Seymour H. Hutner

In vivo effects of α-dl-Difluoromethylornithine on the metabolism and morphology of Trypanosoma brucei brucei

Abstract The EATRO 110 isolate of Trypanosoma brucei brucei was grown in rats for 60 h and the animals treated with the ornithine decarboxylase inhibitor α- dl -difluoromethylornithine 12 h or 36 h prior to sacrifice. Control untreated animals died 72–80 h after infection. Treated parasites were shorter and broader than the predominantly long slender forms found in untreated controls and many had two or more nuclei and kinetoplasts. Trypanosomes were purified from blood and examined for disruption of polyamine metabolism. ODC activity decreased by more than 99% after 12 h treatment and putrescine and spermidine levels also decreased dramatically. Spermine, not normally present in control cells, increased to detectable, low levels ( −1 protein) after 36 h treatment. α- dl -Difluoromethylornithine-treated cells were unable to synthesize putrescine from [ 3 H]ornithine but were able to convert [ 3 H]putrescine + methionine to spermidine. 12-h treated parasites responded to polyamine depletion by assimilating radiolabeled polyamines in vitro at 2- to 4-times the rate of untreated cells. The metabolism of S -adenosylmethionine was also altered in treated parasites: decarboxylated S -adenosylmethionine increased more than 1000-fold over untreated cells while S -adenosylmethionine decarboxylase activity, associated with the formation of spermidine and spermine in other eukaryotes, paradoxically declined in treated cells. Synthesis of macromolecules was perturbed in treated parasites: rates of DNA and RNA synthesis declined 50–100%, while protein synthesis increased up to 4-fold in 36-h treated cells. α- dl -Difluoromethylornithine treatment progressively limits the parasites ability to synthesize nucleic acids and blocks cytokinesis while inducing morphological changes resembling long slender → short stumpy transformation.

Water-soluble vitamins in cells and spent culture supernatants of Poteriochromonas stipitata, Euglena gracilis, and Tetrahymena thermophila

Vitamins B6 and B12, biotin, folates, riboflavin, nicotinate, pantothenate, biopterin, and vitamin C (l-ascorbate) were assayed in Poteriochromonas stipitata, Euglena gracilis, and Tetrahymena thermophila cells grown in defined media and in spent culture supernatants. P. stipitata and E. gracilis synthesized, stored and excreted folates (mainly as 5-methyltetrahydrofolate), B6, riboflavin, pantothenate, nicotinate, biopterin, and ascorbate. E. gracilis synthesized and stored biotin. T. thermophila did not synthesize the above vitamins except for B12, biopterin, and ascorbate; it excreted biopterin and stored B12 and ascorbate. Thiamin was left of consideration because all 3 organisms are thiamin auxotrophs. Possible ecological implications of these findings are considered.

Paradoxical activation of Leptomonas NAD-linked α-glycerophosphate dehydrogenase by ethidium and antrycide

Abstract Antrycide and ethidium bromide — 2 cationic trypanocides — inhibited NAD-linked α-glycerophosphate dehydrogenase from Leptomonas sp. The kinetics of enzyme inhibition was determined by Lineweaver-Burk, Dixon, or direct linear plots. Inhibition by Antrycide was noncompetitive for dihydroxyacetone phosphate in the presence of saturating Mg2+ or spermidine. With dihydroxyacetone phosphate at saturation, Antrycide inhibition was also noncompetitive with respect to Mg2+ (Ki = 115 μM) and spermidine (Ki = 85 μM). Inhibition by ethidium in the presence of saturating dihydroxyacetone phosphate, was noncompetitive for Mg2+ (Ki = 400 μM) but mixed for spermidine (Ki = 495 μM); inhibition was noncompetitive for dihydroxyacetone phosphate in the presence of saturating Mg2+ or spermidine. Rabbit-muscle α-glycerophosphate dehydrogenase was inhibited at all concentrations of Antrycide and ethidium tested, but the Leptomonas enzyme was stimulated up to 3.5-fold by low concentrations of inhibitors in the absence of polyamine. New chemotherapeutic possibilities may thus be opened and an evolutionary distinction between trypanosomatid and mammalian enzyme.

A practical assay of lipoate in biologic fluids and liver in health and disease.

A procedure for assaying lipoic acid concentration in biologic fluids and tissues was devised using a eukaryotic protozoan Tetrahymena thermophila. T.thermophila has a specific and sensitive (30 pg/ml) requirement for lipoic acid. Unlike humans and other microorganisms, T.thermophila can not synthesize lipoic acid; hence, its requirement for exogenous lipoic acid is specific. The lipoic acid supplied to T. thermophila by the processing of biologic fluids and tissues during the assay procedure, permits the derivation of a practical assay for lipoate concentration as described here. Lipoate concentration in biologic fluids and tissue obtained from healthy humans, compared to those obtained from patients with renal and liver disease, indicate deviations from normal during disease. Absorption chartings of 200 mg of DL-alpha-lipoic acid in humans indicate a peak concentration of lipoate in plasma 2 h after ingestion and then a steady descent of lipoate to a baseline level after 24 h. With this practical assay, it is now possible to chart lipoates antioxidant activity and therapeutic action during health and disease.

Unsubstituted agarose as an affinity matrix for isolation of NAD-dependent α-glycerophosphate dehydrogenase from the trypanosomatid Crithidiafasciculata

Summary A soluble, NAD-dependent α-glycerophosphate dehydrogenase from the flagellate Crithidia fasciculata was purified to near homogeneity by substrate elution from DEAE cellulose then affinity chromatography on unsubstituted agarose. This two-step procedure permitted 124-fold purification. The crude enzyme did not bind to unsubstituted agarose, but was retained by n -alkyl-amino agarose derivatives. A molecular weight of 78,000 was obtained from the native enzyme by sucrose gradient ultracentrifugation and 66,000 for the polypeptide chain by SDS gel electrophoresis. The apparent K m for dihydroxyacetone phosphate was 1.17 mM. The enzyme is stimulated by Mg ++ ; low (1 mM) concentrations of spermidine or spermine replaced Mg ++ as activator.

Antioxidant survey to assess antagonism to redox stress using a prokaryotic and an eukaryotic system

Using a prokaryote (Escherichia coli) and a metazoa-resembling eukaryote (Ochromonas danica), we surveyed antioxidants which might overcome redox stress imposed by menadione sodium bisulphite (MD) and buthionine sulphoximine (BSO). BSO oxidant stress was evident only inO. danica; MD oxidant stress was evident in both organisms. Glutathione, its precursors, e.g. cysteine, homocysteine, and 2-oxo-4-thiazolidine carboxylic acid, and red blood cells, emerged as prime antioxidants for relieving BSO and MD oxidant stress. BSO and MD oxidant activity and antioxidant-annulling effect inO. danica were judged comparable to those found in animal cells whereas the resultsE. coli were not entirely equivalent. TheO. danica system emerged as a practical, rapid, and useful system for pinpointing oxidant stressors and antioxidants, and shows promise for studies with mammalian systems.

Role of 5′-Deoxy-5′-Methylthioadenosine in growth of several microbial B12 requirers

5-Deoxy-5-Methylthioadenosine (MTA) figures in cellular methionine and polyamine syntheses. It replaces B12 for growth of the chrysomonad protozoan Poteriochromonas malhamensis at a ratio of MTA:B12 of approximatly 10,000,000:1 (by weight). MTA does not replace B12 for other B12-requirers, e.g.: Euglena gracilis, Lactobacillus leichmannii, and Escherichia coli 113-3. The methionine synergism for P. malhamensis growth is also negated when B12 activity is annulled by alkali treatment; MTA is not inactivated by such treatment. The growth promoting activity of various deoxynucloesides and deoxynucloetides for P. malhamensis and other B12-requirers is reported here due to contamination by cobalamins. Ethionine antagonizes the growth-enhancing effect of MTA, methionine, and B12, individually and collectively -evidence that MTA plays a role in supplying methionine for P. malhamensis growth. MTA concentrations in body fluids and mammalian tissues are too low to interfere with the use of P. malhamensis for estimating only metabolically active B12.

Polyamines antagonize both the antileukemic activity and the reverse transcriptase stimulatory activity of 4,4′-diacetyldiphenylurea bis(guanylhydrazone) (DDUG)

(Diacetyldiphenylurea)bis(guanylhydrazone) (DDUG) functions as a cationic trypanocide antagonized in vivo by exogenous concomitant addition of the biologically active polyamine, spermine. It also inhibits the DNA polymerases of L1210 murine leukemia cells. We have found that DDUG stimulates Rauscher murine leukemia virus DNA polymerase activity in a manner similar to polyamines. Such stimulation does not occur if DNA synthesis is carried out on spermine + activated DNA complexes. We also show that the in vivo antileukemic activity of DDUG in the L1210 ascites mouse model is antagonized by biologically active polyamines. These studies suggest a new intracellular target for the antileukemic activity of DDUG: interference with polyamine function.