Transgenic zebrafish for detecting mutations caused by compounds in aquatic environments.

We have established a transgenic zebrafish line carrying a shuttle vector plasmid (pML4) for detecting mutagens in aquatic environments. The plasmid contains the rpsL gene of Escherichia coli as a mutational target gene, and the kanamycin-resistance gene for recovering the plasmid from the chromosomal DNA. To evaluate the system, we treated embryos of the transgenic fish with N-ethyl-N-nitrosourea (ENU), which induces a dose-dependent increase in the mutation frequency of the target gene. The mutation spectrum was consistent with the proposed mechanism of ENU mutagenesis. Similarly, treating the embryos with benzo[a]pyrene or 2-amino-3, 8-dimethylimidazo[4,5- f]quinoxaline, which are found in naturally polluted water, significantly increased the frequency of mutations in the target gene.

A hyperthermostable bacterial histone-like protein as an efficient mediator for transfection of eukaryotic cells.

Gene delivery has shown potential in a variety of applications, including basic research, therapies for inborn genetic defects, cancer, AIDS, tissue engineering, and vaccination. Most available systems have serious drawbacks, such as safety hazards, inefficiency under in vivo-like conditions, and expensive production. When using naked DNA, for instance, a large amount of ultrapure DNA has to be applied as a result of degradation by nucleases. Similarly, the use of eukaryotic histones, synthetic peptides, or peptide nucleic acids may be limited by high production costs. We have demonstrated a biotechnologically feasible and economical approach for gene delivery using the histone-like protein from the hyperthermostable eubacterium Thermotoga maritima, TmHU as an efficient gene transfer reagent. HU can be easily isolated from recombinant Escherichia coli, is extraordinarily stable, and protects dsDNA from thermal denaturation. This study demonstrates its use as an inexpensive tool for gene delivery.

Phenotype and ontogeny of cells carrying a tumor-associated antigen that is expressed on bovine leukemia virus-induced lymphosarcoma.

The phenotype and ontogeny of cells carrying the tumor-associated antigen (TAA), identified in tumors of cattle with enzootic bovine leukosis (EBL) by use of the monoclonal antibody (MAb) c143, were analyzed by flow cytometry and immunohistochemistry. The TAA recognized by the c143 MAb (c143 TAA) was mainly expressed on B-cells, macrophages, reticular cells, and a minor population of BoCD4-positive T-cells in bovine leukemia virus (BLV)-free normal cattle. When the peripheral blood mononuclear cells from normal cattle were activated in vitro, some populations of BoCD8-positive T- and non-T/non-B-cells also showed expression. Moreover, B-cells expressed the c143 TAA until the antigen was lost from cells at the final stage of B-cell differentiation, namely, the plasma cell stage. The c143 MAb-positive cells in blood of BLV-free normal cattle form heterologous subpopulations, and these cells coexpress other surface markers such as BoCD2, BoCD5, BoCD6, and B-cell-specific molecules B1 low, B1 bright, and B2. In BLV-infected cattle, the proportion of peripheral blood mononuclear cells that express the c143 TAA increased with the progression of EBL, and, in addition, BLV-infected cattle that had no lymphosarcomas showed increased proportions of c143 MAb-positive cells that coexpressed surface IgM (sIgM), BoCD5, B1 low, and B2 but not BoCD2, BoCD4, or BoCD6. Furthermore, most of the c143 MAb-positive tumors from all cattle with EBL appeared to be a monoclonal population derived from a single B-cell and to be divided into two types, c143 TAA+BoCD5+B1 low+ B2+ sIgM+ or sIgM-. Collectively, these results show that the c143 TAA is not only a useful surface marker for identification of EBL but also a marker of differentiation of lymphoid cells.

Tumor-associated M(r) 34,000 and M(r) 32,000 membrane glycoproteins that are serine phosphorylated specifically in bovine leukemia virus-induced lymphosarcoma cells.

Tumor-associated antigens that are expressed in tumor cells of cattle with enzootic bovine leukosis (EBL) were analyzed previously by use of 13 monoclonal antibodies. We biochemically identified one of the tumor-associated antigens, which is recognized by the c143 monoclonal antibody, as two glycoproteins, each having an apparent molecular weight of 32,000 or 34,000. These glycoproteins were found in the plasma membrane of peripheral blood lymphocytes of both bovine leukemia virus (BLV)-free normal and BLV-infected cattle. With the progression of EBL, the proportion and the absolute number of cells positive for the tumor-associated antigen increased. Moreover, the level of the M(r) 34,000 component, which was susceptible to cell-surface labeling, increased over the level of the M(r) 32,000 component. Partial proteolytic peptide mapping with V8 protease and deglycosylation analysis revealed that the two glycoproteins most likely have an identical M(r) 30,000 polypeptide portion but have different N-linked oligosaccharide portions. Both glycoproteins were found to be phosphorylated at serine residue(s) in EBL-derived B-lymphoid cell lines and in tumor cells and peripheral blood lymphocytes from cattle with EBL, but not in peripheral blood lymphocytes from BLV-free normal cattle and BLV-infected cattle without any evidence of tumor, suggesting that the phosphorylation of these glycoproteins is related to the transformed state of the BLV-infected B-lymphoid cells.

Characterization of erythropoietin receptor on erythropoietin-unresponsive mouse erythroleukemia cells.

A membrane receptor for erythropoietin was identified in various erythropoietin-unresponsive mouse erythroleukemia cells. Scatchard analyses of the binding of human 125I-labeled erythropoietin to T3C1-2-0, K-1, GM86 and 707 cells showed the presence of a single class of binding sites with apparent Kd values of 0.27-0.78 nM, which are slightly higher than those of erythropoietin-responsive cells. The number of binding sites varied from 110 to 930 per cell. Crosslinking of 125I-erythropoietin to its binding sites with disuccinimidyl suberate revealed the existence of a single binding protein with molecular mass of 63 kDa. No binding sites with higher molecular mass, as observed in erythropoietin-responsive cells, were detected, nor was any specific binding observed to the non-erythroid hematopoietic cell or to the human erythroleukemia cells examined.

Decrease in glucose transport activity of Friend erythroleukemia cell caused by dimethylsulfoxide, a differentiation-inducing reagent.

A transport system for D-glucose was found in a Friend erythroleukemia cell line, T-3-C1-2-O and was characterized as a facilitated diffusion system. D-Glucose transport activity showed a half-saturation concentration of 2.2 mM and was inhibited by mercuric ions, cytochalasin B, phloretin, and stilbestrol, but was not strongly inhibited by phloridzin. Transport of 3-O-methyl-D-glucose was faster than D-glucose and the intracellular concentration of the sugar was found to reach the concentration in the assay medium. The treatment of cells with a differentiation-inducing reagent, dimethylsulfoxide(Me2SO), for 24 h caused a marked decrease in glucose transport activity due to a decrease in Vmax. In an induction-insensitive Friend cell line, T-3-K-1, D-glucose transport activity was low in untreated cells and Me2SO treatment did not cause a significant decrease in transport activity. The results obtained in this study indicate that the decrease in glucose transport activity is not due to the direct effect of Me2SO on transport activity, but is associated with the induction of differentiation. By immunoblotting cell lysates of T-3-C1-2-O cells using antibody to human erythrocyte glucose transporter, a single major band having a molecular weight of 52,000 was detected, which may be a glucose transporter in Friend cells.

Identification of tumor-associated antigen that is expressed on bovine leukemia virus-induced lymphosarcoma cells and expression of its human homologue in human T-cell lymphotrophic virus I-infected cell lines.

By using the monoclonal antibody (MAb) c143 against tumor-associated antigen that is expressed in tumor cells of cattle with bovine leukemia virus (BLV)-induced enzootic bovine leukosis (EBL), we found a novel bovine MHC class II-related antigen which consists of alpha chain (36-37 kDa) and beta chain (32 and 34 kDa). The nature of the c143 antigen was different from previously identified class II antigens, such as DR and DQ, as indicated by test for reactivities with mouse L cell transfectants expressing human class II antigens, sequential immunoprecipitation and tryptic peptide mapping. With the progression of EBL, the number of cells carrying the c143 antigen increased, and the beta chain was specifically phosphorylated at serine residue(s) in lymphosarcoma cells and the cell lines derived from them. A marked difference between expression patterns of the c143 antigen and the class II antigens was observed in lymph nodes from BLV-free and -infected animals. Although bovine mixed lymphocyte reaction (MLR) was inhibited by the addition of MAbs against class II antigens, the c143 MAb did not inhibit a lymphoproliferative response of T cells in the MLR, suggesting that the function of this antigen is distinct from those of classical class II molecules. Significantly, although the human homologue of the c143 antigen is expressed little if at all in human T-cell lines, such as CEM, Molt-4, Jurkat and HPB-ALL, its expression become apparent in T-cell lines infected with human T-cell lymphotrophic virus I (HTLV-1).

Mutational analysis of the structure-function relationship of the leukemogenic membrane glycoprotein (GP55) of Friend spleen focus-forming virus (F-SFFV).

Friend spleen focus-forming virus (F-SFFV) causes acute erythroleukemia in adult mice. F-SFFV encodes an envelope protein-like membrane glycoprotein called gp55 in its defective env gene. Gp55 is responsible for the early stage of leukemogenesis by F-SFFV by specifically binding to and activating the murine erythropoietin receptor (EPO-R). Gp55 has a polytropic env sequence in its N-terminal portion. This portion probably contains the binding site for the EPO-R. In order to obtain a clue for the structure of the binding site to the EPO-R, we isolated and analyzed many spontaneous revertant F-SFFVs which derived from the non-leukemogenic mutant F-SFFV having an ecotropic env sequence instead of the polytropic env sequence in its gp55 gene.

Peptide-based bovine leukemia virus (BLV) vaccine that induces BLV-Env specific Th-1 type immunity.

In controlling retrovirus infection and replication, cell-mediated immunity (CMI) is considered to be effective. To develop a synthetic peptide vaccine capable of inducing CMI, mannan-coated liposome encapsulating 20-mer synthetic peptide, spanning the 98-117 amino acids of bovine leukemia virus (BLV) envelope glycoprotein (Env) gp51 was constructed and inoculated to BALB/c mice. The liposome induced specific delayed-type hypersensitivity, lymphocyte proliferative responses, and a weak cytotoxic lymphocyte response. The spleen cells from the immunized mice produced a large amount of IFN-gamma and IL-2, whereas they released neither IL-4 or IL-10. Mannan-coated liposome containing two kinds of peptides (the 121-140 and 142-161 regions of BLV Env gp51) also induced peptide-specific lymphocyte proliferative response and IFN-g production in C57BL/6 mice. Thus, the synthetic T-cell epitope peptide-liposome system augmented a strong Th-1 type immunity in mice.

The role of tumor-associated antigen in bovine leukemia virus-induced lymphosarcoma.

Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle. To clarify the way in which BLV-infected cattle progress from the asymptomatic stage to the lymphoma stage, we produced a monoclonal antibody (MAb) c143 which recognized a tumor-associated antigen (TAA) that is phosphorylated in the transformed state of BLV-infected B-lymphoid cells. Since the nature of c143 TAA was likely to be that of the major histocompatibility complex (MHC) class II antigens, we isolated cDNAs for bovine MHC (BoLA) class II a-chains and b-chains, produced transfectants that expressed a single type of BoLA class II molecules and analyzed them by flow cytometry with c143 MAb. The c143 MAb recognized the transfectant expressing BoLA-DR but not BoLA-DQ. However, the treatment of lymphocytes with c143 or anti-BoLA-DR MAb induced different effects. Although mixed lymphocyte reaction (MLR) was inhibited by the addition of anti-BoLA-DR MAb, the c143 MAb did not inhibit a proliferative response of T cells in MLR. Increased spontaneous proliferation of lymphocytes in healthy donors was obtained in the presence of c143 MAb but not anti-BoLA-DR MAb, and was much in lymphocytes from the carrier. Moreover, the patterns of immunohistological staining for c143 MAb in BLV-infected sheep showed distinguishing differences from those of anti-BoLA-DR MAbs.

Isolation and characterization of a genomic DDD mouse interleukin-3 gene.

A chromosomal DNA segment containing the entire gene for interleukin-3 (IL-3) was isolated from DDD mouse erythroleukemia cells, and its gene organization and nucleotide sequence were determined. Enhancer-like sequences in the second intron and G + C-rich sequence in the 5'-flanking region may play a role in the regulation of tissue-specific and inducible gene expression. Southern blot analyses revealed that constitutively IL-3-producing WEHI-3 cells have the rearranged IL-3 gene, and that genomic DNAs prepared from the adult and fetal mouse liver have the same organization of the IL-3 gene. No IL-3 gene transcript was detected in the mouse erythroleukemia cells by Northern blot analysis.

Transport of sugars and amino acids in bacteria. XVI. Theory and evaluation of a model for the membrane transport reaction mediated by a single carrier with three binding sites for substrate.

A novel model is presented for bacterial active transport reactions which show a curvilinear Eadie-Hofstee plot and negative homotropic cooperativity in the kinetics of substrate uptake. Various models of a single carrier with multi-binding sites for substrate were constructed and examined theoretically. The fit of these models with experimental data on the kinetics of branched chain amino acid transport reactions were tested by iterative computation using the non-linear least square method. The transport model which fitted the experimental data best consisted of a single carrier with three binding sites for substrate in which one of the substrate-carrier complexes, CSS, is not active in translocating substrate across the cytoplasmic membrane. The mechanism of homeostatic regulation of the intracellular concentration of amino acids by active transport systems is discussed on the basis of this transport model.

Cross-reactivity between a monoclonal antibody that recognizes a tumor-associated antigen on bovine lymphosarcoma cells and blood lymphocytes from various mammalian species.

Tumor-associated antigens that are expressed in lymphosarcoma B cells of cattle with enzootic bovine leukosis had been analyzed in terms of their reactivity with 13 monoclonal antibodies (MAB). By use of flow cytometry and radioimmunoprecipitation, 1 of the MAB (c143) that recognized a tumor-associated antigen cross-reacted with blood lymphocytes (BL) from various mammalian species. By use of flow cytometry, the c143 MAB reacted with 10 to 49% of BL derived from human beings, mice, dogs, horses, pigs, llamas, sheep, goats, and cattle. Titer of the c143 MAB with BL from horses, pigs, human beings, and llamas ranged between 1:6.0 x 10(4) and 1:5.3 x 10(5); titer associated with BL of goats and sheep was 1:1.6 x 10(6); and that associated with BL of cattle was 1:4.3 x 10(7). The c143 MAB specifically immunoprecipitated 3 homologous proteins from cell extracts of caprine, ovine, and bovine BL (32-, 34-, and 36- to 37-kDa bovine proteins; 31-, 32-, and 36- to 37-kDa caprine proteins; and 31.5-, 33-, and 36- to 37-kDa ovine proteins), but none was immunoprecipitated from human, murine, canine, porcine, and llama BL. These results indicate that the avidity of the c143 MAB in binding to BL from ruminants (eg, goats, sheep, and cattle) is higher than that to BL from human beings, mice, dogs, horses, pigs, and llamas. In sheep, the c143 MAB could immunoprecipitate the aforementioned proteins from BL of the Suffolk breed, but not BL from the Corriedale breed, whereas the c143 MAB immunoprecipitated apparently identical proteins from BL of 4 breeds of cattle.

[Modified env gene in Friend spleen focus forming virus--structure, origin, and its role in leukemogenesis].

Modified env gene or gp55 gene in Spleen Focus Forming Virus, polycythemic strain, K-1 was molecularly cloned and its structure was characterized gp55 is a fusion glycoprotein consisting of N terminal 2/3 of xenotropic virus-related gp70 and C terminal half of F-MuLV p15E. A unique structure at the 3' and of the gp55 gene with 6 base pairs + 1 base pairs insert was identified. Gp55 may be hooked by the lipid bilayer of the cell membrane of the SFFV-intected cells, and additional glycosylation on the cell surface may modify the growth regulation of the cells. Possible origin of SFFV-specific gp55 gene was discussed.