Calcium entry in Trypanosoma brucei is regulated by phospholipase A2 and arachidonic acid.

In contrast with mammalian cells, little is known about the control of Ca2+ entry into primitive protozoans. Here we report that Ca2+ influx in pathogenic Trypanosoma brucei can be regulated by phospholipase A2 (PLA2) and the subsequent release of arachidonic acid (AA). Several PLA2 inhibitors blocked Ca2+ entry; 3-(4-octadecyl)-benzoylacrylic acid (OBAA; IC50 0.

Characterization of a fourth lipoprotein from Pasteurella haemolytica A1 and its homology to the OmpA family of outer membrane proteins.

A fourth lipoprotein gene from Pasteurella haemolytica A1 was cloned and characterized. The plpD gene encodes a 31-kDa lipoprotein (Plp4) which could be recognized in Western immunoblot by sera from calves immunized with the culture supernatant vaccine Presponse. This suggests that Plp4 is one of the immunogenic molecules in the P.

Bovine platelet adhesion is enhanced by leukotoxin and sialoglycoprotease isolated from Pasteurella haemolytica A1 cultures.

Platelet and fibrin deposits are among characteristic changes observed in lung alveoli of cattle with pasteurellosis induced by Pasteurella haemolytica (biotype A, serotype 1). To determine whether the platelet function could be directly affected by protein products produced by the bacterium, the effects of leukotoxin and O-sialoglycoprotease, culture supernatant antigen secreted by Pasteurella haemolytica A1, on bovine platelet activation were examined by evaluating the enhancement of platelet adhesion to a negatively charged surface relative to untreated control samples. The glycoprotease, or the leukotoxin, was added to plasma free suspensions of bovine platelets and platelet adhesion assessed by two parameters: (i) the number of 3H-adenine-labeled adherent platelets and (ii) the morphology of unlabeled platelets adhering to the charged surface under scanning electron microscopy (SEM).

Membrane protein proteolysis assayed by fluorescence quenching: assay of O-sialoglycoprotein endopeptidase.

The assay of the O-sialoglycoprotein endopeptidase of Pasteurella haemolytica has previously used the cleavage of 125I-labeled glycophorin A, measured by SDS-PAGE, autoradiography, gel-slicing, and scintillation counting. A new assay is based on the increased fluorescence which results from proteolytic cleavage of a fluorescence-quenched micellar substrate, 4,4-difluor-5,7-dimethyl-4-bora-3 alpha, 4 alpha-diaza-s-indacene-3-propionic acid conjugated to glycophorin A (BODIPY-FL-glycophorin A). Micellar association of glycophorin A molecules results in 97% fluorescence quenching despite a low molar ratio of BODIPY-FL-glycophorin A.

Synthesis of a radiolabeled zwitterionic detergent and its use in protein purification.

Radiolabeling permits the detection of trace amounts of zwitterionic detergent remaining in extracted hydrophobic or membrane proteins. To develop a sensitive and specific assay for its presence, the commonly used zwitterionic membrane protein detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) was synthesized in a tritiated form. Synthesis via 7-ketodeoxycholic acid gave [7-3H]Chaps in 53% yield with a specific activity of 0.