Mycobacterium tuberculosis survives macrophage bactericidal activities by mechanisms that may include induction of stress proteins. We sought to determine whether the synthesis of any mycobacterial proteins is increased during phagocytosis and whether any of these proteins are also up-regulated during heat shock. Protein synthesis by M. tuberculosis H37Ra during phagocytosis by the mouse macrophage cell line IC-21, and during heat shock at 45 and 48 degrees C, was monitored at various time intervals using 35S-labeled methionine/cysteine and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our data suggest the existence of certain common elements in the stress response of mycobacteria to the three stress stimuli. This apparent similarity was best characterized by the up-regulation of a 25-kDa protein after exposure to each of the stress conditions. Furthermore, this 25-kDa protein and a 37-kDa protein that was also synthesized during phagocytosis appeared to be extracellular because they were preferentially solubilized when infected macrophages were lysed with 0.5% NP-40.

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In situ incubations of natural autotrophic picoplankton populations during a 15 month study were used to test the frequency of dividing cells procedure in estimating phototrophic picoplankton growth rates. These rates were estimated using dilution experiments and compared to the average frequency of dividing cells over the same time interval. The regression equation of p = 2.85 x 10 -3 (FDC) + 0.022 was calculated to relate autotrophic picoplankton growth rate and the frequency of dividing cells in this study. The resulting relationship was compared to 14 C-bicarbonate derived growth rates. Productivity estimates using frequency of dividing cells correlated closely to sodium ' 4 C-bicarbonate results and indicated a range of productivity by autotrophic picoplankton of 55.6% the total phytoplankton primary productivity in July to a January rate of 2.3 %. Annual autotrophic picoplankton abundance varied seasonally in the lower Chesapeake Bay ranging from 7.26 x 106 cells 1-' in winter to 9.28 x 108 cells 1-during late summer.

Two-dimensional immunoelectrophoresis (2D-IEP), in which a complex of antigens is subjected to electrophoresis first through an agarose matrix in one direction and secondly through an antiserum-agarose matrix at right angles to the first direction, was evaluated as a tool for analysis of mycobacterial antigens. Cell extracts from four species of mycobacteria, Mycobacterium tuberculosis (four strains), M. bovis strain BCG, M. scrofulaceum, and M. phlei, were assayed by 2D-IEP with four anti-mycobacterial antisera. Besides displaying the precipitin curves in a more easily interpreted format than did conventional immunoelectrophoresis (IEP), 2D-IEP offered greater sensitivity in terms of numbers of precipitin curves when like reactions were compared with IEP patterns. As many as 60 immunoprecipitates were observed on 2D-IEP slides compared to 18 on comparable IEP plates. Technical reproducibility of patterns from run to run was excellent. Other parameters, such as the influence of using different batches of antigen on the pattern, are discussed. Each of the cell extract antigens gave a unique pattern of precipitin peaks which could be easily differentiated from the patterns given by the other mycobacterial cell extracts when reacted with any of the antisera in 2D-IEP. Since both the species and strains of mycobacteria could be easily and reproducibly differentiated solely on the basis of two-dimensional immunoelectrophoretic patterns obtained with any of the antisera employed in this study, it may be possible, by using IEP, to differentiate and identify all species and strains of mycobacteria with one standard, highly sensitive antiserum, rather than a battery of antisera.

Concentrated culture filtrates and cell extracts were prepared from selected mycobacteria and analyzed by a multistage polyacrylamide electrophoretic procedure. Various staining procedures were used to detect protein, carbohydrate, nucleic acid, and lipid constituents. Visual examination of the more prominently stained components indicated differences and similarities in each species of Mycobacteria. The majority of the individual components were of small molecular weight; however, there appeared in all culture filtrates and extracts examined a slowly moving protein component of relatively large molecular size. Evidence based on immunodiffusion suggests that it is a common mycobacterial antigen.It has been demonstrated by a number of studies that the mycobacterial cell and its metabolic products are of an exceedingly complex nature. The biology of the tubercle bacillus and the relationship of its complex products within the host have been studied for many years to better understand the pathogenesis of tuberculosis. There have been many physicochemical techniques which have been applied to the separation and isolation of these complex cellular products. It is also important that their chemical composition be known so that the mechanism of tuberculous disease and, consequently, how to combat it may be better understood. Moreover, by employing more refined mycobacterial fractions of known composition, the similarities and differences that exist among the various acid-fast organisms could be more sharply defined and thus contribute to a more satisfactory understanding of mycobacterial relationships.The present study is a direct outgrowth of our long-range investigations concerned with developing and standardizing methods for isolating and purifying mycobacterial antigens and elucidating their possible biological effects. Specifically, it has been one of the major aims of this study to identify and compare mycobacterial antigens by utilizing a modified pore gradient polyacrylamide electrophoresis method. The present report characterizes these biologically active components from various selected strains of mycobacteria utilizing this technique. Antigenic profiles from each of them are obtained and compared, and the possible interrelationships are examined. MATERIALS AND METHODS Source of organisms for culture filtrates and bacterial extracts. Selected mycobacterial species were obtained from the American Type Culture Collection and the culture collection of the Microbiology Department of The George Washington University School of Medicine. These included: Mycobacterium tuberculosis H37Ra, M. tutberculosis H37Rv, M. bovis, M. bovis BCG, M. aviuim, M. kansasii, M.intracellitlare, M. scrofulaceum, M. phlei, M. fortuitum, and M. smegmatis. Cells were grown as surface pellicles on Proskauer-Beck medium at 37 C for 6 to 8 weeks before separation from the culture fluid by Seitz filtration. The culture filtrates were concentrated by pervaporation to one-tenth their original volume. The total volumes obtained were each divided ...