Crystallization and preliminary characterization of three different crystal forms of human saposin C heterologously expressed in Pichia pastoris.

The amphiphilic saposin proteins (A, B, C and D) act at the lipid-water interface in lysosomes, mediating the hydrolysis of membrane building blocks by water-soluble exohydrolases. Human saposin C activates glucocerebrosidase and beta-galactosylceramidase. The protein has been expressed in Pichia pastoris, purified and crystallized in three different crystal forms, diffracting to a maximum resolution of 2.5 A. Hexagonal crystals grew from 2-propanol-containing solution and contain a single molecule in the asymmetric unit according to the Matthews coefficient. Orthorhombic and tetragonal crystals were both obtained with pentaerythritol ethoxylate and are predicted to contain two molecules in the asymmetric unit. Attempts to determine the respective crystal structures by molecular replacement using either the known NMR structure of human saposin C or a related crystal structure as search models have so far failed. The failure of the molecular-replacement method is attributed to conformational changes of the protein, which are known to be required for its biological activity. Crystal structures of human saposin C therefore might be the key to mapping out the conformational trajectory of saposin-like proteins.

The murine Kupffer cell. II. Accessory cell function in in vitro primary antibody responses, mitogen-induced proliferation, and stimulation of mixed lymphocyte responses.

The ability of murine Kupffer cells to function in several in vitro immunologic systems was investigated. These cells have been shown previously to function as accessory cells in antigen-stimulated T cell proliferation in response to protein antigens. In the present study it has been demonstrated that murine Kupffer cells also are competent as accessory cells in in vitro primary antibody responses to TNP-KLH and for T cell proliferative responses to concanavalin A. In addition, murine Kupffer cells were found to be potent stimulators of mixed lymphocyte responses. These studies extend previous observations by demonstrating that Kupffer cells are competent accessory cells in several distinct in vitro correlates of in vivo immune responses. The role of Kupffer cells in in vivo immune responses, particularly those to enterically derived antigens, may require re-evaluation in the light of these findings.

The murine Kupffer cell. I. Characterization of the cell serving accessory function in antigen-specific T cell proliferation.

Murine Kupffer cells, the tissue macrophages of the liver, were isolated by collagenase digestion, differential sedimentation over Metrizamide, and glass adherence. The resultant cell population was more than 86% phagocytic, and 95% of cells stained positively for alpha-naphthyl butyrate esterase activity. The cells also had cell surface receptors for complement (C) and the Fc portion of IgG. In addition, a large proportion of Kupffer cells was shown to bear Ia antigens: about half of the cells bore I-A subregion-encoded antigens and about half bore I-BJE or I-EC subregion-encoded antigens. Kupffer cell populations were capable of reconstituting antigen-stimulated proliferative responses of antigen-primed, macrophage-depleted, lymph node T cells. The ability to reconstitute proliferation was enriched in the adherent population and was resistant to radiation and treatment with an anti-Thy antiserum and C. We conclude that isolated murine Kupffer cells bear the Ia phenotype of accessory cells that function in antigen presentation and that Kupffer cells can participate in the induction of antigen-specific immune responses. These data suggest that Kupffer cells may play a role in modulating responses to enterically derived antigens.

The effects of pharmacologic agents on immune-complex-induced redistribution of B-lymphocyte Fc receptors.

The effects of pharmacologic agents on the immune-complex-induced redistribution of B-lymphocyte Fc receptors (and, as a control, the anti-Ig induced redistribution of surface Ig) were examined. Immune-complex-induced capping of B-cell Fc receptors was moderately to markedly inhibited by the combination of colchicine and cytochalasin B, the Ca++ ionophore A23187, and the local anaesthetic lidocaine but was only slightly inhibited by cytochalasin B alone and was not inhibited by colchicine alone. Inhibition of capping was not due to the inhibition of binding of immune complexes to the B-lymphocytes or to decreased cell viability since these effects were absent. Preformed immune complex-Fc receptor caps were disrupted by A23187, lidocaine, and the combination of colchicine and cytochalasin B, but not by either colchicine or cytochalasin B alone. The effects of the pharmacologic agents were similar for Fc receptors and surface Ig in all cases. These results suggest that ligand bound Fc receptors are affected by cytoskeletal structures and that the ligand-induced redistribution of two distinct B lymphocyte surface receptors (fc receptors and surface Ig) occurs by similar or identical mechanisms.

Fat embolism syndrome in delirium tremens.

Delirium tremens in a common feature in the alcoholic population. The Fat Embolism Syndrome (FES) is characterized by fever, encephalopathy, respiratory failure and skin petechiae. Fat embolism has been associated with alcoholics but the diagnosis was apparent only at autopsy. We present an alcoholic male who developed delirium tremens unresponsive to therapy, followed by features of the FES. Asterixis and Korsakoff's psychosis are newly described features of this syndrome. Corticosteroids were a definitive therapy in this case.