Reversible inactivation of thiaminase I of Bacillus thiaminolyticus by its primary substrate, thiamine.

Thiaminase I of Bacillus thiaminolyticus is reversibly inactivated when it is incubated with its primary substrate, thiamine, or with one of several structural analogues of thiamine in the absence of an acceptor base. The inactivation reaction is pH and temperature dependent and is stochiometric with respect to thiamine and thiaminase I concentrations. One molecule of thiamine is cleaved for each molecule of enzyme inactivated. Inactivation is prevented or reversed by sulfhydryl-reducing agents. Active or reactivated thiaminase I migrate as a single band in polyacrylamide electrophoresis gels. Inactive thiaminase I appears to migrate as two separate bands. Active, inactive, and reactivated thiaminase I are immunologically similar. A possible mechanism for the inactivation of thiaminase I by its substrate is discussed.

Intervent dilution chromatography: concept for separation of strongly interacting macromolecules.

Intervent dilution chromatography separates interacting macromolecules by subjecting them to a dynamic environment in which the association constant is continuously varied. The dynamic environment is produced by using the sieving properties of a gel to repeatedly propel the molecular complex across an intervent boundary. Behind the boundary, at high intervent concentrations, the complex dissociates; ahead of the boundary, the component molecules are separated by adsorption processes. By selective adjustment of the intervent composition of the sample and the conditions of column equilibration, the process is adapted to a particular need. This report describes the chromatographic concept and shows how parameters are adjusted to obtain the desired separation. In this study a particularly difficult separation, i.e., the separation of ribosomal proteins from ribosomal RNA, is chosen to illustrate the power of the procedure.

Antimycoplasmal activity of dimethylphenols in a tracheal explant culture system.

Mycoplasma pneumoniae induces pneumonia-like symptoms in hamsters and causes ciliostasis and cytonecrosis in hamster tracheal explants. 2,4-Dimethylphenol and, to a lesser extent, its 2,3-, 2,5-, and 2,6-dimethylphenol isomers protected tracheal explants from these changes after exposure to virulent M. pneumoniae strain PI 1428. The effect was concentration, time, and isomer dependent. At concentrations of 10(-9) M or greater, 2,4-dimethylphenol completely prevented the morphological (loss of ciliated cells) and biochemical (decreased dehydrogenase activity) changes normally observed after exposure to M. pneumoniae. Apparently, 2,4-dimethylphenol interfered with an early event in the infection process. Complete protection required that it be present during the first 2 h of exposure of the explants to the infecting mycoplasmas. These xylenols may prove to be useful tools for helping to define the mechanisms of pathogenesis in certain respiratory infections.

Cell-bound thiaminase I of Bacillus thiaminolyticus.

The distribution of the extracellular enzyme, thiaminase I, was determined for logarithmically growing cultures of Bacillus thiaminolyticus. About 60% of the enzyme is associated with the cells throughout the growth cycle. The remainder of the enzyme is in the culture medium. The release of the cell-bound thiaminase I is examined under a variety of conditions. The rate and extent of release is dependent on the pH and the nature of the incubation solution. The release process appears to be relatively independent of de novo protein synthesis, energy derived from oxidative phosphorylation, or divalent metal ions. The absence of carbon or nitrogen sources has little effect on the release of the enzyme. Cell-bound thiaminase I probably is the immediate precursor for extracellular thiaminase I found in the culture medium. Washed cells continue to release thiaminase I at the expense of cell-bound enzyme. In addition, purified cell-bound thiaminase I is indistinguishable from purified extracellular thiaminase I by a number of physical and kinetic criteria.

Differential distribution of ciliated epithelial cells in the trachea of hamsters: implications for studies of pathogenesis.

The morphology of the inner aspect of the adult hamster trachea was examined by scanning electron microscopy. Relatively large patches of unciliated cells were observed in the epithelial layer. The patches, which covered several hundreds to thousands of square microns, were most conspicuous on the ventral surface of the trachea, especially in the middle third. The frequency of these areas of unciliated cells, both isolated and in patches, was much greater in hamsters than in mice, rats, or cats. Greatest ciliation in the hamster trachea was observed over the strip of trachealis muscle between the open ends of the cartilaginous rings. Areas with the heaviest ciliation also had the greatest activity of cellular metabolism, as measured by the tetrazolium reduction assay. The attachment of tritium-labeled cells of Mycoplasma pneumoniae was inversely correlated with extensive ciliation, since the greatest numbers of counts were found on the middle third and ventral regions of the tracheal surface. The results of this study suggest that the regional differences in ciliation of respiratory epithelium in hamsters may influence studies of pathogenesis and isolation of M. pneumoniae and that these differences should therefore be considered and controlled in the experimental design.

Effects of elevated P02 upon tracheal explants.

Time-dependent effects of elevated PO2 upon hamster tracheal rings in culture were examined. Ciliary activity of rings in 0.20 and 0.40 O2 remained stable for 144 h. Ciliary activity decreased 25% following 120 h exposure to 0.60 O2 and 50% within 72 h exposure to 0.95 O2. Ciliostasis occurred by 144 h at 0.95 O2. [3H]Leucine uptake progressively decreased during 24 to 144 h exposure to 0.40, 0.60, and 0.95 O2 when compared to 0.20 O2. Tracheal explants incubated for 6 days under 0.20 and 0.40 O2 had intact epithelial surface with abundant cilia, and no mucus present (scanning electron microscopy). Explants incubated under 0.60 O2 contained mucus globules by 3 days. Incubation in 0.95 O2 resulted in mucus globules within 3 days and epithelial sloughing, loss of cilia, and increased mucus globules within 6 days. Time and O2 dependent increases in nuclear atypia, leukocytic infiltration into submucosal area, and focal flattening of pseudostratified ciliated epithelium were observed (light microscopy). These changes progressed following 6 days' exposure to 0.95 O2 to complete loss of normal epithelium, with broken-up, epithelial cells along the tracheal lumen.

Development of an improved tracheal explant bioassay for the detection of the ciliary dyskinesia factor in cystic fibrosis serum.

A tracheal ring explant system, when used with 25% cystic fibrosis (CF) serum, displayed obvious ciliostasis. Hamster, rabbit, and guinea pig explants all had measurable decreases in ciliary activity after 24 hr of incubation in the serum. The differential response to CF serum (relative to normal serum) was greatly increased by using explants which were maintained 24-72 hr in minimal essential medium (MEM) with 10% horse serum and which were selected on the basis of optimal ciliary activity and vigor. With such a bioassay system of guinea pig tracheal explants, incubation with 25% normal serum would produce essentially no change in relative ciliary activity (score of 242 of a possible 300), whereas CF serum resulted in an 86% decrease (score of 33). Scanning electron microscopic observation indicated that the explants displaying the CF-ciliostatic effect had significant accumulations of mucous over the ciliated epithelial surface. A biochemical viability assay (dehydrogenase activity) showed no cytonecrosis when CF serum-treated tissues were compared to standard explants (10% horse serum in MEM) or control explants (25% normal human serum).