It has been shown that uracil can be incorporated into ribonucleic acid of the normal rat tissues (2). However, this incorporation becomes significant only when the enzymes associated with the catabolism of uracil are saturated with their substrates. When thii is achieved, the incorporation of uracil into RNA increases with substrate concentration. At high concentrations the extent of incorporation of uracil, uridine, uridylic acid, and of erotic acid into RNA becomes of the same order of magnitude. These experiments showed further that an inverse relationship exists between the capacity of a tissue to synthesize RNA and its capacity to degrade uracil. A group of normal, nondividing tissues (rat and mouse liver) has been shown to have a high catabolic capacity for uracil and a low capacity to incorporate uracil into RNA. In contrast, a group of normal but rapidly dividing tissues (intestinal mucosa, regenerating rat liver) and also a group of abnormal tissues (hepatomas (2) as well as Ehrlich ascites cells (3)) have a low capacity for the degradation of uracil, whereas they actively incorporate this pyrimidine into RNA. An interplay between the degradative and the synthetic pathways for uracil could constitute an important means for the control of RNA synthesis. From a consideration of these facts the possible existence of a cellular homeostatic mechanism involved in the regulation of RNA synthesis was proposed (2). In order to explore further the details of such a homeostatic mechanism we have studied the changes which occur in the anabolic and catabolic capacities of enzymes involved in nucleic acid metabolism. The aim has been to contrast these capacities in normal rat liver with those occurring in rat liver during the course of regeneration induced by partial hepatectomy. Variations in the catabolic and anabolic rates could then be considered to reflect changes in the state of growth in cell types of like origin. The results of these experiments give additional support to the contention that a homeostatic mechanism exists for the control of RNA synthesis. Evidence will also be presented which indicates that similar mechanisms may control the metabolism of thymine as well as that of thymidine 5’-phosphate and consequently exert an effect on DNA synthesis. Based on the results of this and previous work we wish to suggest that a transition from a high catabolic-low anabolic capacity to the inverse situa-

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.

Srivastava A. K. and Jaffe J. J. 1987. Phosphatidylinositol, phosphatidylglycerol, and cardiolipin synthesis in adult Dirofilaria immitis females. International Journal for Parasitology17:917–920. The pathways leading to the formation of phosphatidylinositol (PI), phosphatidylglycerol (PG) and cardiolipin (CL) in adult Dirofilaria immitis females were investigated. PI was synthesized by both de novo as well as via base exchange pathway in the worms. Under specified assay conditions, the respective rates of PI formation by way of these pathways in crude homogenates of the worms in the order given were around 3.0 and 0.75 nmol min−1 mg−1 protein. PG synthesizing activity in the worms was mainly associated with the particulate fractions and the rate of formation by these fractions was around 1.5 nmol min−1mg−1 protein. The worms were unable to synthesize CL by the pathway found in mammals.

Phosphatidylserine synthesis in adult Dirofilria immitis females. International Journal for Parasitology16: 9–11. Phosphatidylserine synthesis in extracts of Dirofilaria immitis females was found to occur exclusively by way of a calcium-stimulated enzyme-catalyzed exchange of l-serine for the base components of preformed phospholipids. Under specified conditions the rate of serine incorporation into phospholipid(s) was around 44 pmol min−1 mg−1 protein.

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.

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.

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.

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.

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.

1.1. 5,10-Methenyltetrahydrofolate cyclohydrolase (CHFH4CH) and 5-formyltetrahydrofolate cyclodehydrase (f5FH4CD) activity was detected in crude extracts of adult male and female Aedes aegypti.2.2. The properties of mosquito CHFH4CH generally were similar to those of its counterparts from mammalian sources, but the former had a lower pH optimum and was more sensitive to inhibition by certain divalent cations.3.3. Mosquito f5FH4CD differed from its counterpart from sheep liver in its stricter requirement for Mg2+ and ATP, its inactivity when K+ replaced Na+, and in its higher sensitivity to heat inactivation.

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.

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.

Thymidylate synthetase (5,10-methylenetetrahydrofolate: dUMP C-methyltransferase; EC 2.1.2.45; TMPS) activity was detected in crude extracts of 4 to 5 day-old adult male and uninfected female Aedes aegypti. Mosquito TMPS was strongly inhibited by Mg2+ and Ca2+ (id50 values of 0.25 and 0.12 μM, respectively) but not by other divalent cations. The Km values for dUMP and 5, 10-methylene-tetrahydrofolate were 55 and 100 μM, respectively. It appeared that mosquito TMPS would not be subject to regulation by physiological concentrations of thymidine mono-, di- and triphosphates or di- and triphosphates of cytidine, uridine, adenosine or guanosine. When subjected to Sephadex G-200 gel filtration, TMPS activity occurred in three peaks, corresponding to molecular weights of 210,000, 310,000 and 450,000. Among a variety of compounds tested as inhibitors of the enzyme, FdUMP was the most potent, with an id50 value of 0.5 nM; by contrast, FUdR was inactive at 1.0 mM. The polycyclic sulfonated anionic drug, suramin, was also a strong inhibitor of mosquito TMPS, with an id50 value of 0.3 μM. Various 2,4-diaminoheterocyclic antifols, including methotrexate, either were inactive or were relatively weak inhibitors of this enzyme. The total activity of TMPS did not change in extracts of female mosquitoes prepared 7 and 12 days after they fed upon either normal or Brugia pahangi-infected jirds.

The activity of 10-formyltetrahydrofolate synthetase (f1oFHS) in crude extracts of 4- to 5-day-old adult male and uninfected female Aedes aegypti was higher than that of other folate-related enzymes studied so far. L-Cysteine, Mg2?, and NH4? were required for optimal activity of this enzyme in the forward direction (forming f1oFH4, ADP and P1). The apparent Km values of the substrates and cofactors for the forward reaction showed correspondence between ATP and Mg2' (both 0.07 mM) and formate and NH? (13 mM and 7 mM); the apparent Km value of FH, (0.26 mM) corresponded closely to the Ki value of the 4-amino analog of folate methotrexate (0.24 mM), the latter being a competitive inhibitor of the enzyme. The estimated molecular weight of A. aegypti PfoFHS was 260,000. Two compounds were relatively strong inhibitors of the enzyme, as indicated by their IDo50 value: metho- trexate (2 CM) and suramin (8 /tM). The activity of PoFHS was approximately 40% lower in unin- fected female mosquitoes more than a week old than in those only 5 days old. The activity of this enzyme in female mosquitoes 7 days after they fed upon Brugia pahangi-infected jirds was approximately the same as that in age-matched uninfected females. However, the activity of fpoFHS in female mos- quitoes 12 days after infection with B. pahangi was 50% lower than that in age-matched uninfected fe- males. The significance of this latter finding is discussed.

N',N'?-Methylenetetrahydrofolate dehydrogenase (MTHFD) and reductase (MTHFR) ac- tivity was detected in crude extracts of 4- to 5-day-old adult male and uninfected female Aedes aegypti. In such extracts, the activity of MTHFD assayed in the forward direction (forming N5,N10- methenylTHF) was approximately 25 times higher than that of MTHFR assayed in the reverse direction (forming N5,N?1-methyleneTHF). MTHFD appeared to be a dimer consisting of 2 subunits, each with a molecular weight of 105,000, had an apparent Km for N5,N10-methyleneTHF of 0.55 mM and a Km for NADP of 0.29 mM. MTHFR had a molecular weight of 220,000, had an apparent Km for N5-methylTHF of 0.2 mM and remained fully active in the absence of added FAD even after exten- sive dialysis. Suramin inhibited MTHFR activity by 50% at a concentration of 0.002 mM; MTHFD was unaffected by this drug at a concentration of 0.1 mM. The activity of MTHFD did not change in extracts of female mosquitoes prepared 7 and 12 days after they fed upon either normal or Brugia pahangi-infected jirds. The activity of MTHFR did not change in extracts of female mosquitoes pre- pared 7 days after they fed upon either normal or B. pahangi-infected jirds. The level of MTHFR was higher in older female mosquitoes. Its activity in extracts of females that were prepared 12 days after they fed upon normal jirds was approximately twice that in extracts of younger adults. Its activ- ity in extracts of age-matched females prepared 12 days after they fed upon B. pahangi-infected jirds was nearly 4 times higher than that in extracts of younger adults. It is probable that an increased amount of MTHFR was produced by the mosquito host in response to an advanced infection with B. pahangi, because MTHFR from adult B. pahangi, unlike mosquito MTHFR, was found to be FAD- dependent after 24 hr dialysis, and all assays were carried out on dialyzed mosquito extracts in the absence of FAD.

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.

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.

In considering the potential usefulness of nucleoside analogs as antiparasitic agents, it is appropriate to recall the words of Ehrlich:’ “What we want is a Chemotherapia specifics, that is, we are looking for chemical agents which, on the one hand, are taken up by certain parasites and are able to kill them and, on the other hand, in the quantities necessary for this lethal action, are tolerated by the host without too great damage.” There are two general means by which antiparasitic agents can be selectively toxic for the pathogenic organisms. They can exploit physiological or biochemical differences between the parasites and their hosts, and/or they can be administered in a manner that could exploit peculiarities of habitat and behavior exhibited by the parasites. The following are cited as illustrative examples: Piperazine (diethylenediamine) produces a flaccid paralysis of the musculature of the parasitic nematode Ascaris lumbricoides, either by neuromuscular blockade’ or by hyperpolari~ation,~ but this drug exerts no such action on mammalian muscle. An outstanding property of plasmodial dihydrofolate reductases is their much greater sensitivity to inhibition by pyrimethamine, trimethoprim, and related diaminopyrimidines than the analogous mammalian enzyme?*’ A close correlation was found between the concentrations of these drugs required to inhibit plasmodia1 dihydrofolate reductases by 50% in a cell-free system and the minimum concentration required to inhibit the growth of these parasites in uitro and in uivo. Niclosamide (N-(2’-chloro-4-nitrophenyl)J-chlorosalicylamide) is very effective in the treatment of various tapeworm infestations of man following its oral administration in relatively large doses6 Under these circumstances, these intestinal parasites are exposed to lethal concentrations of the drug, yet niclosamide is well tolerated and very safe. Its low host toxicity is attributed to the fact that very little is absorbed from the gastrointestinal tract and that it has no direct irritant effect.’ The selective toxicity of tubercidin (7-deazaadenosine) for the blood flukes Schistosoma mansoni and s. japonicum in vivo can be increased when it is administered, after its prior absorption into a portion of the hosts’ total erythrocytes in vitro, by transfusing the drug-laden erythrocytes back into each infected donor.8-1’ Schistosomes feed on erythrocytes,” which renders these cells useful vehicles for delivery of antischistosoma1 agents such as tubercidin, which can be sequestered within them. Because tubercidin is a purine nucleoside analog with antiparasitic activity, it will be discussed later in more detail. Evidence is now at hand to indicate that there are peculiarities in the biochemistry and physiology of diverse kinds of parasites that theoretically are exploitable for chemotherapy by nucleoside analogs.