A 38-kilodalton (kDa) protein antigen from Mycobacterium tuberculosis was purified by monoclonal antibody TB71-based affinity chromatography. This molecule carries two nonoverlapping epitopes recognized by monoclonal antibodies TB71 and TB72, which are expressed substantially more strongly by M. tuberculosis than by Mycobacterium bovis. However, cross-reactive determinants between these two species were revealed on the 38-kDa protein by a rabbit anti-BCG serum. An immunoradiometric assay based on the TB71 and TB72 antibody pair specifically determined 38-kDa-antigen concentrations in 'nycobacterial extracts. Antibodies in sera from tuberculosis patients estimated by binding to 38-kDa-antigen-coated microtiter plates were positively correlated with TB72 competing titers. Unlike antibodies, T-cell proliferative responses to the 38-kDa protein were expressed equally by 60% of tuberculosis patients and healthy BCG-vaccinated subjects. Similarly, delayed-type hypersensitivity skin reactions were elicited in both M. tuberculosisand M. bovis-sensitized guinea pigs. The results suggest the immunodominance of the species-specific B-cell and cross-reactive T-cell stimulatory epitopes.(pH 2.5), and (iii) io % (vol/vol) dioxan in 0.1 M glycine-HCl (pH 2.5). The column was then reequilibrated with PBS and stored. Fractions (5 ml) were collected during the elution procedure and assayed for 38-kDa protein by enzyme-linked 177 on July 11, 2020 by guest

This paper presents the crystal structure of porcine pancreatic carboxypeptidase B (pp-CpB) in complex with a variety of thiol-based inhibitors that were developed as antagonists of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recent studies have indicated that a selective inhibitor of TAFIa could enhance the efficacy of existing thrombolytic agents for the treatment of acute myocardial infarction, one of the most prevalent forms of heart attacks. Unfortunately, activated TAFIa rapidly degrades in solution and cannot be used for crystallographic studies. In contrast, porcine pancreatic CpB is stable at room temperature and is available from commercial sources. Both pancreatic CpB and TAFIa are zinc-based exopeptidases, and the proteins share a 47% sequence identity. The homology improves considerably in the active site where nearly all of the residues are conserved. The inhibitors used in this study were designed to mimic a C-terminal arginine residue, one of the natural substrates of TAFIa. The X-ray structures show that the thiol group chelates the active site zinc, the carboxylic acid forms a salt bridge to Arg145, and the guanidine group forms two hydrogen bonds to Asp255. A meta-substituted phenyl was introduced into our inhibitors to reduce conformational freedom. This modification vastly improved the selectivity of compounds against other exopeptidases that cleave basic residues. Comparisons between structures indicate that selectivity derives from the interaction between the guanidine group in the inhibitors and an acidic active site residue. The location of this acidic residue is not conserved in the various carboxypeptidases.

In vitro, high density monolayer cultures of vascular smooth muscle cells can be induced to form multicellular nodules. The nodular cells appear to be morphologically differentiated smooth muscle cells. Sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis was used to compare the proteins synthesized and secreted by monolayer and nodular cultures of smooth muscle cells. Although most proteins appeared to be similar, the nodular cultures contained a unique heparin binding protein of Mr = 38,000 (38kD protein) (Millis, A.J.T., Hoyle, M., Reich, E., and Mann, D.M., 1985, J. Biol. Chem., 260:3754-3761). The 38kD protein was glycosylated and its apparent molecular weight was shifted to Mr = 32,500 after synthesis in the presence of tunicamycin or digestion with endoglycosidase F. The production of 38kD protein by nodular cell cultures did not appear to result from the degradation of a high molecular weight precursor in nodular conditioned medium. Further, it was not detected in monolayer cell conditioned medium that had been incubated with nodular cells. Finally, its synthesis was not induced in monolayer cell cultures that had been labeled in nodular cell conditioned medium. The 38kD protein appears to be uniquely associated with nodular cultures of smooth muscle cells.

The insertion sequence IS6110 has an important role in diagnostic PCR and typing of Mycobacterium tuberculosis. We have evaluated a one-tube nested PCR which detects IS6110. Positive results were obtained with DNAs from four of four M. tuberculosis isolates, seven of eight M. fortuitum isolates, four of seven M. avium-M. intracellulare complex isolates, four of five M. kansasii isolates, five of five M. xenopi isolates, two of four M. malmoense isolates, and two of two M. chelonei isolates. These results were confirmed by hybridization of genomic DNA from bp 505 to 685 of the IS6110 from M. tuberculosis H37Rv. Dot blot hybridization of genomic DNAs from these isolates with the same probe confirmed the presence of a homologous sequence in these mycobacterial species. These data suggest that false-positive results may be obtained for clinical samples when some methods based on IS6110 are used.

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