RESUMO
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.
Assuntos
Resgate Aéreo , Respiração Artificial/instrumentação , Ventiladores Mecânicos , Aeronaves , Humanos , Umidade , Respiração Artificial/efeitos adversos , TemperaturaRESUMO
Fungal methionine synthase catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine to create methionine. The enzyme, called Met6p in fungi, is required for the growth of the pathogen Candida albicans, and is consequently a reasonable target for antifungal drug design. In order to understand the mechanism of this class of enzyme, we created a three-dimensional model of the C. albicans enzyme based on the known structure of the homologous enzyme from Arabidopsis thaliana. A fusion protein was created and shown to have enzyme activity similar to the wild-type Met6p. Fusion proteins containing mutations at eight key sites were expressed and assayed in this background. The D614 carboxylate appears to ion pair with the amino group of homocysteine and is essential for activity. Similarly, D504 appears to bind to the polar edge of the folate and is also required for activity. Other groups tested have lesser roles in substrate binding and catalysis.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/química , Candida albicans/enzimologia , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Antifúngicos/química , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Catálise , Desenho de Fármacos , Homocisteína/química , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
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.
Assuntos
Candida albicans/enzimologia , Candida albicans/crescimento & desenvolvimento , Sistemas de Liberação de Medicamentos/métodos , Regulação Fúngica da Expressão Gênica/fisiologia , Metiltransferases/genética , Metiltransferases/metabolismo , Antifúngicos/administração & dosagem , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Proliferação de Células/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Inativação GênicaRESUMO
In this study, we investigated methionine synthase from Candida albicans (CaMET 6p) and Saccharomyces cerevisiae (ScMET 6p). We describe the cloning of CaMet 6 and ScMet 6, and the expression of both the enzymes in S. cerevisiae. CaMET 6p is able to complement the disruption of met 6 in S. cerevisiae. Following the purification of ScMET 6p and CaMET 6p, kinetic assays were performed to determine substrate specificity. The Michaelis constants for ScMET 6p with CH(3)-H(4)PteGlu(2), CH(3)-H(4)PteGlu(3), CH(3)-H(4)PteGlu(4), and l-homocysteine are 108, 84, 95, and 13 microM, respectively. The Michaelis constants for CaMET 6p with CH(3)-H(4)PteGlu(2), CH(3)-H(4)PteGlu(3), CH(3)-H(4)PteGlu(4), and l-homocysteine are 113, 129, 120, and 14 microM, respectively. Neither enzyme showed activity with CH(3)-H(4)PteGlu(1) as a substrate. We conclude that ScMET 6p and CaMET 6p require a minimum of two glutamates on the methyltetrahydrofolate substrate, similar to the bacterial metE homologs. The cloning, purification, and characterization of these enzymes lay the groundwork for inhibitor-design studies on the cobalamin-independent fungal methionine synthases.
