Michael W. Clarke

Characterization of Ca2+-dependent neutral protease (calpain) from human blood flukes, Schistosoma mansoni

Calcium-dependent, neutral cysteine-proteases (calpain) were purified from human blood flukes, Schistosoma mansoni. The electrophoretic mobilities, Western blot analyses and high specificity to peptide inhibitors confirmed the presence of both calpain I and II in the purified preparation. The schistosome calpains were localized in the surface syncytial epithelium and underlying musculature. Using peptide inhibitors, calpain was shown to function as a mediator of the surface membrane synthetic process. Since there was also no immunological cross-reactivity between vertebrate and schistosome calpains using antibodies affinity-purified from native and recombinant schistosome calpains, this protease may be usefully investigated as forming the basis of a molecular vaccine against schistosomiasis.

Schistosoma mansoni: Characterization and identification of calcium-binding proteins associated with the apical plasma membrane and envelope

Calcium-binding proteins (CaBPs) of Schistosoma mansoni were purified by hydrophobic affinity chromatography. Metabolically labeled CaBPs were characterized using SDS-polyacrylamide gel electrophoresis followed by fluorography. A number of CaBPs were detected in total tissue extracts, apical plasma membrane, and soluble fractions of the apical bilayer complex, ranging from 15 to 205 kDa in their molecular masses. No CaBPs were discerned in the envelope of the apical bilayer complex. Two CaBPs were positively identified as calmodulin and gelsolin via immunoblot analyses. The possible role of CaBPs in surface signal transduction mechanisms has also been briefly discussed.

DETECTION OF PSP94 AND ITS SPECIFIC BINDING SITES IN THE PROSTATE ADENOCARCINOMA CELL LINE LNCaP

PURPOSE To evaluate the expression of prostate secretory protein of 94 amino acids (PSP94) and PSP94 binding proteins in the LNCaP cell line. MATERIALS AND METHODS The reverse-transcription polymerase chain reaction (RT-PCR) and Southern blot hybridization were employed to assay the expression of PSP94. Immunoprecipitation with specific polyclonal antibodies was used to detect PSP94 secreted by the LNCaP cells. The binding proteins were assayed by equilibrium binding assays. RESULTS PSP94 was expressed and secreted in the LNCaP cells. As well as, LNCaP cells expressed surface membrane proteins capable of binding PSP94 in a specific and saturable manner. Exposure of LNCaP cells to exogenous PSP94 resulted in the up-regulation of PSP94 binding sites, indicating functional interactions for PSP94 and its receptor in this cell line. CONCLUSION The expression of PSP94 and its receptors may be partially regulated by an autocrine pathway in the LNCaP cell line.

Characterization of a small variable surface glycoprotein from Trypanosoma vivax

Several biochemical properties of a variant surface glycoprotein (VSG) from the parasite Trypanosoma (Duttonella) vivax have been determined. ILDat 2.1 VSG is approximately 40 kDa in size making this the smallest trypanosome VSG described to date. The glycolipid anchor of ILDat 2.1 VSG is resistant to treatment with T. brucei-derived phospholipase C and data based on lectin affinity chromatography, incorporation of radiolabelled sugar and treatment with endoglycosidase H suggest that the T. vivax VSG bears little carbohydrate. cDNA to ILDat 2.1 VSG mRNA has been cloned and the encoded protein sequence includes the N-terminal amino acid peptide sequence derived from native VSG. The molecular weight of the VSG predicted from the translated cDNA sequence is similar to that of the native molecule and in support of the biochemical data it is devoid of sites for N-linked glycosylation. Examination of the deduced ILDat 2.1 VSG protein sequence reveals that it is most similar to T. congolense VSGs in the distribution of Cys residues and like the former it does not contain any of the defined VSG C-terminal domain types. However, unlike T. congolense VSGs it does not readily fit into the currently described VSG N-terminal domain types. Our studies suggest that ILDat 2.1 VSG is distinct from any of the previously characterized VSGs.

Identification of binding proteins for PSP94 in human prostate adenocarcinoma cell lines LNCaP and PC‐3

Prostatic secretory protein of 94 amino acids (PSP94) is one of the predominant proteins found in human seminal fluid. Limited information is available regarding a physiological function for PSP94. An important step in the elucidation of this function is the determination of the mechanism of interaction of PSP94 with potential cellular targets.

Isolation of a highly enriched plasma membrane fraction of Trypanosoma brucei by free-flow electrophoresis

A procedure is described whereby a highly enriched plasma membrane fraction was isolated from Trypanosoma brucei by the technique of preparative free-flow electrophoresis. The purity of the plasma membrane fraction was monitored by electron microscopy and by marker enzymology, and is compared to those obtained by previous methods. Proteins associated with plasma membrane fraction were analyzed by SDS-PAGE and phase separated in Triton X-114.

Rat PSP94 Inhibits the Growth and Viability of the Rat Adenocarcinoma Cell Line PAIII In Vitro

Previous studies have shown that human PSP94 can inhibit the growth of prostate cancer cells both in vitro and in vivo. To further validate this potential and investigate the protein within a homologous setting, we examined the effects of rat PSP94 on the growth of the rat prostate adenocarcinoma cell line PAIII in vitro. To generate rat PSP94, we used both a plasmid-based expression system and a recombinant rat PSP molecule. Rat PSP was shown to inhibit the growth and survival of PAIII cells in a dose-dependent manner with > 90 percent reductions in both observed. TUNEL and Annexin-V assays confirmed PAIII cell death to be via apoptosis.

Localization of a 24-kilodalton glycoprotein in adult Schistosoma mansoni using immunofluorescence and immunoelectron microscopy

Studies on schistosome protective immune responses have focused mainly on antigens of the parasites syncytial surface. One of the characterized schistosome antigens, a 24-kDa glycoprotein, has been considered important in mechanisms of immune evasion by the parasites. In the present study, using affinity-purified antibodies to the 24-kDa protein for immunofluorescence and immunoelectron microscopy, we demonstrated an association of the 24-kDa antigen with the discoid bodies (the major syncytial inclusion bodies; DBs) and the surface membrane complex (most likely the apical plasma membrane) of adult Schistosoma mansoni. This is consistent with previous observations that the 24-kDa antigen appeared to be localized to the syncytial membrane and DB fractions. The present results also support the suggestion that the DBs are the precursor organelles of the apical plasma membrane.