Similarities between the antABC-encoded anthranilate dioxygenase and the benABC-encoded benzoate dioxygenase of Acinetobacter sp. strain ADP1.

Acinetobacter sp. strain ADP1 can use benzoate or anthranilate as a sole carbon source. These structurally similar compounds are independently converted to catechol, allowing further degradation to proceed via the beta-ketoadipate pathway. In this study, the first step in anthranilate catabolism was characterized. A mutant unable to grow on anthranilate, ACN26, was selected. The sequence of a wild-type DNA fragment that restored growth revealed the antABC genes, encoding 54-, 19-, and 39-kDa proteins, respectively. The deduced AntABC sequences were homologous to those of class IB multicomponent aromatic ring-dihydroxylating enzymes, including the dioxygenase that initiates benzoate catabolism. Expression of antABC in Escherichia coli, a bacterium that normally does not degrade anthranilate, enabled the conversion of anthranilate to catechol. Unlike benzoate dioxygenase (BenABC), anthranilate dioxygenase (AntABC) catalyzed catechol formation without requiring a dehydrogenase. In Acinetobacter mutants, benC substituted for antC during growth on anthranilate, suggesting relatively broad substrate specificity of the BenC reductase, which transfers electrons from NADH to the terminal oxygenase. In contrast, the benAB genes did not substitute for antAB. An antA point mutation in ACN26 prevented anthranilate degradation, and this mutation was independent of a mucK mutation in the same strain that prevented exogenous muconate degradation. Anthranilate induced expression of antA, although no associated transcriptional regulators were identified. Disruption of three open reading frames in the immediate vicinity of antABC did not prevent the use of anthranilate as a sole carbon source. The antABC genes were mapped on the ADP1 chromosome and were not linked to the two known supraoperonic gene clusters involved in aromatic compound degradation.

Cytotoxic activities of gut mucosal lymphoid cells in guinea pigs.

Populations of lymphoid cells were obtained from the gastrointestinal tract of guinea pigs and compared with splenic cells for effector cell activity in assays of spontaneous cell-mediated cytotoxicity (SCMC), mitogen-induced cellular cytotoxicity (MICC), and antibody-dependent cellular cytotoxicity (ADCC). Lymphoid cells were isolated from mesenteric lymph nodes (MLN), Peyer's patches (PP), and the small intestinal mucosa. Mucosal lymphoid cells were separated into a population of cells containing intraepithelial lymphocytes (IEL), and a second population of cells containing intraepithelial lymphocytes (IEL), and a second population of cells containing lamina propria (LP) lymphocytes by a combination of physical and enzymatic methods. All populations of lymphoid cells from the gastrointestinal tract were capable of mediating MICC. Relative to spleen cells, IEL possessed more MICC effector cell activity whereas cells from MLN, PP, and LP possessed less activity. In contrast, SCMC and ADCC effector cell activities were present only in the IEL population and these cytotoxic activities were comparable to that of splenic cells. These results demonstrate that mucosal lymphoid cells possess cytotoxic effector cell activities that may contribute to local mucosal immunologic reactivity.

Spontaneous cell-mediated cytotoxicity by human peripheral blood lymphocytes in vitro.

With isolated subpopulations of human peripheral blood mononuclear cells, spontaneous cell-mediated cytotoxicity (SCMC) and antibody-dependent cellular cytotoxicity (ADCC) were demonstrated to be functions of a population of surface immunoglobulin (sIg)-negative, Fc receptor-positive lymphocytes. Organ distribution studies revealed that SCMC and ADCC effector cell activity occurred in parallel, being present in spleen and absent in tonsils. Treatment of effector cells with trypsin significantly inhibited SCMC but not ADCC and this inhibition was not reversed by short-term incubation in autologous plasma. These results demonstrate that SCMC and ADCC involve different cytotoxicity mechanisms and are consistent with two subsets of effector cells with separate functional capabilities or a single effector cell mediating both forms of cytotoxicity through separate mechanisms.

Distribution of alloantigens on human Fc receptor-bearing lymphocytes: the presence of B cell alloantigens on sIg-positive but not sIg-negative lymphocytes.

Human peripheral blood lymphocyte subpopulations were analyzed for the presence of B cell alloantigens with a microcytotoxicity assay. B cell alloantigens were found exclusively on sIg-positive lymphocytes and were not present on sIg-negative, Fc receptor-bearing lymphocytes or sIg-negative, Fc receptor-negative T lymphocytes.

The effector cells in human peripheral blood mediating mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity.

The identity of the effector cells in human peripheral blood capable of mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) was investigated utilizing effector cell populations consisting of purified polymorphonuclear leukocytes, macrophages, lymphocytes, and cell surface immunoglobulin (sIg)-negative and sIg-positive lymphocyte subpopulations obtained by Sephadex anti-Fab immunoabsorbent column fractionation techniques. Chicken erythrocytes (CRBC) and Chang liver cells were used as target cells in both cytotoxicity assays. With CRBC targets MICC was mediated by polymorphonuclear leukocytes, macrophages, sIg-positive lymphocytes (B cells), and sIg-negative lymphocytes. On the contrary, with Chang liver cells as targets, MICC was mediated only by lymphocytes, and effector cells occurred exclusively in sIg-negative lymphocyte subpopulations containing thymus-derived lymphocytes (T cells). Further purification of sIg-negative lymphocyte subpopulations on antigen-antibody coated plastic surfaces yielded a nonadherent T lymphocyte population depleted of Fc receptor-bearing lymphocytes that was capable of mediating MICC against both CRBC and Chang cell targets. With use of CRBC targets, ADCC was mediated by polymorphonuclear leukocytes, macrophages, and sIg-negative lymphocyte subpopulations. However, with Chang cell targets, ADCC was mediated only by lymphocytes, and effector cells were present only in sIg-negative lymphocyte subpopulations. SIg-positive lymphocytes (B cells) and T lymphocytes were not effective in mediating ADCC against either CRBC or Chang cell targets. These studies demonstrate that the nature of the target cell employed in MICC and ADCC reactions is of critical imporatnce in defining the effector cell(s) capable of mediating cytotoxicity.