RESUMO
The pel1 mutation in Saccharomyces cerevisiae and the Cgpgs1Delta mutation in Candida glabrata result in deficiency of mitochondrial phosphatidylglycerolphosphate synthase and lack of two anionic phospholipids, phosphatidylglycerol and cardiolipin. DNA sequence analysis of the PCR-amplified pel1 mutant allele revealed that the pel1 mutation resulted from a single amino-acid substitution (Glu(463)Lys) in the C-terminal part of encoded enzyme. The CgPGS1 gene cloned in a centromeric pFL38 vector functionally complemented the pel1 mutation in S. cerevisiae. Likewise, the ScPGS1 gene cloned in pCgACU5 plasmid fully complemented the Cgpgs1Delta mutation in C. glabrata. This mutation increased the cell surface hydrophobicity and decreased biofilm formation. These results support a close evolutionary relatedness of S. cerevisiae and C. glabrata and point to the relationship between expression of virulence factors and anionic phospholipid deficiency in pathogenic C. glabrata.
Assuntos
Mutação , Fosfolipídeos/deficiência , Leveduras/genética , Sequência de Aminoácidos , Biofilmes , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Fenótipo , Fosfolipídeos/química , Alinhamento de Sequência , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Leveduras/química , Leveduras/classificação , Leveduras/fisiologiaRESUMO
The implication of endogenous nitric oxide in the effect of hypoxia on the neurotransmission in the enteric nervous system of guinea-pig ileum was studied in vitro. Three methodological approaches have been used: (i) Stretch-induced phases of peristaltic reflex in ileal segments; (ii) twitch contractions of longitudinal segments, evoked by electrical field stimulation; and (iii) release of [3H]acetylcholine from longitudinal muscle-myenteric plexus preparations, measured by liquid spectrophotometry. The effect of nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NNA, 100 microM) was studied under normoxic conditions. L-NNA did not change significantly the ascending contraction phase of peristaltic reflex and the amplitude of twitch contractions. However, the same concentration of L-NNA increased the stimulation-evoked acetylcholine release. The descending relaxation phase decreased in the presence of L-NNA. In another set of experiments, hypoxia was mimicked by replacement of oxygen from the perfusion medium with nitrogen for a period of 30 min. Hypoxia significantly decreased the ascending contraction phase, the twitch contractions, and the release of acetylcholine from the myenteric plexus. Under hypoxic conditions, pretreatment with L-NNA did not change either the contractile responses, nor the release of acetylcholine. Our results suggest that under conditions of oxygen deprivation, endogenous nitric oxide seems to be inefficient in modulating the cholinergic neurotransmission in guinea-pig ileum.
