Characterization of the Phytochelatin Synthase of Schistosoma mansoni

Treatment for schistosomiasis, which is responsible for more than 280,000 deaths annually, depends exclusively on the use of praziquantel. Millions of people are treated annually with praziquantel and drug resistant parasites are likely to evolve. In order to identify novel drug targets the Schistosoma mansoni sequence databases were queried for proteins involved in glutathione metabolism. One potential target identified was phytochelatin synthase (PCS). Phytochelatins are oligopeptides synthesized enzymatically from glutathione by PCS that sequester toxic heavy metals in many organisms. However, humans do not have a PCS gene and do not synthesize phytochelatins. In this study we have characterized the PCS of S. mansoni (SmPCS). The conserved catalytic triad of cysteine-histidine-aspartate found in PCS proteins and cysteine proteases is also found in SmPCS, as are several cysteine residues thought to be involved in heavy metal binding and enzyme activation. The SmPCS open reading frame is considerably extended at both the N- and C-termini compared to PCS from other organisms. Multiple PCS transcripts are produced from the single encoded gene by alternative splicing, resulting in both mitochondrial and cytoplasmic protein variants. Expression of SmPCS in yeast increased cadmium tolerance from less than 50 µM to more than 1,000 µM. We confirmed the function of SmPCS by identifying PCs in yeast cell extracts using HPLC-mass spectrometry. SmPCS was found to be expressed in all mammalian stages of worm development investigated. Increases in SmPCS expression were seen in ex vivo worms cultured in the presence of iron, copper, cadmium, or zinc. Collectively, these results indicate that SmPCS plays an important role in schistosome response to heavy metals and that PCS is a potential drug target for schistosomiasis treatment. This is the first characterization of a PCS from a parasitic organism

Localization of alpha 2 receptors in ocular tissues

Alpha 2 adrenergic agonists are used for controlling intraocular pressure (IOP) in the treatment of glaucoma. They have also been shown to be neuroprotective to retinal cells in a variety of injury models. Despite this significance, the localization of the three known alpha 2 adrenergic receptors has not been unequivocally established. The aim of this study was to determine the location of the three alpha 2 adrenergic receptors in ocular tissues using immunohistochemical techniques. New antibodies were generated and their specificity was determined using Western blotting and preadsorption. In the anterior segment of the eye alpha 2A immunoreactivity was located in the nonpigmented ciliary epithelium, corneal, and conjunctival epithelia. Alpha 2B staining was not apparent in these tissues. Alpha 2C immunostaining was present in the membrane of pigmented ciliary epithelium and corneal and conjunctival epithelial cells. In the rat retina, all three receptor subtypes were present but were differentially localized. Alpha 2A was present in the somata of ganglion cell layer and inner nuclear layer somas, alpha 2B was located in the dendrites and axons of most of the neurons as well as glia, while alpha 2C was present in the somata and inner segment of the photoreceptors. In human and monkey retinas, similar pattern of labeling for alpha 2A and 2B receptors were observed, while alpha 2B was additionally present in the membranes of many cell somata in addition to dendrites and axons. Alpha 2C labeling was much weaker but exhibited similar pattern to that observed in the rat. These data provide additional information on the location of the alpha 2 receptors in the anterior portion of the eye and present new information on their specific location in the retina. This offers insights into possible targets for adrenergic agonists in a therapeutic contex