Sinefungin, a natural nucleoside isolated from cultures of Streptomyces incarnatus and S. griseolus, is structurally related to S-adenosylhomocysteine and S-adenosylmethionine. Sinefungin has been shown to inhibit the development of various fungi and viruses, but its major attraction to date resides in its potent antiparasitic activity. This compound has been reported to display antiparasitic activity against malarial, trypanosomal, and leishmanial species. Very little is known about the antiparasitic mode of action of sinefungin. We found that S-adenosylmethionine was capable of reversing the inhibitory growth effects of sinefungin in Leishmania mexicana and that dATP was capable of reversing inhibitory effects of the drug on DNA polymerase activity when pyrophosphate release was measured. However, when incorporation of [3H]dTTP was used to measure DNA polymerase activity, no inhibition could be observed. Inhibition of DNA polymerase activity by sinefungin occurred only during the initial stages of purification of this enzyme, and inhibition by aphidicolin, a known DNA polymerase inhibitor, paralleled the inhibition by sinefungin. Neither sinefungin nor aphidicolin inhibited partially purified DNA polymerase. S-Adenosylmethionine synthetase was partially purified, and sinefungin, at levels active in vitro, had no significant effect. Sinefungin was significantly suppressive against both L. donovani and L. braziliensis panamensis infections in hamsters when compared with meglumine antimonate (Glucantime).

Adenine aminohydrolase (EC 3.5.4.2) from four species of Leishmania and from Crithidia fasciculata was examined for specific activities, affinity for substrate (adenine), and stability to heat. All were found to be strongly and noncompetitively inhibited by both coformycin and deoxycoformycin, two tight-binding inhibitors-of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4). Deoxycoformycin is the more potent inhibitor of the two. Neither inhibitor was active against the purine phosphoribosyltransferases. When deoxycoformycin was added to the defined growth medium containing hypoxanthine as the purine source, the. growth of C. fasciculata was unaffected, but when adenine was the purine source for the organism, severe inhibition resulted. This implies that hypoxanthine is the obligatory base for nucleotide synthesis and that the adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) is, in some manner, denied access to exogenous substrate. Since our earlier report (1) on the occurrence and properties of adenine aminohydrolase (EC 3.5.4.2) (adenine deaminase) in the trypanosomid flagellate Crithidia fasciculata, the enzyme has been reported from Leishmania tropica (2), L. donovani, and L. braziliensis (3). We have extended our investigations of this somewhat unusual enzyme and have found it to be present also in L. tarentolae and L. mexicana, and have shown that it is subject to drastic inhibition by two tight-binding adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) inhibitors, coformycin and deoxycoformycin. By the use of these potent inhibitors, it has been possible to assess to some degree the role of the enzyme as a control mechanism in these purine-requiring organisms. MATERIALS AND METHODS[8-14C]Adenine and [8-14C]hypoxanthine (both at 50 Ci/mmol, 1 Ci = 3.70 X 1010 becquerels) were obtained from Amersham/Searle. All chemicals of the defined medium used for C. fasciculata were obtained from Sigma; the ingredients of the semi-defined medium of Berens et al. (4) C. fasciculata, a mosquito parasite, is the strain that has been used in this laboratory for the past 20 years (5-6). L. tarentolae, a lizard parasite, was obtained through the courtesy of Larry Simpson (University of California, Los Angeles). This is the strain originally used by Trager (7). L. donovani, L. brazilhensis, and L. mexicana, all human pathogens, were kindly supplied by J. J. Marr (St. Louis University). These strains have been used by Marr and his associates for the past several years (3).-C. fasciculata was grown in the defined medium of Kidder and Dutta (5), in low profile flasks when large amounts of tissue were needed or in side-arm Nephelo flasks (Bellco, Vineland, NJ) or 25 X 125 mm optically clear tubes for the growth studies.Growth was followed by reading optical densities at 650 nm, with uninoculated medium as the standard. In all cases hemin, glucose, and inhibitors (if any) were filter sterilized and added to the bulk of the heat-sterilized medium.L. tarentolae w...

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In the presence of 100 micrograms of caffeine per ml or 200 micrograms of theobromine per ml, sporulation of Clostridium perfringens NCTC 8679 rose from less than 1 to 80 or 85%. Enterotoxin concentration increased from undetectable levels to 450 micrograms/mg of cell extract protein. Heat-resistant spore levels increased from less than 1,000 to between 1 X 10(7) and 2 X 10(7)/ml. These effects were partially reversible by the addition of adenosine or thymidine. In the case of NCTC 8238, caffeine and theobromine caused a three- to fourfold increase in the percentages of cells possessing refractile spores and a similar increase in enterotoxin concentration. Heat-resistant spore levels, however, were unaffected. Inosine was ineffective in promoting sporulation in NCTC 8679.

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