Functional expression of the ascofuranone-sensitive Trypanosoma brucei brucei alternative oxidase in the cytoplasmic membrane of Escherichia coli

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms (LS forms) of African trypanosoma, which causes sleeping sickness in human and nagana in cattle. TAO is a cytochrome-independent, cyanide-insensitive quinol oxidase and these properties are quite different from those of the bacterial quinol oxidase which belongs to the heme-copper terminal oxidase superfamily. Only little information concerning the molecular structure and enzymatic features of TAO have been available, whereas the bacterial enzyme has been well characterized. In this study, a cDNA encoding TAO from Trypanosoma brucei brucei was cloned into the expression vector pET15b (pTAO) and recombinant TAO was expressed in Escherichia coli. The growth of the transformant carrying pTAO was cyanide-resistant. A peptide with a molecular mass of 37 kDa was found in the cytoplasmic membrane of E. coli, and was recognized by antibodies against plant-type alternative oxidases from Sauromatum guttatum and Hansenula anomala. Both the ubiquinol oxidase and succinate oxidase activities found in the membrane of the transformant were insensitive to cyanide, while those of the control strain, which contained vector alone, were inhibited. This cyanide-insensitive growth of the E. coli carrying pTAO was inhibited by the addition of ascofuranone, a potent and specific inhibitor of TAO ubiquinol oxidase. The ubiquinol oxidase activity of the membrane from the transformant was sensitive to ascofuranone. These results clearly show the functional expression of TAO in E. coli and indicate that ubiquinol-8 in the E. coli membrane is able to serve as an electron donor to the recombinant enzyme and confer cyanide-resistant and ascofuranone-sensitive growth to E. coli. This system will facilitate the biochemical characterization of the novel terminal oxidase, TAO, and the understanding on the mechanism of the trypanocidal effect of ascofuranone.     

Malignant histiocytosis. A report of three cases.

Three cases of malignant histiocytosis were immunohistochemically studied. The cases included the following three patients: a 38-year-old man, a 44-year-old man, and a girl aged 5 years 9 months. All three patients died within 3 months of hospitalization. They had a high fever (temperature over 38.5 degrees C), lymph node swelling, hepatosplenomegaly, and pancytopenia. Blastoid and hemophagocytic cells proliferated in the bone marrow and lymph nodes, especially in the sinuses of the latter. We diagnosed malignant histiocytosis in the three cases based on clinical features, extremely poor prognoses, and the morphologic features and growth pattern of blastoid and hemophagocytic cells. Blastoid and hemophagocytic cells expressed phenotype Mac-387+/KP1+/lysozyme+/polyclonal CD3+. The Mac-387 and KP1 antigens and lysozyme are markers for monocytes/macrophages, and polyclonal CD3 is a marker for T lymphocytes. Therefore, we suggest that a certain number of cases of malignant histiocytosis have a biphenotypic nature, namely, the T cell and macrophage, although many cases of malignant histiocytosis have been reported as expressing only T-lymphocyte antigens.     

Primary pulmonary low-grade marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue type with prominent hyalinosis. A case report

We report a case of primary pulmonary low-grade marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT)-type with prominent sclerosis, which morphologically resembled pulmonary hyalinizing granuloma (PHG) or inflammatory pseudotumor (IPT) of the lung. The patient, a 66-year-old Japanese female with a history of Sj?gren's syndrome and primary biliary cirrhosis, presented with a lower left lobe mass 6.8 cm in diameter. Histologically, the lesion is characterized by dense bundles of collagen with scattered plasma cells, mature small lymphocytes, and histiocytes among the collagen bundles. Only the peripheral area of the nodule contained dense lymphoplasmacytoid and histiocytoid infiltrates. A few centrocyte-like cells were obscured by the numerous plasma cells and plasmacytoid cells. In addition, lymphoepithelial lesions and colonalized lymphoid follicles were identified by immunohistochemistry alone. Although PHG and IPT are unlikely to be confused with pulmonary MALT-type lymphomas, the present case suggests that MALT-type lymphoma should be added to the list of differential diagnoses for PHG and IPT.     

CEROID-LIKE HISTIOCYTIC GRANULOMA OF GALL-BLADDER — A PREVIOUSLY UNDESCRIBED LESION —

In the present study, 13 cases of a peculiar gall-bladder granuloma characterized by marked proliferation of ceroid-fllled brown histiocytes were pathomorphologically, histochemically and ultrastructurally examined to define the pathologic features of such a lesion previously undescribed in the literature. The lesion grossly showed a granulomatous appearance of yellow brown to dark brown color developing in the wall of gall-bladder. Histologically, there was proliferation of histiocytes containing abundant brown pigment granules In their cytoplasm. The pigment granules proved to have staining characteristics closely resembling those of lipogenic ceroid-like pigment. Ultrastructurally, these granules showed membrane-bound, pleomorphic osmiophilic inclusions of heterogenous materials. With regard to the pathogenesis of this granuloma, it may be suggested that lipid components of bile juice, particularly unsaturated fatty acids and phospholipids, play an Important role as a source of ceroidogenesis In the proliferating histiocytes.     

Plasmacytoma of Lymph Node Recurrent Lymphadenopathy Terminating in Plasma Cell Dyscrasia with Polyneuropathy and Endocrine Disturbances

A case with lymphadenopathy of the left side of the neck in a 38-year-old male is described. He had a history of several relapses of about 10 years duration. Swollen lymph nodes were histologically similar to those of the hyaline-vascular type of Castleman's disease, but contained clear-cut lymph sinus and a sheet-like proliferation of plasma cells. Lymph follicles showed proliferation and atrophic germinal centers, in which cellular hypertrophy in the wall of ramifying small blood vessels, called angiosclerosis, was frequently encountered. During its progress, the patient developed plasmacytoma of the lymph nodes with varied clinical manifestations such as polyneuropathy, disturbance of gait, unusual perspiration, hirsuitism, gynecomastia, bilateral papilledema, and albumino-cytologic dissociation in cerebrospinal fluid.     

Kappa-type light chain crystal storage histiocytosis.

An autopsy case of systemic histiocytosis with excessive deposition of kappa-type light chain crystals was reported in a 58 year-old man who had consistently showed kappa-type light chain paraproteinemia, Bence Jones proteinuria and hypogammaglobulinemia for about 10 years until his death. However, no bony destruction was found by repeated X-ray examinations. At autopsy, extensive hyperplasia of crystal-storing histiocytes was observed in the bone marrow, spleen, liver, lymph nodes, interstitial tissues of visceral organs and loose connective tissues. In the bone marrow and some other tissues, mild proliferation of plasmocytoid cells containing small crystals were found. Histochemically the crystals positively stained with various methods for amino acids and proteins, especially with Weigerts' method for fibrin. Ultrastructurally intralysosomal crystal deposition was confirmed in the storage histiocytes and derivation of the crystals from Golgi's sacculi in the plasmocytoid cells was suggested. Biochemically the crystals were regarded as mainly consisting of dimers of a variable half of light chain immunoglobulin and immunochemically and immunohistochemically reacted to anti-kappa type light chain serum. Such a generalized storage histiocytosis may be secondarily induced by immunoglobulin synthesized in plasmocytoid cells.     

Bacterial superantigen-treated intestinal epithelial cells upregulate heat shock proteins 25 and 72 and are resistant to oxidant cytotoxicity

While the pathological events evoked by infection are commonly described, effective host responses to bacteria and their products should primarily be protective. Heat shock protein (Hsp) expression is upregulated by many stimuli and serves to maintain intracellular protein integrity. The ability of the prototypic superantigen, Staphylococcus aureus enterotoxin B (SEB) to induce Hsps was investigated with BALB/c mice and by in vitro addition to the murine small intestinal epithelial cell line MSIE. SEB-treated (5 or 100 microg intraperitoneally) mice revealed increased Hsp25 and Hsp72, but not Hsc73, in jejunal lymphocytes and epithelial cells. A similar Hsp response to SEB occurred in MSIE cells and was preceded by activation of the ERK1/2 and p38 mitogen-activated protein kinases but not the SAPK/JNK pathway; pharmacological inhibition of ERK1/2, but not p38, significantly reduced SEB-induced Hsps. Moreover, SEB-treated MSIE cells were protected against oxidant-induced cytotoxicity (measured by 51Cr release) and F-actin depolymerization. Thus, SEB exposure results in a rapid induction of the Hsp25 and Hsp72 in intestinal epithelial cells, both directly and through lymphocyte activation, and we suggest that this event is important in protecting the gut from damage by Staphylococcus infection or in the reparatory process and may be a generalized response to lumen-derived bacterial toxins.     

The combination of IFN-alpha2 and IFN-alpha8 exhibits synergistic antiproliferative activity on renal cell carcinoma (RCC) cell lines through increased binding affinity for IFNAR-2.

Although there are at least 13 interferon-alpha (IFN-alpha) subtypes in humans, interactions between the subtypes remain unknown. To understand IFN-alpha interactions, we examined the antiproliferative activities and the receptor binding affinities of different combinations of IFN-alpha2 and IFN-alpha8 using six renal cell carcinoma (RCC) cell lines. Although IFN-alpha8 was the more potent subtype, synergistic and antagonistic antiproliferative effects were also observed in certain combinations of IFN-alpha2 and IFN-alpha8. To analyze the interactions between IFN-alpha2 and IFN-alpha8, the receptor-binding kinetics of different combinations of IFN-alpha2 and IFN- alpha8 to the IFN-alpha receptors, IFNAR-1 or IFNAR-2, were measured using a surface plasmon resonance-based biosensor. Unexpectedly, the receptor binding kinetics to IFNAR-2 but not to IFNAR-1 were mutually related to antiproliferative activity and increase in the binding speed (K(a)) for IFNAR-2. Moreover, we observed the increased fluorescence intensity (FI) of biotin-labeled IFN-alpha8 to IFNAR-2 by receptor binding inhibition assay with unlabeled IFN-alpha2 but not the other combinations. These findings indicate that the binding manner of IFN-alpha8 for IFNAR-2 is different from that of IFN-alpha2, suggesting that binding of IFN-alpha8 rather than binding of IFN-alpha2 to IFNAR-2 leads to activation and subsequent antiproliferative activity despite the same antiviral activity in RCC.     

Inhibition of MAP kinase activity moderates changes in expression and function of Cx32 but not claudin-1 during DNA synthesis in primary cultures of rat hepatocytes

The signal transduction pathways and activation of the MAP kinase or PI3 kinase signaling cascade regulate a variety of cellular processes, including proliferation and differentiation in hepatocytes. To elucidate the mechanisms of signal transmission required for the regulation of gap and tight junctions during DNA synthesis in rat hepatocytes, we determined changes of expression and function of gap and tight junctions of cells grown in primary culture, using inhibitors of signaling pathways for MAP kinase (PD98059) and PI3 kinase (LY294002). During the stimulation of DNA synthesis induced by epidermal growth factor (EGF), immunoreactivity and mRNAs of gap junction protein Cx32 and of tight junction protein claudin-1 markedly decreased with reduction of gap junctional intercellular communication (GJIC) and the fence function of tight junctions. In Western blots, whole-cell lysate of claudin-1 protein decreased and phosphorylated Cx32 protein in the insoluble fraction of Triton X-100 increased during the stimulation of DNA synthesis. During reinhibition of DNA synthesis, the changes of Cx32 and claudin-1 returned to control levels, as did both functions. In treatment with the inhibitors before DNA synthesis, PD98059 inhibited the changes of expression and function of Cx32, but not claudin-1, without inhibition of cell growth, whereas LY294002 completely inhibited cell growth. These findings indicate that the PI3 kinase pathway rather than the MAP kinase pathway plays an important role for EGF-induced proliferation of rat hepatocytes, and that changes of Cx32 in hepatocytes during the stimulation of DNA synthesis may be in part controlled through MAP kinase. Furthermore, Cx32, but not claudin-1, protein may be a target of activated MAP kinase in hepatocytes.     

Immune response to Trichomonas vaginalis. IV. Immunochemical and immunobiological analyses of T. vaginalis antigen

The immune response of patients with trichomoniasis vaginitis to Trichomonas vaginalis was examined by using passive hemagglutination assay and skin reactions. Trichomonas-specific immune responses could hardly be detected in the patients by these assay methods. In order to develop a simple and reliable assay for demonstration of T. vaginalis-specific immune responses in the patients, proliferation responses of peripheral mononuclear cells from the patients were examined. Lymphocytes obtained from the patients were shown to incorporate 3H-methyl-thymidine when stimulated with T. vaginalis antigen in vitro. Furthermore, immunochemical analysis of T. vaginalis antigen has been done by Sephadex chromatography and sodium dodecyl sulfate gel electrophoresis. The data revealed that the murine and human IgM antibodies specific to T. vaginalis recognize various antigens with a wide range of molecular weights (between 13,000 and 100,000 daltons), while human and murine IgG antibodies recognize molecules with a much narrower range of molecular weights (50,000-100,000 daltons).