Methionine synthase catalyzes the transfer of a methyl group from tetrahydrofolate to homocysteine to produce methionine. Although mammalian enzymes are cobalamin-dependent, fungal methionine synthases are cobalamin-independent. The opportunistic pathogen Candida albicans is a diploid and carries two copies of the methionine synthase gene, MET6. Homologous recombination was used to disrupt a single MET6 gene. MET6/met6 knock-outs, deleted with either the URA3 or ARG4 cassette, grew as well as the wild-type strain. However, we were unable to obtain a viable met6/met6 deletion strain, even on media supplemented with exogenous methionine. This suggests that methionine synthase is essential to C. albicans. To explore this further, a C. albicans strain was constructed in which one MET6 locus was deleted and the second placed under a regulatable promoter. The conditional mutant grew well under inducing conditions, even in the absence of methionine. It would not grow under repressing conditions in the absence of methionine, but would grow when the media was supplemented with exogenous methionine. A western blot showed that a small amount of enzyme was expressed under repressing conditions. Taken together, these data reveal that methionine is necessary for growth of C. albicans, but not sufficient -a minimal level of methionine synthase expression is required, perhaps to limit homocysteine toxicity. Furthermore, these results suggest that cobalamin-independent methionine synthase is a plausible target for the design of antifungal agents.

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Heat and moisture exchangers (HMEs) are used for airway humidification in mechanically ventilated patients and have been evaluated only under hospital conditions. U.S. Air Force aeromedical evacuation transports are performed under rugged conditions further complicated by the cold and dry environment in military aircrafts, and HMEs are used to provide airway humidification for patients. This study evaluated 10 commercial HMEs using a test system that simulated aeromedical evacuation conditions. Although the American National Standards Institute recommends inspired air to be at an absolute humidity value of > or = 30 mg/L for mechanically ventilated patients, the highest absolute humidity by any HME was approximately 20 mg/L. Although none of the HMEs were able to maintain a temperature high enough to achieve the humidity standard of the American National Standards Institute, the clinical significance of this standard may be less important than the relative humidity maintained in the respired air, especially on evacuation flights of short duration.