The microsporidia are obligate intracellular protozoan parasites of increasing importance as human pathogens, which are characterized by a small resistant spore with a single polar filament that coils around the sporoplasm. When stimulated, the polar filament rapidly everts out of the spore to form a hollow polar tube through which the sporoplasm passes, thus serving as a unique mechanism of transmission. A genomic library of the human microsporidium Encephalitozoon hellem was screened using a polyclonal rabbit antibody (anti-PTP Eh55) produced to the major HPLC purified polar tube protein (PTP) of E. hellem. This antibody localized to intrasporal polar filaments and extrasporal polar tubes of E. hellem by immunogold electron microscopy confirming the polar tube specificity of the antibody. A total of 14 anti-PTP Eh55 reactive genomic clones were identified and purified. A PTP gene was identified consisting of 1362 bp coding for 453 amino acids. The N-terminus of the translated protein consists of aputative N-terminal signal sequence of 22 amino acids, which when cleaved results in a mature protein of 431 amino acids with a predicted molecular mass of 43 kDa. The protein has a high proline content (14.6%) and contains a central domain of six alternating tandem repeats of 20 amino acids. After ligation of the gene into a glutathione S-transferase (GST) expression vector, a fusion protein was produced that reacted by immunoblotting with the polar tube specific anti-PTP Eh55. The gene was present as a single copy in the genome and there was no homology with other known genes. As the polar tube is a critical structure for the transmission of this organism to a new host cell, further study of PTPs may lead to the development of new therapeutic strategies and diagnostic tests.

Microsporidia are parasitic protists of all classes of vertebrates and most invertebrates. They recently emerged as important infections in various immunosuppressed and immunocompetent patient populations. They are also important veterinary and agricultural pathogens. Current therapies for microsporidiosis include benzimidazoles, which bind tubulin-inhibiting microtubule assembly, and fumagillin and its derivatives, which bind and inhibit methionine amino peptidase type 2 (MetAP2). Benzimidazoles are not active against Enterocytozoon bieneusi, the most common cause of human microsporidiosis. Fumagillin is active against most microsporidia, including E. bieneusi, but thrombocytopenia has been a problem in clinical trials. There is a pressing need for more-specific microsporidian MetAP2 inhibitors. To expedite and facilitate the discovery of safe and effective MetAP2 inhibitors, we have engineered Saccharomyces cerevisiae to be dependent on Encephalitozoon cuniculi MetAP2 (EcMetAP2) for its growth, where EcMetAP2 is harbored on an episomal uracilselectable tetracycline-regulated plasmid. We have also constructed a leucine-selectable tetracycline-regulated expression plasmid into which any MetAP2 gene can be cloned. By utilizing a 5-fluoroorotic acid-mediated plasmid shuffle in the EcMetAP2 yeast strain, a yeast strain can be generated whose growth is dependent on MetAP2 from any organism. The level of heterologous MetAP2 gene expression can be controlled by the addition of tetracycline to the growth medium. These yeast strains should permit high-throughput screening for the identification of new inhibitors with high specificity and activity toward microsporidian MetAP2.

Trypomastigotes of Trypanosoma cruzi maintain an intracellular Ca2+ concentration ([Ca2+]i) of 64 +/- 30 nM. Equilibration of trypomastigotes in an extracellular buffer containing 0.5 mM [Ca2+]o (preloaded cells) increased [Ca2+]i < 20 nM whereas total cell Ca2+ increased by 1.5 to 2.0 pmole/cell. This amount of Ca2+ would be expected to increase [Ca2+]i to > 10 microM suggesting active sequestration of Ca2+. We tested the hypothesis that maintenance of [Ca2+]i involved both the sequestration into intracellular storage sites and extrusion into the extracellular space. Pharmacological probes known to influence [Ca2+]i through well characterized pathways in higher eukaryotic cells were employed. [Ca2+]i responses in the presence or absence of [Ca2+]o were measured to asses the relative contribution of sequestration or extrusion processes in [Ca2+]i homeostasis. In the presence of 0.5 mM [Ca2+]o, the ability of several agents to increase [Ca2+]i was magnified in the order ionomycin >>> nigericin > thapsigargin > monensin > valinomycin. In contrast, preloading markedly enhanced the increase in [Ca2+]i observed only in response to monensin. Manoalide, an inhibitor of phospholipase A2, enhanced the accumulation of [Ca2+]i due to all agents tested, particularly ionomycin and thapsigargin. Our results suggest that sequestration of [Ca2+]i involved storage sites sensitive to monensin and ionomycin whereas extrusion of Ca2+ may involve phospholipase A2 activity. A Na+/Ca2+ exchange mechanism did not appear to contribute to Ca2+ homeostasis.

The aim of this paper is to elucidate the drainage gradient method's feasibility in evaluating and screening anti-scaltants. Nine different anti-scalants were selected as research object, the results show that KCa、KJD、pH and Langelier's saturation index (LSI) can be used as indicators and KJD、pH are more sutible indicators.with the relative standard deviations (RSD) of repeated experiments were from 0.24% to 3.04%, the drainage gradient method has good repeatability.