Alfred W. Senft

Purine metabolism in Schistosoma mansoni

Abstract Schistosoma mansoni has been found to have a spectrum of purine nucleotides which is similar, but not identical to mammalian cells. The principal component of this system is ATP, which is present at a level of about 5·5 × 10 −9 moles/mg worm pairs. The ATP concentration falls if worms are incubated in a defined medium containing no purines. When worms are incubated in the presence of adenine or adenosine, ATP levels rise sharply and may be maintained at higher than normal levels. Schistosomes were found not to incorporate labelled glycine or glucose into purine nucleotide bases. In contrast, 14 C-8-adenine is taken up from the medium and can be detected in nucleotides, principally as ATP. In comparison with various mammalian cell systems, schistosomes show a much higher rate of uptake of free adenine. These findings suggest that schistosomes do not utilize the de novo pathway for the synthesis of nucleotides. It is postulated that the salvage mechanism is required to fulfil the worms purine requirements. A new rational chemotherapeutic approach for schistosomiasis, based on interference with their specific nucleotide pathways, may thus become possible.

A study of Schistosoma mansoni transferred into permissive and nonpermissive hosts

Abstract A study of Schistosoma mansoni transferred into permissive and nonpermissive hosts. The International journal for Parasitology 7: 293–297. A study was made of the survival, growth and egg laying capacity of S. mansoni worms surgically transplanted from mice into rats or from rats into hamsters. Worms in the rat are stunted, remain localized in the liver and lay non-fertile eggs in small numbers. When transferred to the hamster, they increase in size, localize in the mesenteric veins and produce numbers of eggs approaching normal hamster-grown worms within 3 weeks following transplantation. Conversely, when adult mouse worms are injected into rats, they regress in size, remain in the liver and produce small numbers of incompletely developed eggs. These studies indicate that the physiological and reproductive status of the worm is strongly influenced by the host. Furthermore, in the nonpermissive host (the rat), the suppression of growth and egg laying is not permanent, since on transfer into a permissive environment (the hamster), stunted worms can resume growth and oviposition.

A chemically defined medium for maintenance of Schistosoma mansoni.

A completely synthetic medium containing amino acids, nucleic acid derivatives, vitamins, salts, and glucose for the maintenance of schistosomes in vitro is herein described. This medium appears adequate for short-term survival of these parasites, since they can be kept alive for periods somewhat more than a month. During early in vitro maintenance, the worms lay eggs and otherwise demonstrate normal behavior patterns. However, after about 20 days they begin to deteriorate, indicating that this artificial medium is far from optimal. The fundamental importance of determining the nutritional requirements of parasites hardly needs to be emphasized. Although the classical treatments of fluke infestation have depended on the administration of poisonous heavy metals, it seems reasonable to suggest that such methods should be supplanted by more reliable and less toxic procedures. A beginning step towards a more rational approach to the therapy of schistosomiasis might be the determination of the essential amino acids required by this parasite. Until recent years most of the in vitro maintenance fluids for schistosomes have been composed of a balanced salt solution to which various mammalian sera and/or embryonic extracts, amniotic fluids, or ascitic fluids were added (Lee and Chu, 1935; Hoeppli, Feng and Chu, 1938; Newsome and Robinson, 1954; Senft and Weller, 1956). Such fluids allowed prolonged survival of the worms and, in some cases, growth and regeneration of young adults. Limited egg production in vitro, using a variety of diluted sera (Cheever and Weller, 1958; Robinson, 1960) has also been reported. However, these successes have not adequately advanced our understanding of the nutritional requirements of Bilharzia. Although many of the above media can be reproducibly prepared, their components are variable, and either unidentified or vaguely defined. Few investigators have employed chemically defined media for the in vitro studies, a notable exception being Ross and Bueding (1950), who found survival of S. mansoni limited to 18 hours in a holidic medium. The addition of Received for publication 9 February 1961. This work was supported by the National Institutes of Health, Grant No. E-1689(C-3). muscle extract derivatives (largely unidentified substances called protogens) enhanced survival time considerably. Mao et al. (1957) found that S. japonicum would survive about 6 days in a Tyrode-Glucose-Vitamin mixture. Such short-term survivals are, however, quite inadequate for growth, regeneration, or egg production studies. The apparent ease with which certain strains of mouse fibroblasts can be maintained in a protein-free chemically defined medium (Evans et al., 1956), encouraged a trial of such a solution in schistosome work. It seemed logical that a synthetic medium modeled on previous chemical analyses of chick embryo extract (Westfall, Peppers, Sanford, and Earle, 1954) and horse serum (Westfall, Peppers, and Earle, 1954) might be tolerable for schistosomes, especially since schistosomes can be grown to maturity in laboratory mice. This paper will record moderate success in both survival and egg-production in such a completely synthetic medium. MATERIALS AND METHODS

Pathways of nucleotide metabolism in Schistosoma mansoni—III: Identification of enzymes in cell-free extracts☆

Abstract A survey of purine anabolic and catabolic enzymes has resulted in identification of major pathways in schistosome nucleotide biosynthesis. It is shown that multiple pathways for the incorporation of purine bases and nucleosides exist. The evidence suggests that adenosine phosphoribosyltransferase (APRT) activity is about ten times greater than adenosine kinase activity. Furthermore adenosine is converted to AMP principally via the pathway of adenosine deaminase, followed by conversion of inosine to hypoxanthine. In this sequence hypoxanthine phosphoribosyltransferase (HPRT) activity is rate limiting. On the basis of enzyme activities determined, one can suggest candidates of nucleotide analogs which might be useful chemotherapeutic agents.

Schistosoma mansoni: purification and characterization of the major acidic proteinase from adult worms.

We report purification of the major digestive proteinase from adult worms of Schistosoma mansoni. This enzyme is a thiol proteinase with a pH optimum of 5 and is activated by thiol reagents. It was purified 300-fold using a combination of gel chromatography and chromatofocusing. It readily hydrolyzed hemoglobin with an apparent Km of 0.29 microM and a specific activity of 27 micrograms degraded/min/mg enzyme at 37 C. Peptides with positively charged amino acids were preferentially cleaved. The enzyme degraded Boc-Arg-Arg-7-amino-4-methyl coumarin with a kcat/Km of 9083 M-1 sec-1. Lengthening the peptide chain to 3 amino acids or substituting glycine for the amino terminal arginine resulted in decreased activity. The enzyme was inhibited by chloromethylketone-derivatized peptides of similar sequence and by leupeptin. The purified proteinase exhibits microheterogeneity in different preparations with forms ranging in molecular weight from 30,000 to 35,000, and pI 5.7-6.0.

Pathways of nucleotide metabolism in schistosoma mansoni—VI adenosine phosphorylase

Abstract Schistosoma mansoni worms have been found to have an adenosine phosphorylase enzyme which requires inorganic phosphate during cleavage and α-ribose-1-phosphate for synthesis of adenosine from adenine. Kinetics of the reaction show a K m of 5.6 × 10 −5 M for adenosine and a broad pH activity range from 6.0 to 8.0. The adenosine phosphorylase activity can be distinguished from purine nucleoside phosphorylase by substrate specificity and by product inhibition studies.

Pathways of nucleotide metabolism in schistosoma mansoni—IV: Incorporation of adenosine analogs in vitro☆

Abstract The incorporation in vitro of adenine or adenosine analogs into schistosome nucleotides is demonstrated. Tubercidin, 2-fluoroadenosine and 2-fluoroadenine were all shown to be converted into analog triphosphate nucleotides. Since tubercidin and 2-fluoroadenosine are not substrates for adenosine deaminase or purine nucleoside phosphorylase and are not susceptible to degradation to the free base level, it is assumed that they are converted to nucleotides by reaction with adenosine kinase. The incorporation of 2-fluoroadenine into the nucleotide pools indicates that it serves as a substrate for adenine phosphoribosyltransferase. Tubercidin, added to the culture medium, interferes with the maintenance of normal ATP levels. When the concentration of the analog greatly exceeded that of adenine or adenosine in the medium, virtual shutdown of adenosine triphosphate synthesis followed. It is suggested that stoichiometric competition for enzyme sites may determine the relative amounts of nucleotides formed.

Pathways of nucleotide metabolism in Schistosoma mansoni—V Adenosine cleavage enzyme and effects of purine analogues on adenosine metabolism in vitro☆

Abstract Schistosoma mansoni extracts have been found to possess an active anabolic pathway for nucleotide biosynthesis in which adenosine is cleaved to adenine followed by conversion of adenine to AMP via adenine phosphoribosyltransferase. A significant fraction of labeled adenosine was found to enter the nucleotide pool by this pathway; howeverm, most of the nucleoside was converted to nucleotides by a pathway which employs adenosine deaminase, purine nucleoside phosphorylase and hypoxanthine phosphoribosyltransferase enzymes. Formycin A has been found to be a potent blocker of adenosine cleavage when tested in worm extracts. Arabinosyl-6-mercaptopurine and 6-thioguanosine are inhibitors of worm adenosine deaminase, and formycin B and 6-thioguanosine were found to inhibit the purine nucleoside phosphorylase of this parasite. Combinations of arabinosyl-6-mercaptopurine with either formycin A or formycin B result in substantial blockage of adenosine utilization for nucleotide synthesis. These studies thus suggest that adenosine analogues in combination might be useful in vivo for the chemotherapy of schistosomiasis.

Pathways of nucleotide metabolism in schistosoma mansoni—VII: Inhibition of adenine and guanine nucleotide synthesis by purine analogs in intact worms

Abstract Twelve analogs of adenosine or its related purines have been tested as single agents or in combinations as possible antischistosomal compounds. Measurements were made of the effect of these drugs on the anabolism and catabolism of [8- 14 C]adenosine by Schistosoma mansoni in vitro . Based on the degree of inhibition of biosynthesis of adenine and guanine nucleotides by intact worms. the best currently available purine analogs are 7-deaza-adenosine (tubercidin) and N 6 -phenyladenosine. These drugs reduced the total synthesis of nueleotides to 30 and 25 per cent, respectively, of controls. Blockade of the catabolic pathway (adenosine deaminase) by coformycin resulted in significantly increased synthesis of adenine nucleotides rather than the expected decrease. Thus, adenosine kinase must play a more prominent role in nucleotide synthesis than had been previously estimated. The implications of these findings in the development of new anti-schistosomal drugs are discussed.

Strategies in the chemotherapy of schistosomiasis

Abstract In contrast to previous generations, when essentially the only antischistosome compounds were the antimonials, a series of effective drugs is now available. Thus, theoretically, the millions of people in the world who suffer from schistosomiasis could be cured. The obstacles to treatment are the cost of the medicines, the absence of medical personnel who could diagnose and treat each case, and the pernicious likelihood of eventual reinfection.