MRI demonstrates glutamine antagonist-mediated reversal of cerebral malaria pathology in mice.

The deadliest complication of Plasmodium falciparum infection is cerebral malaria (CM), with a case fatality rate of 15 to 25% in African children despite effective antimalarial chemotherapy. No adjunctive treatments are yet available for this devastating disease. We previously reported that the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) rescued mice from experimental CM (ECM) when administered late in the infection, a time by which mice had already suffered blood-brain barrier (BBB) dysfunction, brain swelling, and hemorrhaging. Herein, we used longitudinal MR imaging to visualize brain pathology in ECM and the impact of a new DON prodrug, JHU-083, on disease progression in mice. We demonstrate in vivo the reversal of disease markers in symptomatic, infected mice following treatment, including the resolution of edema and BBB disruption, findings usually associated with a fatal outcome in children and adults with CM. Our results support the premise that JHU-083 is a potential adjunctive treatment that could rescue children and adults from fatal CM.

DNA strand cleavage by tumor-inhibiting antibiotic 6-diazo-5-oxo-L-norleucine.

A tumor-inhibiting antibiotic, 6-diazo-5-oxo-L-norleucine (DON), caused DNA single-strand breaks. Thus, supercoiled plasmid DNA was transformed into an open circular relaxed form DNA by incubation with DON at pH 7.4. DNA strand cleavage by DON was not inhibited by superoxide dismutase, but inhibited by catalase. The inhibition by catalase may not be due to the destruction of hydrogen peroxide, but to the masking DON by the interaction with the heme moiety of the enzyme. DNA strand cleavage by DON was inhibited by azide, mannitol, ethanol, cysteine and 2-mercaptoethanol, suggesting the involvement of radical species in the cleavage. The cleavage, however, was not suppressed by removal of dissolved oxygen from the reaction mixture, indicating that no oxygen-derived radicals participated in the cleavage. Electron spin resonance spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN) elucidated the generation of a carbon-centered radical from DON. Hence, the carbon-centered radical may participate in DNA strand cleavage by DON.

Limb development in mouse embryos: protection against teratogenic effects of 6-diazo-5-ox-L-norleucine (DON) in vivo and in vitro.

The glutamine analogue, 6-diazo-5-oxo-L-norleucine (DON), has been shown to inhibit biosynthesis of purines and glycosaminoglycans, presumably by blocking the glutamine-dependent steps in the biosynthetic pathways. The teratogenic potential of DON on the developing mouse limb-bud in vivo and in vitro was studies in an attempt to discriminate whether DON is exerting its teratogenic effect by interfering with glycosaminoglycan or purine metabolism. A single intramuscular injection of DON (0-5 mg/kg) to ICR/DUB mice on day 10 of gestation resulted in 76% resorption, while fetuses surviving to day 17 exhibited growth retardation, median cleft lip, and limb malformation. Concurrent administration of L-glutamine (250 mg/kg) provided no protection against resorption of malformations, while 5-aminoimidazolecarboxamide (AIC, 250 mg/kg) decreased the resorption rate to 34% without significantly altering the incidence of malformations. Injection of DON alone on day 11 resulted in 87% of fetuses exhibiting limb formations, with only 2% resorption. Concurrent injection of AIC decreased the frequency of limb malformations to 32% L-Glutamine, D-glucosamine, or inosinic acid were without any protective effect in vivo. DON (5mug/ml medium) added in vitro to organ cultures of day 11 mouse limb-buds caused all limbs to evidence cartilage abnormalities. In this system, either L-glutamine ro D-glucosamine (0-5 mg/ml medium) provided protection against DON effects while AIC (0-5 mg/ml medium) offered no protection in vitro. These data suggest that DON exerts its effects in vivo by interfering with purine metabolism while in vitro its teratogenic action may be interruption of glycosaminoglycan biosynthesis. This may reflect upon the relative importance of growth and differentiation to limb development in vivo and in vitro. These data infer that limb development in vitro relies more on the differentiative process (differentiation of cartilage) than on growth, whereas limb development in vivo is dependent, at this stage, to a greater extent on growth for normal phenotypic expression.