at 20 to 30 microM. Intracellular concentrations of reduced trypanothione, the apparent target of arsenicals, were similar for all isolates, approximately 1.02 +/- 0.28 nmol/10(8) cells, as detected by monobromobimane derivitization and HPLC analysis. Uptake of melarsen oxide was found to be reduced in arsenical refractory strains. Uptake was judged by reduction of free reduced trypanothione as a result of formation of the trypanothione-arsenic complex Mel T. Little change was found in arsenical-resistant strains, but sensitive strains had 50 to 70% reductions in trypanothione levels after incubation with a low (1 microM) level of melarsen oxide.

Combination chemotherapy of drug-resistant Trypanosoma brucei rhodesiense infections in mice using DL-alpha-difluoromethylornithine and standard trypanocides.

Combinations of DL-alpha-difluoromethylornithine (DFMO; eflornithine; Ornidyl) with either suramin or melarsen oxide were found to be effective against acute laboratory model infections with Trypanosoma brucei rhodesiense. We used clinical isolates known to be resistant to these drugs when used singly. An infection with a melarsen oxide-refractory isolate was cured by a combination of low-dose DFMO (0.5% in the drinking water) plus low-dose suramin (1 mg/kg of body weight given intraperitoneally). Another strain, moderately resistant to arsenical drugs, was cured with combinations of 4% DFMO with 5 mg of melarsen oxide per kg. Furthermore, a combination of DFMO (2% in the drinking water) and suramin (20 mg/kg) provided a 100% cure rate in a central nervous system model, although the same doses of these drugs used singly were completely ineffective. The synergism of DFMO and suramin against an acute infection was improved when suramin was given at the end of the DFMO administration. No adverse interactions were observed when high doses of DFMO combined with high doses of suramin were administered to uninfected mice. These results suggest that combinations of DFMO and suramin should be examined clinically for activity in arsenical-drug-refractory cases of East African sleeping sickness.

Trypanocidal activity of dicationic compounds related to pentamidine

Eight dicationic compounds related to pentamidine were studied for trypanocidal activity in seven trypanosome isolates. In vitro studies revealed that diamidines are more potent than diimidazolines. For example, 2 (a diamidine) and 4 (a diimidazoline) inhibited the growth of KETRI 243 with IC50 values of 2.3 and 900 nM, respectively. Introduction of polar groups into the linker decreased the effectiveness of the compounds against drug-resistant trypanosomes. In compounds with a 2-butene linker between the cationic groups, trans-isomers were more potent than cis-isomers. The cis- and trans-buteneamidines cured infection caused by Trypanosoma brucei brucei (EATRO Lab 110) and protected mice against infection by Trypanosoma brucei rhodesiense isolates, some of which are resistant to diamidines and melarsoprol.

Inhibition of Trichomonas vaginalis ornithine decarboxylase by amino acid analogs.

Ornithine decarboxylase (ODC) from Trichomonas vaginalis was inhibited irreversibly by several substrate analogs. Of these, DL-alpha-monofluoromethyldehydroornithine (MFMDO) and DL-alpha-monofluoromethylornithine (MFMO) were the most potent. The enzyme was unaffected by putrescine analogs suggesting that differences exist between the regulation of the trichomonad enzyme and that in other eukaryotes. In culture the ornithine analogs strongly inhibited putrescine synthesis and increased the generation time after 24 hr of exposure. In a semi-defined growth medium MFMDO methyl and ethyl esters increased the generation time from 4.5 hr to 9.0 and 8.2 hr, respectively. In standard undefined growth medium the trichomonad ODC was fully induced only after 15 hr (late log) and had an extended half-life of greater than 8 hr.

The Microaerophilic Flagellate, Trichomonas vaginalis, Contains Unusual Acyl Lipids but no Detectable Cardiolipin

ABSTRACT. Previous lipid analysis of trichomonads has led to controversy as to whether these hydrogenosome‐containing organisms contain cardiolipin (CL), which is a characteristic component of mitochondria. Here we report a careful lipid analysis of the sexually transmitted protist Trichomonas vaginalis. Major lipids were phosphatidylethanolamine (42%) and phosphatidylcholine (20%) with lesser amounts of phosphatidylglycerol (PG) (12%) and non‐polar components. Two unusual lipids, acyl‐PG (8%) and ceramide phosphorylethanolamine (2%), were also significant components. The structures of these lipids were confirmed by tandem mass spectrometry following reverse‐phase high‐performance liquid chromatography. This is the first time ceramide phosphorylethanolamine has been reported in a trichomonad. In contrast, CL (diphosphatidylglycerol) could not be detected either by two‐dimensional thin‐layer chromatography or by mass spectrometry. These data are discussed in relation to the organisms phylogenetic origin as a parasite showing secondary adaptation to microaerobic conditions.

Trichomonas vaginalis and Giardia intestinalis Produce Nitric Oxide and Display NO‐Synthase Activity

TRICHOMONAS vaginalis is a microaerophilic parasite of the human urogenital tract which is the causative agent of the sexually transmitted disease trichomoniasis. In vivo, T. vaginalis is exposed to polyamines (White et al. 1983), which are cationic molecules essential for cell proliferation and differentiation. The arginine dihydrolase pathway in T. vaginalis provides these amitochondriate parasites with an important source of energy, and some steps in this energy-yielding pathway take place in the redox-balancing organelle: the hydrogenosome (Yarlett and Bacchi 1994; Yarlett et al. 1996). Nitric oxide (NO) is an important signaling molecule, a weapon used in the immune system’s response to pathogens; it is also responsible for vasodilation of blood vessels, a property which makes this volatile gas important for the control of blood pressure. The metabolic conversion of arginine to citrulline via the action of nitric oxide synthase, (NOS) with NO as a by-product, has been studied in numerous cell types, tissues, and organs. In studies of NO and parasitic protozoa, however, this topic has heretofore been dominated by a one-sided exploration. These studies focused on the effects of the release of NO from the host’s immune system, and provided evidence about its cytotoxic manifestations in the parasites. However, only a few studies have explored the possibility that these parasites themselves produce NO. NOS activity was discovered in Trypanosoma cruzi and found to be sensitive to typical NOS inhibitors, such as N-methyl-L-arginine (Paveto et al. 1995). It was also shown that Leishmania donovani produces NO and contains a NOS similar to that of the mammalian enzyme (Basu et al. 1996). Entamoeba histolytica was shown to have NOS activity and responded to typical NOS inhibitors (Hernandez-Campos et al. 2003). The polyamine metabolic pathway in T. vaginalis has been characterized, specifying the presence of arginine deiminase and the production of ammonia by the reaction converting arginine to citrulline (Yarlett et al. 2000). We have found that under aerobic conditions this reaction is also catalyzed by NOS, which produces NO rather than ammonia. Giardia intestinalis, another microaerophilic parasitic protozoan, is the causative agent of one of the world’s most common intestinal infections. G. intestinalis has been termed ‘‘amitochondriate’’ and produces energy solely by substrate-level phosphorylation. However, redox active organelles and mitosomes have recently been found in G. intestinalis (Lloyd et al. 2002; Tovar et al. 2003). The location of both Giardia and trichomonads in evolutionary phylogeny has been debated mostly due to their lack of mitochondria. We have found that NO is produced by G. intestinalis, and also by hydrogenosomes in T. vaginalis. As mitochondria have been shown to produce NO; these results add to the evidence that both T. vaginalis and G. intestinalis have evolved from organisms containing fully-functional mitochondria (Bates et al. 1996; Ghafourifar and Richter 1997). MATERIALS AND METHODS