and GAM82, is integral to oocyst wall formation, tyrosine rich pe

and GAM82, is integral to oocyst wall formation, tyrosine rich peptides formed by the degradation of these two proteins are believed to be subsequently cross linked via dityrosine bonds, giving the oocyst wall its renowned strength and resistance to environmental and chemical insults. To test this hypothesis, we designed inhibitor expert an assay to fol examined in E. tenella is upregulated in merozoites fur ther underscores the importance of proteases in the biol ogy of the asexual stages of apicomplexan parasites. Not surprisingly, therefore, an eimepsin, several cathepsins, a calpain, a trypsin like protease, subtilisins, Clp and a rhomboid protease are upregulated in the asexual stages of E. tenella.

Likewise, eimepsin1 and insulysin 3 are expressed specifically in oocysts and may play an important role in the first steps of the parasite lifecycle, such as host cell invasion, they are, therefore, worthy of further research. The downregulation of several pro teases in sporu lated oocysts may Inhibitors,Modulators,Libraries be, in part, attributed to the dormancy of this lifecycle stage, yet still warrants further investigation. Perhaps the most significant finding of our stage specific expression study was the relatively large Inhibitors,Modulators,Libraries number of protease genes whose expression is upregulated spe cifically in the gametocytes stage a total of at least 13 genes, including six that are only expressed in gameto cyte. This observation becomes even more intriguing when examined in the context of the low the degradation of GAM56 in freshly harvested gametocytes.

This assay has certain inherent limitations, first, it relies on sensitive antibodies Inhibitors,Modulators,Libraries for de tection of specific degradation of GAM56 and, unfortu nately, the lack of suitable antibodies for detection of GAM82 in E. tenella meant that we were unable to run confirmatory experiments with this protein, and, second, the only controls possible are a zero time point and a cocktail of protease inhibitors designed to prevent all proteolytic activity. These limitations require us to be cautious in our interpretations, none the less, the inhib ition of degradation of native GAM56 by a very specific group of protease inhibitors reveals that this function may be carried out by subtilisin like proteases. Inhibitors,Modulators,Libraries Thus, degradation of GAM56 was inhibited by the serine cyst eine protease inhibitors, chymostatin and leupeptin, and the serine protease specific inhibitors, benzamidine HCL, soybean trypsin inhibitor and aprotinin but not by AEBSF.

Intriguingly, the metal chelating Cilengitide agent, EDTA, also inhibited degradation of GAM56. This profile indicates that serine proteases are critical for degradation of GAM56 but it seems to rule out participation of rhomboid pro teases, which are unaffected by EDTA, aprotonin, leupeptin and chymostatin. Trypsin like proteases can, perhaps, not be completely ruled out of this process but the inhibi tory profile, particularly the lack of inhibition by AEBSF coupled with the inhibitory effect of EDTA, points to a sub tilisin or subtilisins as

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