Tadayoshi Taniyama

Direct augmentation of cytolytic activity of tumor-derived macrophages and macrophage cell lines by muramyl dipeptide.

Abstract Macrophages derived from MSV-induced tumors and several macrophage cell lines showed direct cytolytic activity in an 18-hr 51 Cr release assay against tumor target cells. The cytolytic activity of these macrophages was augmented by the addition of muramyl dipeptide (MDP) to the cytotoxicity assay, an effect similar to that observed with bacterial lipopolysaccharide. The stimulation of macrophage-mediated cytotoxicity by MDP appeared to be under genetic control since macrophages from BALB/c mice were augmented with MDP while those from C57BL/6 animals were not. MDP appears to act directly on the macrophage without the participation of any other cell type, since MDP increased the activity of the macrophage cell lines.

Rapid and sensitive detection method of a bacterium by using a GFP reporter phage.

A rapid, sensitive, and convenient method for detecting a specific bacterium was developed by using a GFP phage. Here we describe a model system that utilizes the temperate Escherichia coli‐restricted bacteriophage lambda, which was genetically modified to express a reporter gene for GFP to identify the colon bacillus E. coli in the specimen. E. coli infected with GFP phage was detected by GFP fluorescence after 4–6 hr of incubation. The results show that a few bacteria in a specimen can be detected under fluorescence microscopy equipped with a sensitive cooled CCD camera. When E. coli and Mycobacterium smegmatis were mixed in a solution containing GFP phage, only E. coli was infected, indicating the specificity of this method. The method has the following advantages: 1) Bacteria from biological samples need not be purified unless they contain fluorescent impurities; 2) The infection of GFP phage to bacteria is specific; 3) The fluorescence of GFP within infected bacteria enables highly sensitive detection; 4) Exogenous substrates and cofactors are not required for fluorescence. Therefore this method is suitable for any phage‐bacterium system when bacteria‐specific phages are available.

Immunologic Reactivity of Lymphoid Cells in Tumors

There have been many studies of immune responses against tumors and almost all of these have focused on the reactivity in the blood or spleen. From such studies, it has become clear that a wide variety of effector cells and types of immune functions may be involved in antitumor responses. Particular attention has been directed toward T cells that may be directly cytotoxic against tumor cells or may proliferate or produce lymphokines upon stimulation with tumor antigens. However, other effector mechanisms may be involved and need to be considered. These include B cells, which can produce antibodies that affect tumor cells directly or that interact with K cells or macrophages and thereby mediate antibody-dependent cell-mediated cytotoxicity; macrophages and monocytes, which are spontaneously cytotoxic or can be activated to become cytotoxic against tumor cells; and natural killer (NK) cells, a subpopulation of lymphocytes with spontaneous cytotoxic reactivity against tumor cells.

The human interleukin-10 receptor gene maps to chromosome 11q23.3

The human interleukin-10 receptor (IL-10R) gene has previously been mapped to chromosome 11. Here, we have determined the precise location of the human IL-10R gene by the fluorescence in situ hybridization method, and have found that the IL-10R gene maps to chromosome 11q23.3.

Assignment of SH3KBP1 to human chromosome band Xp22.1→p21.3 by in situ hybridization

Many tyrosine kinases have been proven to play important roles in signal transduction pathways by which extracellular ligands induce proliferation and differentiation. Among these tyrosine kinases, the Src-family members (Src, Hck, Lck, Lyn, Fyn, Yes, Fgr, Blk, and Rak) have been well characterized (Bolen et al., 1992; Cooper and Howell, 1993). Recently, the Src family of tyrosine kinases has also been found to be involved in the pathogenesis of human immunodeficiency virus (HIV) (Saksela et al., 1995). Especially, the binding of the Src homology 3 (SH3) domains of Hck to the Nef protein of HIV is an order of magnitude tighter that those of other Src-family kinases (Fyn, Lck, and Src) (Arold et al., 1998). To determine the signal transduction cascade downstream from the Hck kinase, we used the SH3 domain of the Hck kinase as bait for a yeast two-hybrid assay, and cloned a novel protein (human Srcfamily kinase binding protein-l, HSB-1) that binds to the SH3 domain of the Src-family kinase. This HSB-1 protein has been shown to enhance tumor necrosis factor (TNF)-mediated but not Fas-induced apoptotic cell death (Narita et al., in preparation). We also found that the human macrophage-like cell clone (U937/pEF1C-HSB-1), ectopically expressing the HSB-1 protein, is resistant to HIV-1 infection (Amano et al., in preparation). Thus, this HSB-1 protein is important in the regulation of apoptosis by TNF and in the pathogenesis of HIV. In the present paper, we have determined the chromosome location of the human SH3KBP1 gene that encodes HSB-1 protein. To determine the chromosome location of SH3KBPl, metaphases were prepared from human lymphocytes using standard techniques. After in situ hybridization with the HSB-l cDNA insert, 100 metaphases were analyzed. DAPI banding was used to identify the specific chromosome and probe signals were assigned to the short arm of the X chromosome. Therefore, this SH3KBPl gene is mapped to human chromosome X, region p22.1→p21.3.

Mapping of the human gene encoding the mutual signal-transducing subunit (β-chain) of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5) receptor complexes to chromosome 22q13.1

The human gene encoding the mutual signal-transducing subunit (β-chain) of granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5 receptor complexes has been mapped to chromosome 22q13.1 by the fluorescence in situ hybridization method.

CIN85 is required for Cbl-mediated regulation of antigen receptor signaling in human B cells.

The aberrant regulation of B-cell receptor (BCR) signaling allows unwanted B cells to persist, thereby potentially leading to autoimmunity and B-cell malignancies. Casitas B-lineage lymphoma (Cbl) proteins suppress BCR signaling; however, the molecular mechanisms that control Cbl function in human B cells remain unclear. Here, we demonstrate that CIN85 (c-Cbl interacting protein of 85 kDa) is constitutively associated with c-Cbl, Cbl-b, and B-cell linker in B cells. Experiments using CIN85-overexpressing and CIN85-knockdown B-cell lines revealed that CIN85 increased c-Cbl phosphorylation and inhibited BCR-induced calcium flux and phosphorylation of Syk and PLCγ2, whereas it did not affect BCR internalization. The Syk phosphorylation in CIN85-overexpressing and CIN85-knockdown cells was inversely correlated with the ubiquitination and degradation of Syk. Moreover, CIN85 knockdown in primary B cells enhanced BCR-induced survival and growth, and increased the expression of BcLxL, A1, cyclin D2, and myc. Following the stimulation of BCR and Toll-like receptor 9, B-cell differentiation- associated molecules were up-regulated in CIN85-knockdown cells. Together, these results suggest that CIN85 is required for Cbl-mediated regulation of BCR signaling and for downstream events such as survival, growth, and differentiation of human B cells.

Human high-affinity FcγRI (CD64) gene mapped to chromosome 1q21.2-q21.3 by fluorescence in situ hybridization

The human FcγRI gene encodes for a highaffinity Fcγ receptor that plays pivotal roles in the immune response. We have used fluorescence in situ hybridization analysis to localize the FcγRI gene to human chromosome 1. The human FcγRI (CD64) gene has been assigned to human chromosome 1q21.2-q21.3 using R-banded human (pro)metaphase chromosomes.

Phospholipases of Leptospira

The hydrolysis of phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and trioleoylglycerol by Leptospira biflexa strain Urawa was studied in vitro.