Trichloroethylene biodegradation by a methane-oxidizing bacterium.

Trichloroethylene (TCE), a common groundwater contaminant, is a suspected carcinogen that is highly resistant to aerobic biodegradation. An aerobic, methane-oxidizing bacterium was isolated that degrades TCE in pure culture at concentrations commonly observed in contaminated groundwater. Strain 46-1, a type I methanotrophic bacterium, degraded TCE if grown on methane or methanol, producing CO(2) and water-soluble products. Gas chromatography and C radiotracer techniques were used to determine the rate, methane dependence, and mechanism of TCE biodegradation. TCE biodegradation by strain 46-1 appears to be a cometabolic process that occurs when the organism is actively metabolizing a suitable growth substrate such as methane or methanol. It is proposed that TCE biodegradation by methanotrophs occurs by formation of TCE epoxide, which breaks down spontaneously in water to form dichloroacetic and glyoxylic acids and one-carbon products.

Membrane-reconstituted, purified H-2Kb specifically inhibits T-cell-target cell conjugate formation.

We report the use of a sensitive microassay to detect purified H-2Kb antigens which have been functionally reconstituted into membrane vesicles of defined composition. The histocompatibility antigens have been purified by monoclonal antibody affinity chromatography. The assay utilizes inhibition of specific conjugate formation between allogeneically primed (H-2d anti- H-2b) cytotoxic T cells and H-2b target cells by the membrane-reconstituted H-2Kb antigens. Cytoskeletal proteins were added to the H-2Kb (and control H-2k) antigens. Sucrose density fractionation of reconstituted vesicles and Pronase E cleavage studies suggested that the cytoskeletal proteins aided in the incorporation and vectorial orientation of the antigens into large, cholesterol-containing membrane vesicles. As little as 6 ng purified H-2Kb plus 28 ng cytoskeletal proteins in vesicles of defined lipid composition (0.28, 0.25, 0.47 mol fraction cholesterol, dimyristoylphosphatidylcholine, and dipalmitoylphosphatidylcholine, respectively) inhibited specific conjugate formation to 50% of the maximum inhibition observed. This inhibition was shown to be specific in two ways: (i) the same H-2Kb-containing vesicles did not inhibit nonspecific conjugate formation, and (ii) control vesicles containing the same amounts of lipid, cytoskeletal proteins, and purified H-2k proteins inhibited conjugate formation but only at significantly higher H-2k concentrations, indicating the specificity of the response with the vesicles containing H-2Kb.

Differential recognition and lysis of EL4 target cells by cytotoxic T cells: differences in H-2Kb antigenic density and cytoskeletal proteins.

Direct binding of 125I-iodinated anti-H-2Kb monoclonal antibody (B8-24-3) to EL4 cells indicated a similar association constant but a 2.7-fold higher H-2Kb antigenic density when the EL4 cells were grown in ascites (2.0 X 10(5) sites/cell) than in tissue culture (7.5 X 10(4) sites/cell). The membrane fluidity of the isolated plasma membrane fractions, measured by using a membrane-sensitive spin label, was essentially identical in the two differently cultured EL4 cells. There were some differences in the cytoskeletal proteins that were isolated from the plasma membrane fractions of the EL4 lines and analyzed by two-dimensional gel electrophoresis. These differences in the H-2Kb antigenic density and the cytoskeletal proteins may contribute to the 2.3-fold higher Vmax and the increased binding of the EL4 ascites target cells to allogeneically primed anti-H-2Kb cytotoxic T cells.

Wheat germ agglutinin-resistant variant of EL4 containing altered oligosaccharides as a target cell for cytotoxic T cells.

A lectin-resistant variant of the murine EL4 lymphocytic leukemia cell line was selected in the presence of wheat germ agglutinin for low levels of cell-surface sialic acid. H-2Kb was the major internally radiolabeled H-2b molecule on the cell-surface of WD1, and it was not sialylated, as determined by two-dimensional gel analysis. Endo-beta-N-acetylglucosaminidase H treatment of the WD1 membrane fractions suggested that the oligosaccharides on the cell-surface H-2Kb molecule were complex, but nonsialylated. Monoclonal antibody inhibition of the allogeneically primed cell-mediated cytotoxicity (CMC) reaction indicated that the T cells (BALB/c anti-EL4; H-2d anti-H-2b) were specific only for the H-2Kb target cell antigen. These WD1 variant cells were used as targets in the CMC assay using anti-H-2Kb T cells and compared with the parent EL4 in vitro line. The change in the cell-surface oligosaccharide did not affect the susceptibility to lysis by the cytotoxic T lymphocytes even though there were 2.5-fold more H-2Kb antigens on the WD1 variant cell (1.5 X 10(5) sites/cell) than on the parent EL4 in vitro cell (5.9 X 10(4) sites/cell). It was possible to isolate highly purified preparations of H-2Kb from either the EL4 or the WD1 line using a monoclonal antibody affinity column. Interestingly, the variant WD1 cell would no longer grow in the peritoneal cavity of the syngeneic C57BL/6 mouse.

Physical separation and biochemical characterization of H-2b-encoded proteins on target cell plasma membranes and endoplasmic reticulum.

Plasma membranes and endoplasmic reticulum were purified on the basis of their physical properties, and their identity was confirmed by membrane marker activities. Comparative adsorption analysis indicated that plasma membrane and endoplasmic reticulum vesicle fractions adsorbed 25% and 5%, respectively, as much anti-D6 alloantiserum as intact cells. Two-dimensional gel analyses of immunoprecipitates of the H-2b-encoded proteins from the EL4 tumor cell line indicated a series of proteins containing complex carbohydrates in the plasma membrane and two additional N-asparagine-linked proteins in the endoplasmic reticulum. Both neuraminidase and protein phosphatase treatments were required to convert the mobilities of the plasma membrane forms of the H-2Kb and H-2Db antigens in two-dimensional gel electrophoresis to the mobilities of the endoplasmic reticulum forms. The endoplasmic reticulum form of H-2Db was shown to contain three N-asparagine-linked carbohydrates by endoglycosidase H treatment.

Cellular mechanisms of prostaglandin action.

The human platelet and erythrocyte differ quite dramatically in relation to the arachidonic acid cascade. The platelet synthesizes its own characteristic products, while the erythrocyte lacks cyclo-oxygenase activity but possesses other metabolic enzymes. In these and other cell types the metabolites are potentially determined by the cascade enzymes, the availability of cofactors, the presence of specific activators and inhibitors, and the selective binding or transport of intermediates. Endogenously synthesized metabolites may have intracellular actions or may be released and exert their effects extracellularly. Study of the cellular mechanisms mediating these effects, like study of the fast-acting hormones, has focused primarily on the cyclic nucleotides and calcium. Considering the diverse activities of several metabolites in the platelet however, these mechanisms seem to need reevaluation or refining. The released cascade metabolites may also act as intercellular signals over a short range. The range depends on their chemical stability in the absence of protective carriers and their selective uptake and metabolism by surrounding cells. Additionally, the effects will reflect the selective interaction of responsive cells with the spectrum of metabolites released. Answering these questions of complex intercellular interactions requires the identification and classification of characteristic responses and the metabolic profile of individual cell types in each tissue. Consequently, this type of analysis may best be done with isolated cells, such as the platelet and erythrocyte.

Inhibition of specific effector T cell-target cell conjugates by cell plasma membranes.

Conjugate formation between effector T cells raised in vivo in a BALB/c hose had fluorescein diacetate-labeled allogeneic EL4 tumor target cells was observed and scored by using a fluorescence mirroscope. This conjugate formation was found to be specifically inhibited by plasma membrane vesicles isolated from the EL4 cells.

Pyridoxamine-pyruvate transaminase. 1. Determination of the active site stoichiometry and the pH dependence of the dissociation constant for 5'-deoxypyridoxal.

Spectrophotometric titration of pyridoxamine-pyruvate transaminase (EC 2.6.1.30) with pyridoxal at pH 7.15 gives four equivalent binding sites per tetramer. The pH dependence of the equilibrium constant for the association of 5'-deoxypyridoxal with the active site lysine residue was determined spectrophotometrically. These dissociation constants increase with increasing pH over the range pH 7.5-9 and are correlated with the values obtained from fast reactions kinetics (Gilmer, P. J., and Kirsch, J. F. (1977), Biochemistry 16 (following paper in this issue)). In addition to this specific reaction at an active site lysine residue, a second slower reaction at non-active site residues is observable at pH values greater than 8. The pH dependencies of the association and dissociation rate constants for this slow reaction were studied over the pH range 8 to 9 after blocking the active site by NaBH4 reduction of the pyridoxal adduct. The enzyme is stabilized and markedly activated by potassium ion.