Expected and actual cDNA amplicon sizes and their corresponding sequence accession num bers are shown in Table 2. The majority of the protease genes were expressed in more than one of the four parasite stages investigated. However, stage specific up or downregulation of protease gene expression was evident. Thus, taking into account that merozoite cDNA http://www.selleckchem.com/products/Tipifarnib(R115777).html contaminates the ase and rhomboid protease 1. Aminopeptidase N1 appeared to be downregulated specifically in merozoites. Gametocyte specific or gametocyte upregulated pro teases were also common, with thirteen in all, also dis tributed across the four groups of proteases, including eimepsin 2, cathepsin C2, ubiquitinyl hydrolase 2 and 5, the pyroglutamyl peptidase, aminopeptidase N2, insuly sin 4, the S2P like metalloprotease, two trypsin like proteases and three of the subtilisins.
Additionally, two other proteases were upregulated or specific for the sexual phase of the lifecycle, namely, cathepsin C3 and subtili sin 4. Cathepsin L appeared to be downregulated specifically in gametocytes. Only two protease genes, a pepsin like protease with high homology to eimepsin and an insulysin, were switched on exclu sively in oocyst lifecycle stages. In contrast, Inhibitors,Modulators,Libraries numerous protease genes appeared to be downregulated in sporu lated oocysts. Protease processing of GAM56 Gametocytes from E. tenella infected caeca were lysed and immediately incubated with or without protease inhibitors for various lengths of time, and the native GAM56 protein analysed by SDS PAGE and western blotting with anti GAM56 antibodies, as described previously, to track the disappearance of the pro tein to determine whether any inhibitors could prevent the degradation observed in the presence of native gam etocyte proteases.
The precise epitopes recognised by the anti GAM56 polyclonal antibodies are not known for E. tenella though there is some evidence, from work with E. maxima, Inhibitors,Modulators,Libraries that they are located in the con Inhibitors,Modulators,Libraries served amino terminus of the protein. The anti GAM56 antibodies are, thus, very useful for sensitive and specific tracking of the degradation of GAM56. No disappear ance Inhibitors,Modulators,Libraries of GAM56 was apparent after 2, 4, 6, 8, 10, 12 or 16 h but was obvious at 24h. The 24 h assay was therefore repeated three times with a comprehensive range of protease inhibitors targeting the four protease families identified in the gen ome.
The aspartyl protease inhibitor, pepstatin A, had no effect on GAM56 disappearance. None of three cysteine protease inhibitors investigated, Z Phe Ala diazomethylketone, N ethylmalemide or E64 inhibited GAM56 disappearance. The serine cysteine protease Inhibitors,Modulators,Libraries inhibitor, chymostatin and leupeptin, inhibited GAM56 disappearance but another inhibitor with the same specificity, antipain, did not. The serine protease specific inhibitors, benzamidine HCL, soybean trypsin inhibitor and aprotinin Ruxolitinib clinical all inhibited the disappearance of GAM56 but AEBSF did not.