Trichomonas vaginalis grown for 16 h in the presence of [(14)C]spermine formed a high intracellul... more Trichomonas vaginalis grown for 16 h in the presence of [(14)C]spermine formed a high intracellular pool of [(14)C]spermidine and a small but detectable pool of [(14)C]putrescine. When [(3)H]putrescine was added to the growth medium, a large intracellular pool of [(3)H]putrescine was found, but it was not further metabolized, confirming previous studies suggesting the absence of a forward-directed polyamine synthetic pathway in T. vaginalis. Spermidine:spermineN:(1)-acetyltransferase (SSAT) and polyamine oxidase enzyme activities were detected which collectively converted spermine to spermidine. Polyamine oxidase was localized in the hydrogenosome-enriched fraction, whereas SSAT was found predominantly in the cytosolic fraction. In the presence of saturating substrate, the trichomonad SSAT had an activity of 0. 39+/-0.09 nmol min(-1) (mg protein)(-1) (the mean of five analyses) and an apparent K:(m) for spermine of 1.7 microM. The enzyme was competitively inhibited by di(ethyl)norsp...
A recently developed diaminotriazine derivative [O,O'-bis(1, 2-dihydro-2,2-tetramethylene-4,6... more A recently developed diaminotriazine derivative [O,O'-bis(1, 2-dihydro-2,2-tetramethylene-4,6-diamino-S-triazin-1-yl)-1, 6-hexanediol dihydrochloride; T-46; SIPI 1029] was examined for activity against African trypanosomes in in vitro and in vivo model systems. In vitro, SIPI 1029 was 50% inhibitory for growth of bloodstream trypomastigotes of four strains of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense at 0.15 to 2.15 nM (50% inhibitory concentrations). In in vivo mouse laboratory models of T. b. rhodesiense clinical isolate infections, SIPI 1029 was curative for 12 of 13 isolates at </=10 mg/kg of body weight/day for 3 days. In eight infections, a single dose was >/=60% curative, and in six of these, a dose of </=5 mg/kg was sufficient for >/=60% cure rates. A number of these isolates were resistant to the standard trypanocide melarsoprol (Arsobal) and/or the diamidines diminazene aceturate (Berenil) and pentamidine. SIPI 1029 was also curative in c...
5*-Deoxy-5*-(methylthio)adenosine (MTA), a key by-product of polyamine biosynthesis, is cleaved b... more 5*-Deoxy-5*-(methylthio)adenosine (MTA), a key by-product of polyamine biosynthesis, is cleaved by MTA phosphorylase and is salvaged as adenine and, through conversion of the ribose moiety, methionine. An analog of MTA, 5*-deoxy-5*-(hydroxyethylthio)adenosine (HETA), is a substrate for trypanosome MTA phosphorylase and is active in vitro and in vivo against Trypanosoma brucei brucei, an agent of bovine trypanosomiasis. In this study, HETA
A series of conformationally restricted congeners of pentamidine in which the flexible pentyl bri... more A series of conformationally restricted congeners of pentamidine in which the flexible pentyl bridge of pentamidine was replaced by trans-1,2-bismethylenecyclopropyl, phenyl, pyridinyl, piperazinyl, homopiperazinyl, and piperidinyl groups were synthesized. The compounds were evaluated for trypanocidal activity in vitro and in vivo against one drug-sensitive and three drug-resistant trypanosome isolates. The DNA binding affinity of the compounds was also studied using calf thymus DNA and poly(dA-dT). The nature of the linker influenced the DNA binding affinity as well as the trypanocidal activity of the compounds. trans-1,2-Bis(4-amidinophenoxymethylene)cyclopropane (1) was over 25-fold more potent than pentamidine against the drug-resistant isolate KETRI 243As-10-3, albeit with comparable DNA binding affinity. N,N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-Bis(4-amidinophenyl)homopiperazine (8) was the most potent trypanocide in vitro against all four trypanosome isolates studied, but N,N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-bis(4-amidinophenyl)piperazine (6) was the most effective agent in vivo against both drug-sensitive and drug-resistant trypanosomes.
Clinical isolates of Trypanosoma brucei rhodesiense, which were resistant to arsenical drugs in m... more 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.
Eight dicationic compounds related to pentamidine were studied for trypanocidal activity in seven... more 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.
Anti-idiotype (Id) antibodies against anti-a1 rabbit allotype antibodies were produced in rabbits... more Anti-idiotype (Id) antibodies against anti-a1 rabbit allotype antibodies were produced in rabbits and mice by immunization with polyclonal rabbit anti-a1 allotype or with a monoclonal murine anti-a1 allotype antibody, respectively. These antibodies recognize an interspecies cross-reactive idiotope on anti-a1 allotype antibodies. However, these anti-Id antibodies also react with various subclasses of human IgG. In multiple systems, it was shown that these anti-Id antibodies interact with the Fc fragment of human IgG and, therefore, exhibit a rheumatoid factor-like activity. These results support the hypothesis that gamma-globulin may not be the only stimulus to clones producing rheumatoid factors and that perhaps other antibodies, especially anti-Id antibodies, also bind gamma-globulin secondarily.
... In situ kinetic characterization of methylthioadenosine transport by the adenosine transporte... more ... In situ kinetic characterization of methylthioadenosine transport by the adenosine transporter (P 2 ) of the African Trypanosoma brucei brucei and ... MTA is then cleaved phosphorolytically by MTA phosphorylase to methylthioribose-1-phosphate (MTR-1-P) and adenine. ...
The effects of 5&#39;-deoxy-5&#39;-(hydroxyethylthio)adenosine (HETA), a trypanocidal ana... more The effects of 5&#39;-deoxy-5&#39;-(hydroxyethylthio)adenosine (HETA), a trypanocidal analog of 5&#39;-deoxy-5&#39;-(methylthio)adenosine (MTA), on polyamine synthesis and S-adenosylmethionine (AdoMet) metabolism were examined in bloodstream forms of Trypanosoma brucei brucei. HETA was cleaved by trypanosome MTA phosphorylase at the same rate as the natural substrate, MTA, in a phosphate-dependent reaction. Fluorine substitution at the 2-position of the purine ring increased activity by approximately 50%, whereas substitution with an amino group reduced activity to about one-third of the control. HETA was accumulated by trypanosomes with internal concentrations of 100-250 microM and &gt;800 microM after a 15-min incubation with 1 and 10 microM, respectively. Trypanosomes preincubated with HETA metabolized it at a rate of 21.9 nmol/hr/mg protein. Preincubation of cells with HETA at 1 or 10 microM inhibited spermidine synthesis from [3H]ornithine by 22-37%, and increased the cytosolic levels of AdoMet by 2- to 5-fold and that of MTA by up to 8-fold. S-Adenosylhomocysteine (AdoHcy) levels also increased 1.5- to 7-fold in treated cells, whereas decarboxylated AdoMet decreased 65%. Preincubation of trypanosomes with HETA for 4 hr also reduced the incorporation of [35S]methionine in trichloroacetic acid-precipitable material by 50-60%, and reduced the methyl group incorporation into protein from [U-14C]methionine by 65-70%. Thus, HETA interferes with a series of biochemical events involving the participation of AdoMet and methionine in polyamine synthesis, protein synthesis, and transmethylation reactions.
Trichomonas vaginalis grown for 16 h in the presence of [(14)C]spermine formed a high intracellul... more Trichomonas vaginalis grown for 16 h in the presence of [(14)C]spermine formed a high intracellular pool of [(14)C]spermidine and a small but detectable pool of [(14)C]putrescine. When [(3)H]putrescine was added to the growth medium, a large intracellular pool of [(3)H]putrescine was found, but it was not further metabolized, confirming previous studies suggesting the absence of a forward-directed polyamine synthetic pathway in T. vaginalis. Spermidine:spermineN:(1)-acetyltransferase (SSAT) and polyamine oxidase enzyme activities were detected which collectively converted spermine to spermidine. Polyamine oxidase was localized in the hydrogenosome-enriched fraction, whereas SSAT was found predominantly in the cytosolic fraction. In the presence of saturating substrate, the trichomonad SSAT had an activity of 0. 39+/-0.09 nmol min(-1) (mg protein)(-1) (the mean of five analyses) and an apparent K:(m) for spermine of 1.7 microM. The enzyme was competitively inhibited by di(ethyl)norsp...
A recently developed diaminotriazine derivative [O,O'-bis(1, 2-dihydro-2,2-tetramethylene-4,6... more A recently developed diaminotriazine derivative [O,O'-bis(1, 2-dihydro-2,2-tetramethylene-4,6-diamino-S-triazin-1-yl)-1, 6-hexanediol dihydrochloride; T-46; SIPI 1029] was examined for activity against African trypanosomes in in vitro and in vivo model systems. In vitro, SIPI 1029 was 50% inhibitory for growth of bloodstream trypomastigotes of four strains of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense at 0.15 to 2.15 nM (50% inhibitory concentrations). In in vivo mouse laboratory models of T. b. rhodesiense clinical isolate infections, SIPI 1029 was curative for 12 of 13 isolates at </=10 mg/kg of body weight/day for 3 days. In eight infections, a single dose was >/=60% curative, and in six of these, a dose of </=5 mg/kg was sufficient for >/=60% cure rates. A number of these isolates were resistant to the standard trypanocide melarsoprol (Arsobal) and/or the diamidines diminazene aceturate (Berenil) and pentamidine. SIPI 1029 was also curative in c...
5*-Deoxy-5*-(methylthio)adenosine (MTA), a key by-product of polyamine biosynthesis, is cleaved b... more 5*-Deoxy-5*-(methylthio)adenosine (MTA), a key by-product of polyamine biosynthesis, is cleaved by MTA phosphorylase and is salvaged as adenine and, through conversion of the ribose moiety, methionine. An analog of MTA, 5*-deoxy-5*-(hydroxyethylthio)adenosine (HETA), is a substrate for trypanosome MTA phosphorylase and is active in vitro and in vivo against Trypanosoma brucei brucei, an agent of bovine trypanosomiasis. In this study, HETA
A series of conformationally restricted congeners of pentamidine in which the flexible pentyl bri... more A series of conformationally restricted congeners of pentamidine in which the flexible pentyl bridge of pentamidine was replaced by trans-1,2-bismethylenecyclopropyl, phenyl, pyridinyl, piperazinyl, homopiperazinyl, and piperidinyl groups were synthesized. The compounds were evaluated for trypanocidal activity in vitro and in vivo against one drug-sensitive and three drug-resistant trypanosome isolates. The DNA binding affinity of the compounds was also studied using calf thymus DNA and poly(dA-dT). The nature of the linker influenced the DNA binding affinity as well as the trypanocidal activity of the compounds. trans-1,2-Bis(4-amidinophenoxymethylene)cyclopropane (1) was over 25-fold more potent than pentamidine against the drug-resistant isolate KETRI 243As-10-3, albeit with comparable DNA binding affinity. N,N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-Bis(4-amidinophenyl)homopiperazine (8) was the most potent trypanocide in vitro against all four trypanosome isolates studied, but N,N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-bis(4-amidinophenyl)piperazine (6) was the most effective agent in vivo against both drug-sensitive and drug-resistant trypanosomes.
Clinical isolates of Trypanosoma brucei rhodesiense, which were resistant to arsenical drugs in m... more 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.
Eight dicationic compounds related to pentamidine were studied for trypanocidal activity in seven... more 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.
Anti-idiotype (Id) antibodies against anti-a1 rabbit allotype antibodies were produced in rabbits... more Anti-idiotype (Id) antibodies against anti-a1 rabbit allotype antibodies were produced in rabbits and mice by immunization with polyclonal rabbit anti-a1 allotype or with a monoclonal murine anti-a1 allotype antibody, respectively. These antibodies recognize an interspecies cross-reactive idiotope on anti-a1 allotype antibodies. However, these anti-Id antibodies also react with various subclasses of human IgG. In multiple systems, it was shown that these anti-Id antibodies interact with the Fc fragment of human IgG and, therefore, exhibit a rheumatoid factor-like activity. These results support the hypothesis that gamma-globulin may not be the only stimulus to clones producing rheumatoid factors and that perhaps other antibodies, especially anti-Id antibodies, also bind gamma-globulin secondarily.
... In situ kinetic characterization of methylthioadenosine transport by the adenosine transporte... more ... In situ kinetic characterization of methylthioadenosine transport by the adenosine transporter (P 2 ) of the African Trypanosoma brucei brucei and ... MTA is then cleaved phosphorolytically by MTA phosphorylase to methylthioribose-1-phosphate (MTR-1-P) and adenine. ...
The effects of 5&#39;-deoxy-5&#39;-(hydroxyethylthio)adenosine (HETA), a trypanocidal ana... more The effects of 5&#39;-deoxy-5&#39;-(hydroxyethylthio)adenosine (HETA), a trypanocidal analog of 5&#39;-deoxy-5&#39;-(methylthio)adenosine (MTA), on polyamine synthesis and S-adenosylmethionine (AdoMet) metabolism were examined in bloodstream forms of Trypanosoma brucei brucei. HETA was cleaved by trypanosome MTA phosphorylase at the same rate as the natural substrate, MTA, in a phosphate-dependent reaction. Fluorine substitution at the 2-position of the purine ring increased activity by approximately 50%, whereas substitution with an amino group reduced activity to about one-third of the control. HETA was accumulated by trypanosomes with internal concentrations of 100-250 microM and &gt;800 microM after a 15-min incubation with 1 and 10 microM, respectively. Trypanosomes preincubated with HETA metabolized it at a rate of 21.9 nmol/hr/mg protein. Preincubation of cells with HETA at 1 or 10 microM inhibited spermidine synthesis from [3H]ornithine by 22-37%, and increased the cytosolic levels of AdoMet by 2- to 5-fold and that of MTA by up to 8-fold. S-Adenosylhomocysteine (AdoHcy) levels also increased 1.5- to 7-fold in treated cells, whereas decarboxylated AdoMet decreased 65%. Preincubation of trypanosomes with HETA for 4 hr also reduced the incorporation of [35S]methionine in trichloroacetic acid-precipitable material by 50-60%, and reduced the methyl group incorporation into protein from [U-14C]methionine by 65-70%. Thus, HETA interferes with a series of biochemical events involving the participation of AdoMet and methionine in polyamine synthesis, protein synthesis, and transmethylation reactions.
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