CD77-dependent retrograde transport of CD19 to the nuclear membrane: functional relationship between CD77 and CD19 during germinal center B-cell apoptosis.

A region of the N-terminal extracellular domain of the B-cell restricted cell differentiation antigen, CD19, has high amino acid sequence similarity to the receptor binding subunit B of verotoxin 1 (VT), an Escherichia coli elaborated cytotoxin, which specifically binds to the cell surface glycolipid, globotriaosylceramide, also known as the germinal center (GC) B-cell differentiation antigen, CD77. We have previously provided evidence of the association of CD19 and CD77 on the cell surface and in CD19-mediated homotypic adhesion of the Daudi Burkitt Lymphoma cell line, one normal counterpart of which is a subset of GC B cells. Evidence for the role of CD77 in CD19-induced apoptosis is now presented. Initial cell surface distribution, antibody-induced redistribution, internalization, and intracellular routing of CD19 were studied by confocal microscopy, IF, and postembedding IEM in CD77+ve and CD77-ve cells to investigate the possible role of CD77 in CD19 internalization and signaling. Daudi Burkitt's lymphoma cells were used as CD77+ve cells and as CD77-ve cells, Daudi mutant VT500 cells, and Daudi cells treated with PPMP, an inhibitor of CD77 synthesis, were used. Antibody ligated CD19 surface redistribution, internalization, and subcellular distribution of internalized CD19 was found to be different in CD77+ve and CD77-ve cells. A delay in internalization of antibody-CD19 complex was observed in CD77-ve cells. Internalized CD19 was targeted to the nuclear envelope in CD77+ve cells in a manner similar to that reported for VT, but not in CD77-ve cells. Internalization of CD77 by ligation with verotoxin prevented the internalization of ligated CD19. Induction of apoptosis following crosslinking of cell surface CD19 was greater in CD77+ve cells than in CD77-ve cells. The nuclear targeting of internalized CD19 and induction of apoptosis following CD19 crosslinking only in CD77+ve cells indicates a role for CD77-dependent CD19 retrograde transport from the B cell surface via the ER to the nuclear envelope in CD19-mediated signal transduction for apoptosis.

Modeling and measuring the elastic properties of an archaeal surface, the sheath of Methanospirillum hungatei, and the implication of methane production.

We describe a technique for probing the elastic properties of biological membranes by using an atomic force microscope (AFM) tip to press the biological material into a groove in a solid surface. A simple model is developed to relate the applied force and observed depression distance to the elastic modulus of the material. A measurement on the proteinaceous sheath of the archaebacterium Methanospirillum hungatei GP1 gave a Young's modulus of 2 x 10(10) to 4 x 10(10) N/m2. The measurements suggested that the maximum sustainable tension in the sheath was 3.5 to 5 N/m. This finding implied a maximum possible internal pressure for the bacterium of between 300 and 400 atm. Since the cell membrane and S-layer (wall) which surround each cell should be freely permeable to methane and since we demonstrate that the sheath undergoes creep (expansion) with pressure increase, it is possible that the sheath acts as a pressure regulator by stretching, allowing the gas to escape only after a certain pressure is reached. This creep would increase the permeability of the sheath to diffusible substances.

Scanning probe microscopy in microbiology.

Scanning probe microscopy (SPM) is emerging as an important alternative to electron microscopy as a technique for analyzing submicron details on biological surfaces. Microbiological specimens such as viruses, bacteriophages, and ordered bacterial surface layers and membranes have played an important role in the development of scanning tunnelling microscopy (STM) and atomic force microscopy (AFM) in cellular and molecular biology. Early STM studies involving metal-coated bacteriophage T4 polyheads, Methanospirillum hungatei, and Deinococcus radiodurans HPI layer clearly demonstrated that resolution was comparable to TEM on similarly prepared specimens and only limited by metal graininess. However, except for thin films or layers, novel biological information has been difficult to obtain since imaging native surfaces of such biomaterials as proteins or nucleic acids by STM proved to be unreliable. With the development of atomic force microscopes, which allow imaging of similar native structures, SPM applications have widened to include straightforward surface structure analysis, analysis of surface elastic and inelastic properties, bonding force measurements between molecules, and micro-manipulations of such individual molecules as DNA. AFM images have progressed from relatively crude representations of specimen topography to nanometer scale representations of native hydrated surfaces. It appears from the study of microbiological specimens that direct visualization of dynamic molecular events or processes may soon become a reality.

Comparison of the sensitivities of Salmonella typhimurium oxyR and katG mutants to killing by human neutrophils.

The respiratory burst of neutrophils is believed to kill bacteria by generating oxidative species, such as superoxide anion, hydrogen peroxide, and oxidized halogen species. The oxyR gene of Salmonella typhimurium controls a regulon induced by oxidative stress, such as exposure to hydrogen peroxide. Some researchers have suggested that oxyR may play a key role in bacterial survival following phagocytosis. We have tested this possibility by comparing the survival, following exposure to human neutrophils, of isogenic strains bearing different oxyR alleles. Neither inactivation of the oxyR gene nor constitutive overexpression of the oxyR-regulated proteins (oxyR1 allele) greatly alters bacterial resistance to neutrophils. The katG gene, encoding the oxyR-regulated enzyme hydroperoxidase I, was also without effect on survival following exposure to neutrophils. We conclude that the oxyR response does not play a significant role in the resistance of S. typhimurium to phagocytic killing in vitro.

Characterization of the cell wall of the sheathed methanogen Methanospirillum hungatei GP1 as an S layer.

The cell wall of Methanospirillum hungatei GP1 is a labile structure that has been difficult to isolate and characterize because the cells which it encases are contained within a sheath. Cell-sized fragments, 560 nm wide by several micrometers long, of cell wall were extracted by a novel method involving the gradual drying of the filaments in 2% (wt/vol) sodium dodecyl sulfate and 10% (wt/vol) sucrose in 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer containing 10 mM EDTA. The surface was a hexagonal array (a = b = 15.1 nm) possessing a helical superstructure with a ca. 2.5 degrees pitch angle. In shadowed relief, the smooth outer face was punctuated with deep pits, whereas the inner face was relatively featureless. Computer-based two-dimensional reconstructed views of the negatively stained layer demonstrated 4.0- and 2.0-nm-wide electron-dense regions on opposite sides of the layer likely corresponding to the openings of funnel-shaped channels. The face featuring the larger openings best corresponds to the outer face of the layer. The smaller opening was encircled by a stalk-like mass from which 2.2-nm-wide protrusions were resolved. The cell wall in situ was degraded at pH 9.6 at 56 degrees C but was unaffected at pH 7.4 at the same temperature. The cell wall was composed of two nonglycosylated polypeptides (114 and 110 kDa). The cell wall resembled an archaeal S layer and may function in regulating the passage of small (< 10-kDa) sheath precursor proteins.

Transmission electron microscopy, scanning tunneling microscopy, and atomic force microscopy of the cell envelope layers of the archaeobacterium Methanospirillum hungatei GP1.

Methanospirillum hungatei GP1 possesses paracrystalline cell envelope components including end plugs and a sheath formed from stacked hoops. Both negative-stain transmission electron microscopy (TEM) and scanning tunneling microscopy (STM) distinguished the 2.8-nm repeat on the outer surface of the sheath, while negative-stain TEM alone demonstrated this repeat around the outer circumference of individual hoops. Thin sections revealed a wave-like outer sheath surface, while STM showed the presence of deep grooves that precisely defined the hoop-to-hoop boundaries at the waveform nodes. Atomic force microscopy of sheath tubes containing entrapped end plugs emphasized the end plug structure, suggesting that the sheath was malleable enough to collapse over the end plugs and deform to mimic the shape of the underlying structure. High-resolution atomic force microscopy has revised the former idea of end plug structure so that we believe each plug consists of at least four discs, each of which is approximately 3.5 nm thick. PT shadow TEM and STM both demonstrated the 14-nm hexagonal, particulate surface of an end plug, and STM showed the constituent particles to be lobed structures with numerous smaller projections, presumably corresponding to the molecular folding of the particle.

Distribution of antigenic determinants between Actinomyces viscosus and Actinomyces naeslundii.

A total of 18 monoclonal antibodies was raised against whole cells of Actinomyces viscosus and Actinomyces naeslundii. The monoclonal antibodies were used to determine the cross-reacting patterns among 26 strains of these species. Eleven different antigenic determinants were found. The specificity profiles of the antibodies indicated that the antigenic determinants of A. viscosus and A. naeslundii were arranged in a complicated mosaic. Extensive cross-reactions occurred between A. viscosus strains and strains of "typical" and "atypical" A. naeslundii. However, cross-reactions were rare between the two groups of A. naeslundii. A. viscosus appears to occupy a "middle position" between the two A. naeslundii groups. In addition to their value in seroclassification, some of the monoclonal antibodies were found to be useful in the identification of these species. One monoclonal antibody appeared to be selective for the "typical" A. naeslundii group. A. viscosus and "atypical" A. naeslundii-specific antibodies were also found, though they did not label every strain in their respective clusters. A. viscosus detection might be improved if mixtures of monoclonal antibodies were used.