William H. Andrews

Oxidation of a specific methionine in thrombomodulin by activated neutrophil products blocks cofactor activity. A potential rapid mechanism for modulation of coagulation.

Endothelial thrombomodulin (TM) plays a critical role in hemostasis as a cofactor for thrombin-dependent formation of activated protein C, a potent anticoagulant. Chloramine T, H2O2, or hypochlorous acid generated from H2O2 by myeloperoxidase rapidly destroy 75-90% of TM cofactor activity. Activated PMN, the primary in vivo source of biological oxidants, also rapidly inactivate TM. Oxidation of TM by PMN is inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase. Both Met291 and Met388 in the six epidermal growth factor-like repeat domain are oxidized; however, only substitutions of Met388 lead to TM analogues that resist oxidative inactivation. We suggest that in inflamed tissues activated PMN may inactivate TM and demonstrate further evidence of the interaction between the inflammatory process and induction of thrombotic potential.

Characterization of a surface antigen of Eimeria tenella sporozoites and synthesis from a cloned cDNA in Escherichia coli

An antigenic surface protein of Eimeria tenella sporozoites has been identified that is the target of two neutralizing monoclonal antibodies Ptn 7.2A4/4 and Ptn 9.9D12. The antigen as isolated from the parasite is composed of a 17 kDa polypeptide and a 8 kDa polypeptide linked by a disulfide bridge. De novo synthesis of the antigen does not begin until approximately 16-20 h after the initiation of oocyst sporulation. A cDNA library was constructed using mRNA from sporulated oocysts and a clone encoding the antigen was isolated. The Ta4 gene encodes a single polypeptide of 25 kDa which contains the 17 and 8 kDa polypeptides. The protein has been synthesized in Escherichia coli either directly or as part of a beta-galactosidase fusion protein. The products synthesized in E. coli are single polypeptides and are not cleaved to two polypeptides as is seen in the parasite. The products accumulate in bacteria in an insoluble form which can be solubilized and renatured to an immunoreactive form.

Enzymatic properties and processing of bovine prochymosin synthesized in Escherichia coli

Abstract Active chymosin has been produced from calf prochymosin synthesized in E. coli . A preprochymosin cDNA clone was used to construct a plasmid, pWHA43, which expresses methionyl-prochymosin. This zymogen protein is synthesized in the bacteria in a stable but insoluble form localized in cytoplasmic inclusion bodies. Partially purified and solubilized prochymosin from E. coli can be processed by the same apparent mechanism as authentic calf prochymosin upon activation with acid. The chymosin thus derived from E. coli showed the same substrate specificity and the same kinetics of activation as that of chymosin derived from purified calf stomach prochymosin. These results also demonstrate that the bovine prochymosin synthesized in E. coli can be reconstituted and activated to a form having the functional properties necessary for an industrial milk coagulant to be used in cheese manufacturing.