Re-examination of inhibitor resistance conferred by Qo-site mutations in cytochrome b using yeast as a model system

Cytochrome b from yeast (Saccharomyces cerevisiae Meyer ex Hansen) provides a convenient model system for the study of Qo-site inhibitor (QoI) resistance mutations from a variety of organisms. QoI resistance mutations from fungal plant pathogens (G143A and F129L), malaria agent Plasmodium sp (Y279C/S), and Pneumocystis carinii (L275F), an opportunistic pathogenic fungus of man, were introduced into yeast cytochrome b and their effect on the binding of a variety of natural (myxothiazol and stigmatellin) and synthetic (atovaquone, azoxystrobin and pyraclostrobin) inhibitors to the bc1 complex monitored. L275S (from a myxothiazol-resistant yeast) was also re-examined. Stigmatellin binding was relatively unaffected by the introduction of these mutations. Significant increases in resistance were observed for the strobilurin-class inhibitors myxothiazol, azoxystrobin and pyraclostrobin, with the largest increase in resistance conferred by G143A. In contrast, atovaquone binding was most effected by Y279C/S and L275S. Notably, F129L, G143A and L275S had a minor effect on bc1 activity, and so are unlikely to confer significant fitness penalties in vivo. These data are discussed in the light of the atomic structures for myxothiazol- and azoxystrobin-inhibited bovine bc1 which have recently become available. We propose that QoI resistance due to G143A arises from steric hindrance between the inhibitor and cytochrome b, whereas the mechanism of resistance for the other mutations is due to an increase in binding energy between the protein and inhibitor molecule. Site-directed mutagenesis was also used to model selected regions of the mammalian Qo site in yeast cytochrome b in order to further understand the differential efficacy of these QoI in the mammalian and pathogen bc1 complexes.     

Seasonal reproduction in the mare: possible role of plasma leptin, body weight and immune status

The primary objective of this study was to evaluate the possible role of leptin, body weight and immune status on reproductive activity throughout the transition period from cyclicity to seasonal anestrus, during anestrus and resumption of ovarian activity in Lusitano mares. Mares in good body condition were monthly monitored throughout 2 years (10 mares in each year) for evaluation of their reproductive status by sequential ultrasonography and plasma progesterone determinations. On the second year, all mares were weighed. Progesterone and leptin were assayed by radioimmunoassay (RIA). Parameters of the immune status (phagocytosis and oxidative burst of neutrophils, characterisation of circulating lymphocyte subsets) were also evaluated. Phagocytosis and oxidative burst in blood neutrophils were measured by flow cytometry using commercially available kits. Lymphocyte subsets were assessed by indirect immunofluorescence staining after incubation with monoclonal antibodies specific for CD2, CD19, CD4, CD8 cells markers by flow cytometry. Natural killer cells and B cells were estimated mathematically. No significant difference was found in phagocytosis, oxidative burst and circulating lymphocyte subsets at anestrus and at either phase of the estrous cycle (p>0.05), suggesting that the immune status of the mare was not influenced by the seasonal changes in ovarian activity. This study also suggests that body weight has a direct relationship with plasma leptin levels. Increased concentrations of this hormone in circulation might be associated with the restart or maintenance of ovarian cyclicity in Lusitano mares.