Cobalamin and folate metabolism in helminths.

Knowledge about the source, metabolism and functions of cobalamins and folates in helminths remains fragmentary. It is likely that all helminths, whether free-living or parasitic, cannot synthesize cobalamins and folates de novo. Folates, but not cobalamins, appear to be ubiquitous in helminths. Of the parasitic helminths that take up free cobalamins in vitro, all but one species showed no uptake of cobalamin bound to transport proteins, although the latter type of cobalamin by far predominates in vivo. Certain free-living and parasitic helminths in vitro and in vivo took up a variety of folyl and antifolyl monoglutamates, but it is not known whether helminths can take up any folyl polyglutamates. Helminths that have any folate-dependent metabolism appear able to produce polyglutamylated forms of the required tetrahydrofolate coenzymes. Helminths that possess a functional cobalamin-dependent pathway from succinyl CoA to propionyl CoA appear able to form the required adenosylcobalamin coenzyme. Only free-living helminths may possess a cobalamin (and 5-methyltetrahydrofolate)-dependent pathway from homocysteine to methionine. It is likely that all helminths possess the 5,10-methylenetetrahydrofolate-dependent pathway from deoxyuridylate to thymidylate. All helminths appear able to salvage purine bases and nucleosides, but 10-formyltetrahydrofolate-dependent de novo purine ribonucleotide synthesis has been demonstrated unequivocally only in nematodes. The primitive parasitic groups of helminths exhibit cobalamin metabolism, whereas the more highly evolved ones seem to have lost the mechanisms for uptake and the associated biochemical pathways utilizing cobalamins.

Glutathione S-transferase in adult Dirofilaria immitis and Brugia pahangi.

Glutathione S-transferase (EC 2.5.1.18) was detected in the cytosolic and microsomal fractions of adult Dirofilaria immitis females at respective levels of 30 nmol and 3 nmol min-1 (mg protein)-1 activity with the substrate 1-chloro-2,4-dinitrobenzene (CDNB). The transferase activity in the cytosolic fraction of adult Brugia pahangi females was 10 nmol min-1 mg-1 with CDNB; determination of its activity in the microsomal fraction of this filariid was not attempted. These filarial glutathione S-transferases were further characterized after their purification by glutathione-affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cytosolic transferase from D. immitis, molecular weight 47000, yielded a single subunit of around 28 kDa. The cytosolic and microsomal transferases from D. immitis differed in their activity with CDNB, 1,2-dichloro-4-nitrobenzene, 4-benzylchloride and ethacrynic acid. The cytosolic transferase from B. pahangi was distinguished by its high activity with ethacrynic acid. Both glutathione S-transferases from D. immitis also functioned as a glutathione peroxidase, strongly preferring cumene hydroperoxide as a substrate over hydrogen peroxide. Both were equiactive inhibitors of malonaldehyde formation in the NADPH-microsomal lipid peroxidation system. Thus, in addition to the ability of filarial glutathione S-transferases to detoxify electrophilic xenobiotics, at least those from D. immitis also exhibited selenium-independent glutathione peroxidase activity. Their glutathione S-transferase function suggests a potential role for these enzymes in the leukotriene synthetic pathway, if filariae can form such eicosanoids from arachidonate. Functioning as a glutathione peroxidase, they could serve to protect filarial membrane lipids from peroxidation.

Isolation, partial purification and some properties of two acid proteases from adult Dirofilaria immitis.

Two acid proteases were isolated from the soluble extracts of adult Dirofilaria immitis, the filarial heartworm of canines. Activity of these proteases was detected using 3H-labeled bovine alpha-casein as substrate, and they were designated Fp-I and Fp-II in order of their elution from a CM-cellulose column. The molecular weight of partially purified Fp-I was approximately 170000, and it was active between pH 4.6-5.8. The activity of Fp-I doubled in the presence of various sulfhydryl reagents at 5 mM, and it was inhibited 50-60% by the sulfhydryl inhibitors p-hydroxymercuribenzoate and iodoacetate at 1 mM, the heavy metal chelating agent o-phenanthroline at 1 mM and the peptide aldehyde protease inhibitors pepstatin (10 microM), leupeptin, antipain and chymostatin (50 microM). The molecular weight of the more extensively purified Fp-II is approximately 48000. This protease was active between pH 2.6-3.4 and was highly sensitive to inhibition by pepstatin (80% inhibition at 10 nM). Fp-II was not significantly affected by sulfhydryl reagents, sulfhydryl inhibitors, metal chelating agents or peptide aldehyde protease inhibitors other than pepstatin. These properties of dirofilarial Fp-II resemble those of mammalian cathepsin D.

Involvement of tetrahydrofolate cofactors in de novo purine ribonucleotide synthesis by adult Brugia pahangi and Dirofilaria immitis.

Adult Brugia pahangi in vitro, unlike mouse leukemia L1210 cells, converted 5-methyltetrahydrofolate (CH3FH4) directly to 5,10-methylenetetrahydrofolate and thence to other FH4 cofactors. The excreted CO2 that was derived from CH3FH4 was due to the presence within the filariae of 10-formyltetrahydrofolate dehydrogenase (EC 1.5.1.6) which catalyzes the deformylation of 10-formyl-tetrahydrofolate. Adult B. pahangi and Dirofilaria immitis, incubated in a purine-free medium containing [5-14C]CH3FH4 as the only form of folate, synthesized purine ribonucleotides radiolabeled at positions 2 and 8 of the purine ring. Presumably, 10-formyl[14C]FH4 donated Carbon 2 during the synthesis de novo of the purine ring and 5,10-methenyl[14C]FH4 donated Carbon 8.

Glycosyl transferase activity in homogenates of adult Dirofilaria immitis.

Homogenates of adult Dirofilaria immitis possess a microsomal enzyme system able to transfer mannose from GDPmannose to endogenous lipid intermediate(s) and exogenous dolichol monophosphate. A divalent metal was required with Mn2+ being the most effective; other requirements for optimal activity included Triton X-100, EDTA and either ATP or NaF. The maximal rate of mannose transfer to the lipid acceptor by the filarial system, 1.6 pmol.min-1.mg-1 protein, occurred at 37 degrees C and pH 7.0, and this was inhibited 50% by 8 microM diumycin and not at all by 100 microM tunicamycin. D. immitis microsomes also were shown to promote the transfer of mannose to derivatives of alpha-lactalbumin, resulting in the synthesis of a mannose-labeled glycoprotein.

Thymidine kinase activity and thymidine salvage in adult Brugia pahangi and Dirofilaria immitis.

Cytosolic thymidine kinase (EC 2.7.1.75), the initial enzyme in the thymidine salvage pathway, was detected in crude homogenates of adult female Brugia pahangi and Dirofilaria immitis, with respective specific activities of 100 and 460 nmol/h/mg protein. Partially purified filarial thymidine kinases were found to have molecular weights of approximately 180 000, to be most active in the presence of Mg2+ and ATP, to have a sharp pH optimum (pH 7.0) and to be heat-labile in the absence of added thymidine. For both, the respective Km values for thymidine and ATP were 60 muM and 1.6 mM, and 5-iodo-2'-deoxyuridine was as good a substrate as thymidine. A distinguishing property was the 3-fold higher sensitivity of the B. pahangi enzyme to feedback inhibition by thymidine 5'-triphosphate. Adult female B. pahangi took up and incorporated [methyl-3 H] thymidine into DNA when they were exposed to this radiolabeled deoxynucleoside in vivo, but the thymidine salvage pathway in these worms was essentially nonfunctional in vitro.

Synthesis of ubiquinone 9 by adult Brugia pahangi and Dirofilaria immitis: evidence against its involvement in the oxidation of 5-methyltetrahydrofolate.

Among various ubiquinone (Q) isoprenologues tested, only Q7 was more efficient than menadione in promoting the oxidation of 5-methyltetrahydrofolate (CH3FH4) by 5,10-methylenetetrahydrofolate reductase isolated from adult Brugia pahangi, whereas Q10 was the best cofactor in the same reaction catalysed by the analogous enzyme from adult Dirofilaria immitis. Menoctone (3-[1-cyclohexyloctyl] -2-hydroxy-1,4-naphthoquinone) was a strong competitive inhibitor of both these ubiquinone isoprenologues in the respective reactions. When incubated in the presence of D,L-[14C]-mevalonate, adult B. pahangi and D. immitis synthesized radiolabelled Q9 only, in addition to other isoprenoid derivatives in the neutral lipid fraction. In view of the inability of Q9 to promote the oxidation of CH3FH4 by 5,10-methylenetetrahydrofolate reductase from B. pahangi, it seems unlikely that this filaria uses Q9 as a cofactor in this reaction. Conceivably, D. immitis could use Q9 as a cofactor in its enzymatic oxidation of CH3FH4, since in this circumstance, it was a better cofactor than menadione.

In vitro antifilarial activity of organometallic complexes against infective larvae of Molinema dessetae and adult females of Brugia pahangi.

New organometallic complexes having protozoocidal properties were evaluated for their in vitro antifilarial activity using two models: infective larvae of Molinema dessetae and adult females of Brugia pahangi. The compound most active on the M. dessetae model was Ir(I)-COD-pentamidine tetraphenylborate with an EC50 = 6 +/- 1 microM after 7-day-incubation. In the 2-aminobenzothiazole series, Ruthenium was more potent than Iridium for antifilarial activity. A dithiocarbamate function significantly enhanced the antifilarial activity. The compounds derived from benzimidazole were inactive whatever the metal (Iridium or Rhodium). The other compounds exhibited EC50 ranging from 10 to 31 microM. On adult female Brugia pahangi in vitro, Pt-DDH-N-acetylleucine, Pt-diminazene and Pd-Cl4-piperazine at 20 microM began to kill both microfilariae and the developing embryos within the mothers on day 2. The compounds, except for Pd-Cl4-piperazine, killed the adults after 5 days. Rh-Cl-2-chloropyridine caused obvious slowing of the adults from day 3 onward but did not affect the viability of adults, microfilariae or developing embryos. In vivo antifilarial investigations are necessary to appreciate the real advantage of heavy metal complexes in the experimental treatment of filariasis.

Leukotriene C4 synthesis catalyzed by Dirofilaria immitis glutathione S-transferase.

The biologically active sulfidopeptide leukotriene, leukotriene C4, is formed by the enzymatic action of leukotriene C4 synthase, which conjugates glutathione with leukotriene A4. We have found that a filarial glutathione S-transferase can function as a leukotriene C4 synthase. Glutathione S-transferase was purified from the cytosol of adult Dirofilaria immitis by glutathione-agarose affinity chromatography and was reacted with 25 microM leukotriene A4 methyl ester and 10 mM glutathione. The filarial enzyme catalyzed the formation of leukotriene C4 methyl ester, as shown by reverse phase high pressure liquid chromatographic analyses. The finding that filarial glutathione S-transferase can function as leukotriene C4 synthase provides a mechanism whereby filarial parasites could form lipoxygenase pathway derived sulfidopeptide leukotrienes.

Long-term efficacy of tubercidin against schistosomiasis japonica and mansoni in primates.

Schistosomiasis japonica in capuchin msnkeys (Cebus apella) and schistosomiasis mansoni in baboons (Papio cyanocephalus and P. hamadryas) were completely arrested for 6 months in every infected primate receiving a single treatment with tubercidin (Tu), administered after prior absorption into 20% of their red cells. It is very likely that a single treatment with Tu sequestered in only 15% of the hosts' red cells would also be 100% effective for prolonged periods of time, but that with lower doses some relapses would be expected. Babbons with patent Schistosoma mansoni infections were rechallenged with S. mansoni cercariae 4 months after treatment with Tu. Although Tu eliminated almost all the sexually mature female worms from the primary infection but spared most of the males for continuing sojourn within their hosts, the baboons retained their full susceptibility to reinfection, as indicated by worm burdens and fecal egg excretion. However, the granulomatous reaction in the rechallenged Tu-treated baboons to new masses of eggs trapped in their livers appeared to be less intense than was seen in animals with primary infections.

Treatment of schistosomiasis mansoni and japonica in baboons with tubercidin given by direct intravenous injection.

Baboons (Papio cyanocephalus and P. anubis), infected with Schistosoma mansoni or S. japonicum, were treated with single doses of tubercidin (7-deazaadenosine; Tu), 1, 3, and 5 mg per kg of body weight, administered by intravenous drip. Crystalline Tu was dissolved in sterile 0.9% NaCl solution (1 mg per ml), and the solution was delivered at a rate of 4 ml per minute. Detectable short-term host toxicity was limited to the 5 mg per kg dose, mainly in the form of reversible mild to moderate kidney damage. Only this 5 mg per kg dose administered to baboons with relatively heavy S. mansoni infections was capable of completely suppressing fecal egg excretion for 6 to 8 weeks, eliminating the female worms, and terminating active disease, as indicated by histopathological findings. Comparable effects were achieved following the administration of the 3 mg per kg dose to baboons with moderate to heavy S. japonicum infections.

Folate metabolism in filariae: enzymes associated with 5,10-methylenetetrahydrofolate.

Adult Brugia pahangi and Dirofilaria immitis were found to possess the following four enzymes that are associated with the cofactor 5,10-methylenetetrahydrofolate (CH2FH4): serine hydroxymethyltransferase, thymidylate synthetase, CH2FH4 dehydrogenase, and CH2FH4 reductase. The properties of the isoenzymes from the two filariae were virtually indistinguishable, except that diethylcarbamazine inhibited CH2FH4 reductase from B. pahangi 50% at 10 muM, but did not not affect the isoenzyme from D. immitis at 100 muM. The properties of these four filarial enzymes generally were similar to their counterparts from mosquitoes and mammalian sources, but several notable differences were identified.

De novo synthesis of methionine in normal and Brugia-infected Aedes aegypti.

Crude extracts of normal, adult Aedes aegypti were able to form methionine from homocysteine in the presence of 5-methyltetrahydrofolate (MeFH4) but not betaine. The requirements for the reaction, including a need for vitamin B12, S-adenosylmethionine (SAM), and a reducing system, indicated that it was catalyzed by MeFH4:homocysteine transmethylase (methionine synthetase). The general properties of A. aegypti methionine synthetase were found to be similar to those of the analogous enzyme from bacterial and mammalian sources, except that its apparent affinity for SAM was significantly lower. Extracts of normal, adult A. aegypti females (5 days after emergence, as well as 7 and 12 days after they fed upon uninfected jirds) synthesized methionine at a rate of 0.6 nmole per hr per mg protein. Extracts of female mosquitoes prepared 7 and 12 days after they fed upon Brugia pahangi-infected jirds synthesized methionine at double the normal rate. Because methionine formation by extracts of adult B. pahangi could not be detected, it is probable that methionine synthetase activity increased in the arthropod host in response to filarial infection.

Isoprenoid biosynthesis in adult Brugia pahangi and Dirofilaria immitis.

The ability of adult B. pahangi and D. immitis to utilize [14C]-mevalonate for the biosynthesis of isoprenoid compounds was investigated. Both filariae appeared to be unable to synthesize squalene and sterols de novo. They did, however, synthesize ubiquinone 9, a family of dolichol isoprenologs, and predominantly, the short-chain isoprenoid alcohol, geranyl geraniol. In addition, B. pahangi and D. immitis apparently were unable to synthesize a menaquinone (Vitamin K2) from [14C]-menadione.

Activity of tubercidin against immature Fasciola hepatica in mice.

HaM/ICR females mice (20 to 25 g), infected with from 3 to 5 Fasciola hepatica metacercariae per os, were treated with a single dose of thepurine nucleoside analog tubercidin (7-deaza-adenosine; Tu) 7, 14, 21, and 28 days postexposure.Tu was administered either by direct intravenous injection or intraerythrocytically. Most of the flukes were killed and host survival rates were markedly increased if treatment was started within 3 weeks postexposure. The minimal single intravenous dose of Tu that was maximally effective was between 10 and 20 mg/kg.

Folate metabolism in filariae. Enzymes associated with 5,10-methenyltetrahydrofolate and 10-formyltetrahydrofolate.

Adult Dirofilaria immitis and Brugia pahangi were found to possess the following folate-related enzymes that catalyze the formation of 5,10-methenyltetrahydrofolate (methenylFH4) or 10-formylFH4 (f10FH4): f10FH4 synthetase, methenylFH4 cyclohydrolase, f5FH4 cyclodehydrase, and a bifunctional complex composed of formiminoglutamate: FH4 formiminotransferase and 5-fomiminoFH4 cyclodeaminase. The properties of these filarial enzymes were generally similar to those of their counterparts from invertebrate and vertebrate sources, although each possessed one or more distinctive characteristics.

Comparative activity and properties of lactate dehydrogenase, xanthine dehydrogenase, and dihydrofolate reductase in normal and Brugia pagangi-infected Aedes aegypti.

The amount of xanthine dehydrogenase (XDH), dihydrofolate reductase (DHFR), and lactate dehydrogenase (LDH) in crude extracts of 4- to 5-day-old adult Aedes aegypti was determined, and the properties of these enzymes were partially characterized. It was then found that the amount and other selected characteristics of XDH and LDH in extracts of female Ae. aegypti processed 5 to 7 days and 12 to 14 days after they had fed upon either normal or Brugia pahangi-infected jirds were indistinguishable from those of these two enzymes in extracts of female mosquitoes that did not have a blood meal. Under the same circumstances, the selected characteristics of DHFR were also unaffected. However, there was a suggestion that the amount of DHFR was slightly increased in extracts of female Ae. aegypti processed 5 to 7 days after they had fed upon B. pahangi-infected jirds; by 12 to 14 days after the blood meal, there was a consistent 30% to 60% increase in the amount of DHFR inextracts of infected mosquitoes. DHFR activity could not be detected in a similarly prepared extract of 4,000 to 5,000 infective (L-3) B. pagangi larvae, the approximate number present in the infected mosquito extracts. It would appear, therefore, that the increased amount of turnover of DHFR in the mosquito host occurs in response to advanced infection with B. pahangi.

Evidence that sulfisoxazole, an antibacterial sulfonamide, can adversely affect the development of Brugia pahangi in Aedes aegypti mosquitoes.

The average number of infective larvae recovered from Brugia pahangi-infected Aedes aegypti was approximately one-half that recovered from the controls after the former group of infected mosquitoes had ingested a 1.0% solution of sulfisoxazole diolamine (SXZ) in 10% sucrose-water for 4 consecutive days, beginning 4 days after infection. Most of the filarial larvae from the SXZ-treated mosquitoes were small and sluggish compared with those from the controls. There was no increased mortality of mosquitoes that ingested 1.0% SXZ in sugar-water for 4 days. Average filarial larval burdens were not decreased in mosquitoes that ingested a solution of 10(-6) M methotrexate (MTX), a potent dihydrofolate reductase inhibitor, in sugar-water for 4 days, beginning 4 days after infection. The distributional pattern of larval burdens in mosquitoes that ingested combined 1.0% SXZ and 10(-6) M MTX in sugar-water for 4 days closely resembled that seen in mosquitoes that had imbibed 1.0% SXZ only. Average filarial larval burdens were not decreased in mosquitoes with 4-day-old B. pahangi infections that fed upon jirds which received intraperitoneal injections of SXZ (2 g/kg) and MTX (1 mh/kh), alone and in combination, 1 hr previously. Survival of the mosquitoes that fed upon the drug-treated hosts was unaffected, as was the hatchability of their eggs and subsequent growth and development of the mosquito larvae.