[Safety issues of retrovirus vectors].
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Animals↗
Biomedical subjects
H Amanuma
Publications and source records attributed to H Amanuma.
Characterization of three different transgenic mouse lines that carry human poliovirus receptor gene--influence of the transgene expression on pathogenesis.
Three transgenic mouse lines, ICR-PVRTg1, ICR-PVRTg5, and ICR-PVRTg21, which are susceptible to poliovirus, have been established by introducing the human gene for poliovirus receptor (PVR) into the genome of mouse strain ICR. Genetic characterizations of the PVR gene were carried out on these mouse lines to define the approximate copy number, insertion site, and expression of the transgene in the central nervous system (CNS). The transgene was integrated in the chromosome 4, 12, and 13 of ICR-PVRTg1, ICR-PVRTg5 and ICR-PVRTg21 mice, respectively, and was stably transmitted to progeny mice. ICR-PVRTg1 appeared to have the most abundant copy numbers of the transgene and showed the highest level of PVR mRNA and membrane associated PVR protein in the CNS among the three mouse lines. Those in ICR-PVRTg21 and ICR-PVRTg5 were at intermediate and lowest levels, respectively. In the CNS, PVR mRNA was detected at high levels only in neurons of the spinal cord and brain stem where poliovirus can replicate, suggesting that the PVR mRNA expression confers cell specificity to poliovirus in the CNS. ICR-PVRTg1 and ICR-PVRTg5 showed the highest and the lowest sensitivity to poliovirus, respectively, whereas ICR-PVRTg21 was in-between. These results may suggest that poliovirus sensitivity of the mice is attributed to relative levels of PVR expression.
Animals↗
A novel POU domain gene, zebrafish pou2: expression and roles of two alternatively spliced twin products in early development.
POU domain proteins are a large family of transcriptional regulatory proteins, many of which are implicated in the control of gene expression during early development. We describe here the cloning and expression of zebrafish pou2, a novel POU domain gene related to the mouse germ-line-specific transcription factor oct-3. Zebrafish pou2 is maternally expressed, and the transcripts are present from the one-cell stage to the gastrula stage. In situ hybridization analyses revealed that the transcripts were present in all blastomeres until the midblastula stage and that the expression was restricted to the epiblast during gastrulation. We found that alternatively spliced transcripts, t-pou2 RNAs, were also expressed in the embryos. In contrast to the Pou2 product, the t-Pou2 product lacks DNA-binding activity because of its incomplete POU domain structure. To examine the roles of the Pou2 and t-Pou2 products, we increased their expression in the embryo by microinjection of synthetic pou2 and t-pou2 RNAs into the fertilized eggs at the one-cell stage. Most embryos that developed from the eggs injected with pou2 RNA did not show any obvious developmental defects. In contrast, overexpression of the t-Pou2 product greatly affected the embryonic development: There was strong developmental retardation or arrest due to the incomplete gastrulation. In the affected embryos, expression of zebrafish T gene was reduced and the hypoblast formation was disturbed. Temporal and spatial expression patterns and the effects of overexpression of these products on development are consistent with the idea that the Pou2 and t-Pou2 proteins are involved in early development of zebrafish embryos. They may be involved in the proliferation of blastomeres in undetermined state at the blastula stage and/or the early cell commitment events at the gastrula stage. Also, our results indicate that different products generated as a result of alternative splicing from the same gene possess distinct functional capacities.
Alternative Splicing↗
The gamma delta T cell population in sheep experimentally infected with bovine leukemia virus.
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Animals↗
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).
Animals↗
Erythropoietin receptor binds to Friend virus gp55 through other membrane components.
Direct interactions of Friend spleen focus-forming virus glycoprotein gp55 with either erythropoietin receptor (EpoR) or interleukin (IL)2 receptor beta chain (IL2R) (but not with IL3 receptor) have been reported to induce factor-independent prolonged proliferation of erythroid or lymphoid cells. In order to clarify the molecular mechanism by which EpoR-gp55 complex transmits an aberrant growth signal in the absence of erythropoietin, various chimeric receptors constituted with IL2R, EpoR or IL3 receptor were constructed. It was found that coexpression of gp55 and the chimeric receptors containing the cytoplasmic domains of EpoR and the extracellular domains of IL3 (or IL2) receptor in IL3-dependent Ba/F3 cells results in factor-independent growth. Since gp55 in cell membrane has only a two amino acid tail in the cytoplasmic domains and thus cannot interact with EpoR in cytoplasm, our data suggest that gp55 does not bind EpoR directly but interacts with EpoR through third membrane component(s).
Animals↗
Tyrosine kinase activation through the extracellular domains of cytokine receptors.
Interaction of cytokines with their membrane receptors induces the proliferation and differentiation of a specific lineage of haematopoietic progenitors. The molecular mechanism of cytokine receptor-mediated signal transduction is unclear because these receptors do not have tyrosine kinase activity. Interleukin-3 and erythropoietin, however, induce transient tyrosine phosphorylation of a common set of proteins as a growth signal, and interleukin-2 induces phosphorylation of an overlapping but distinct set of proteins. Here we show that chimaeric receptors consisting of the extracellular domains of the erythropoietin receptor and the cytoplasmic domains of the interleukin-2 (or interleukin-3) receptor induce an erythropoietin-dependent tyrosine phosphorylation in interleukin-3-dependent Ba/F3 cells; however, chimaeric receptors composed of the extracellular domains of the interleukin-2 receptor and the cytoplasmic domains of the erythropoietin (or interleukin-3) receptor apparently transmit an interleukin-2-dependent signal. Our results indicate that these cytokines transmit distinct signals for activation of specific tyrosine kinases through the extracellular rather than cytoplasmic domains of the receptors.
Amino Acid Sequence↗
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.
Animals↗
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.
Animals↗
A POU-domain gene of zebrafish, ZFPOU1, specifically expressed in the developing neural tissues.
We have isolated a POU domain-containing cDNA (ZFPOU1) from a cDNA library of zebrafish (Brachydanio rerio). The ZFPOU1 cDNA contained an open reading frame encoding a 425 amino acid peptide. The conserved POU domain was located near the carboxy terminus. The deduced amino acid sequence of the reading frame was most similar to that of the mouse class III POU-domain gene, Brain-1. Northern blot analysis revealed that the ZFPOU1 transcripts first appeared at the early neurula stage of embryogenesis and transiently increased thereafter. A significant level of expression, however, was not found in adult tissues except in the brain. In situ hybridization analysis demonstrated that the ZFPOU1 transcripts were localized in the neural tissues of embryos, but not in mesodermal, endodermal or ectodermal tissues. In adult zebrafish, the ZFPOU1 transcripts were detected in the restricted regions of the brain. Spatial and temporal expression patterns suggest that ZFPOU1 has distinct roles in the early neural development of zebrafish.
Amino Acid Sequence↗
Functionally essential cytoplasmic domain of the erythropoietin receptor.
The cytoplasmic domains of the erythropoietin receptor essential for signal transduction were identified by assessing a series of truncated and deletional mutant receptors. A 91-amino acid region proximal to the transmembrane domain was required for growth signaling. In this region, residues between 353Pro and 362His and between 278Gln and 308Leu appeared to constitute the essential cytoplasmic domains. These two domains contain the conserved amino acids common in the cytokine receptor superfamily, which indicates that these domains in the cytoplasmic regions of the erythropoietin receptor may be important for interaction with common signal transducers or protein tyrosine kinases.
Amino Acid Sequence↗
Tryptophan residue of Trp-Ser-X-Trp-Ser motif in extracellular domains of erythropoietin receptor is essential for signal transduction.
The Trp-Ser-X-Trp-Ser motif commonly exists just outside the transmembrane domains of all cytokine receptors so far isolated. The role of this conserved motif in erythropoietin receptor was examined by assessing a series of mutant receptors on erythropoietin-induced signal transduction. Replacement of one of the two conserved Trp residues in the motif to Gly was found to completely abolish the binding of erythropoietin to the receptor and also to lose the ability to transduce the factor-dependent growth signal. While the mutants with one Ser residue converted to Gly or Ala retained full biological activities, the replacement of both conserved Ser residues diminished the functions of the receptor. Furthermore, the receptors lacking a part or all of the Trp-Ser-X-Trp-Ser motif did not respond to erythropoietin. The Trp-Ser-X-Trp-Ser motif, especially Trp residue, located in extracellular domains of the erythropoietin receptor thus appears to play a critical role in receptor-mediated signal transduction.
Amino Acid Sequence↗
Expression of c-kit, a proto-oncogene of the murine W locus, in cerebella of normal and neurological mutant mice: immunohistochemical and in situ hybridization analysis.
The c-kit proto-oncogene encodes a receptor tyrosine kinase and is allelic with the murine white-spoting (W) locus. Although no apparent defects in the brain have been reported in W mutant mice, brain tissue, especially cerebellum, shows a high level of c-kit transcription. In the present study, sites of c-kit expression in the cerebellum were exained by immunohistochemical and in situ hybridization techniques. Immunohistochemistry with a monoclonal antibody against c-Kit protein revealed that the c-Kit protein was localized close to the Purkinje cell soma in the region facing the granular cell layer. Similar distribution of the c-Kit protein was observed in cerebella of mutant mice in which the Purkinje cell (pcd) or the granular cell layer (weaver) is missing. These data suggest that the c-Kit protein is produced not by the Purkinje cell nor by the granular cell but by the cells present in the molecular layer and that the protein is then transported to the region around the Purkinje cell soma. This interpretation was supported by in situ hybridization analysis: cells containing the c-kit transcripts were found only in the molecular layer, while the granular and Purkinje cells were negative.
Alleles↗
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.
Animals↗
Growth signal erythropoietin activates the same tyrosine kinases as interleukin 3, but activates only one tyrosine kinase as differentiation signal.
By Western blotting with anti-phosphotyrosine-specific antibody, we demonstrated that both erythropoietin (Epo) and interleukin 3 (IL3) induce rapid and transient tyrosine phosphorylation of a common set of proteins of 45, 55, 69, 87, 90, 95 and 160 KDa as a growth signal in Epo- and IL3-dependent FD-M6 cells. In contrast, only two proteins of 87 and 90 KDa were transiently phosphorylated in Epo-induced erythroid differentiation of SKT6 cells. Furthermore, no tyrosine phosphorylation was observed in dimethyl sulfoxide-induced differentiation of SKT6 cells. Taken together with other observations, these results indicate that Epo, IL3 and GM-CSF activate the same tyrosine protein kinases as growth signal and that Epo-induced differentiation signal uses only a part of the tyrosine kinase pathway.
Animals↗
Conversion of Friend mink cell focus-forming virus to Friend spleen focus-forming virus by modification of the 3' half of the env gene.
The 3' half of the env gene of the dualtropic Friend mink cell focus-forming virus was modified by replacing the restriction enzyme fragment of the genome DNA with the corresponding fragment of the acutely leukemogenic, polycythemia-inducing strain of Friend spleen focus-forming virus (F-SFFVP) genome DNA. Replacement with the fragment of F-SFFVP env containing the 585-bp deletion, the 6-bp duplication, and the single-base insertion converted the resulting chimeric genome so that the mutant had a pathogenic activity like that of F-SFFVP. Replacement with the fragment containing only the 585-bp deletion did not result in a pathogenic virus. However, when this virus pseudotyped by Friend murine leukemia virus was passaged in newborn DBA/2 mice, we could recover weakly pathogenic viruses with a high frequency. Molecular analysis of the genome of the recovered virus revealed the presence of a single-base insertion in the same T5 stretch where the wild-type F-SFFV env has the single-base insertion. These results provided evidence that the unique genomic structures present in the 3' half of F-SFFV env are the sole determinants that distinguish the pathogenicity of F-SFFV from that of Friend mink cell focus-forming virus. The importance of the dualtropic env-specific sequence present in the 5' half of F-SFFV env for the pathogenic activity was evaluated by constructing a mutant F-SFFV genome in which this sequence was replaced by the ecotropic env sequence of Friend murine leukemia virus and by examining its pathogenicity. The results indicated that the dualtropic env-specific sequence was essential to pathogenic activity.
Animals↗
Oligomerization of Friend spleen focus-forming virus (SFFV) env glycoproteins.
The glycoprotein gp55 and its processed form gp65, which are encoded by the env gene of Friend spleen focus-forming virus (SFFV), have been implicated in the initiation of the murine acute erythroleukemia induced by Friend virus (FV). Analyses of these glycoproteins by chemical crosslinking and nonreducing/reducing two-dimensional electrophoresis showed that both gp55 and gp65 exist as monomer and disulfide-bonded dimer and trimer. These oligomers could be detected in various FV-infected erythroleukemia cell lines, as well as in the spleen cells of FV-induced erythroleukemic mice, suggesting that oligomerization is an intrinsic feature of SFFV env glycoproteins.
Animals↗
A deletion in the Friend spleen focus-forming virus env gene is necessary for its product (gp55) to be leukemogenic.
To determine the biological significance of the 585-base-pair deletion in the env gene of Friend spleen focus-forming virus (SFFV) encoding a leukemogenic glycoprotein (gp55), we examined the pathogenicity of a constructed mutant SFFV (SFFVDF). In the SFFVDF genome, the env deletion was filled in with the corresponding env sequence of Friend mink cell focus-forming virus, whereas the 6-base-pair duplication and the single base insertion near the 3' terminus of SFFV env remained intact. SFFVDF was nonpathogenic in adult mice. During passage of SFFVDF through newborn mice, we recovered various pathogenic variant SFFVs. Molecular analyses of variant SFFV genome DNAs revealed the presence of a distinct deletion in each env gene, which was similar but not identical to that in the wild-type SFFV env. Starting with the SFFVDF genome DNA, other mutant SFFV genome DNAs were constructed in which the sequence coding for the gp70/p15E proteolytic cleavage site present in the SFFVDF genome was modified by oligonucleotide-directed site-specific mutagenesis to prevent the cleavage. These mutant SFFVs were also nonpathogenic. These results indicate that for the pathogenic activity of gp55, a certain env deletion is necessary which causes production of a gp70-p15E fusion protein with an absence of at least the N-terminal one-third of the p15E-coding region.
Animals↗