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Biomedical subjects

J Miyazaki

Publications and source records attributed to J Miyazaki.

At least 199 records · Page 11Linked to original sources
PubMed1991

Efficient selection for high-expression transfectants with a novel eukaryotic vector.

We have developed a new expression vector which allows efficient selection for transfectants that express foreign genes at high levels. The vector is composed of a ubiquitously strong promoter based on the beta-actin promoter, a 69% subregion of the bovine papilloma virus genome, and a mutant neomycin phosphotransferase II-encoding gene driven by a weak promoter, which confers only marginal resistance to G418. Thus, high concentrations of G418 (approx. 800 micrograms/ml) effectively select for transfectants containing a high vector copy number (greater than 300). We tested this system by producing human interleukin-2 (IL-2) in L cells and Chinese hamster ovary (CHO) cells, and the results showed that high concentrations of G418 efficiently yielded L cell and CHO cell transfectants stably producing IL-2 at levels comparable with those previously attained using gene amplification. The vector sequences were found to have integrated into the host chromosome, and were stably maintained in the transfectants for several months.

Actins↗
PubMed1991

Specific depletion of the B-cell population induced by aberrant expression of human interferon regulatory factor 1 gene in transgenic mice.

Interferons (IFNs) are well known both as antiviral proteins and as potent regulators of cell growth and differentiation. In fact, IFNs inhibit growth of various normal and transformed cell types. Previously, a nuclear factor, IRF-1 (interferon regulatory factor 1), which binds to type I IFN and some IFN-inducible gene promoters, was identified and cloned. Since the IRF-1 gene is both virus and IFN inducible, an intriguing issue is raised as to whether the IRF-1 gene is functioning in IFN-mediated regulation of cell growth and differentiation. In this study, we generated transgenic mice carrying the human IRF-1 gene linked to the human immunoglobulin heavy-chain enhancer. In the transgenic mice, all the lymphoid tissues examined showed a dramatic reduction in the number of B lymphocytes (B cells). Preparation and analysis of bone marrow cells from the chimeric mice indicated that the bone marrow is the effective site for specific depletion of the B-cell population. In fact, transgenic bone marrow cells cocultured with a bone marrow-derived stromal cell line revealed an altered B-cell maturation pattern.

Animals↗
PubMed1991

Differentiation of growth signal requirement of B lymphocyte precursor is directed by expression of immunoglobulin.

During B cell differentiation, at least three stages can be defined in terms of their growth signal requirement by using two different growth signals, which are recombinant interleukin 7 (IL-7) and a stromal cell clone PA6 which does not produce IL-7; first a PA6 dependent stage, second a PA6 + IL-7 dependent stage and third an IL-7 dependent stage. In order to test the possibility that this differentiation of growth signal requirement is controlled by the expression of functional immunoglobulin molecules, we have investigated the frequencies of PA6 + IL-7 dependent and IL-7 dependent cells which are present in the bone marrow of either mu-chain or kappa-chain gene transgenic mice. In a mu-chain gene transgenic mouse, the frequency of PA6 + IL-7 dependent cells is selectively reduced, while that of IL-7 dependent cells is selectively reduced in a kappa-chain gene transgenic mouse. This result suggests that expression of a functional mu-chain gene drives PA6 + IL-7 dependent cells to differentiate into the subsequent IL-7 dependent stage. Likewise, when mu-chain positive IL-7 dependent cells express a functional light-chain gene, their growth signal requirement changes into an IL-7 unreactive stage.

Animals↗
PubMed1991

Characterization and sequence of a novel nitrate reductase from barley.

Barley (Hordeum vulgare L.) has both NADH-specific and NAD(P)H-bispecific nitrate reductases. Genomic and cDNA clones of the NADH nitrate reductase have been sequenced. In this study, a genomic clone (pMJ4.1) of a second type of nitrate reductase was isolated from barley by homology to a partial-length NADH nitrate reductase cDNA and the sequence determined. The open reading frame encodes a polypeptide of 891 amino acids and its interrupted by two small introns. The deduced amino acid sequence has 70% identity to the barley NADH-specific nitrate reductase. The non-coding regions of the pMJ4.1 gene have low homology (ca. 40%) to the corresponding regions of the NADH nitrate reductase gene. Expression of the pMJ4.1 nitrate reductase gene is induced by nitrate in root tissues which corresponds to the induction of NAD(P)H nitrate reductase activity. The pMJ4.1 nitrate reductase gene is sufficiently different from all previously reported higher plant nitrate reductase genes to suggest that it encodes the barley NAD(P)H-bispecific nitrate reductase.

Amino Acid Sequence↗
PubMed1991

Suppression of murine leukemia virus-mediated 3Y1 cell fusion by expression of mouse MHC class I.

Rat 3Y1 fibroblasts transformed by adenovirus type 12 or its E1A gene formed syncytia by cocultivation with Friend murine leukemia virus (MuLV)-producing cells. On the other hand, parental 3Y1 cells and those derivatives induced by other tumor viruses or chemical carcinogen showed no MuLV-mediated syncytium formation [N. Momozaki et al. (1990) Arch. Virol. 115: 123-126]. The expression of major histocompatibility complex (MHC) class I mRNA and antigens was significantly reduced in these Ad12- and E1A-transformed 3Y1 cells. In contrast, other tumor virus-and chemical carcinogen-transformed 3Y1 cells expressed MHC class I almost in normal levels as did parental 3Y1 cells. Furthermore, Ad12-transformed 3Y1 cells which started to express the transfected exogenous MHC class I gene, H-2Ld, showed no more MuLV-mediated 3Y1 cell fusion. These results indicate that the expression of MHC class I on the cell membrane is closely related to the inhibition of 3Y1 cell fusion by MuLV.

Actins↗
PubMed1991

Development of two types of hepatocellular carcinoma in transgenic mice carrying the SV40 large T-antigen gene.

We produced transgenic mice by introducing a fusion gene (ST) comprising of the promoter for human serum amyloid P component (SAP) and the coding region of the simian virus 40 (SV40) large tumor antigen (Tag). The ST transgenic mice developed hepatocellular carcinomas (HCC) between 7 and 12 weeks of age. Clinically and pathologically these HCC were classified into two types: diffuse and focal. In the diffuse type Tag is expressed in almost all hepatocytes and HCC has presumably developed from these T-antigen-positive cells. The focal type, which resembles human HCC, is only Tag positive in the neoplastic nodules. These are surrounded by Tag-negative normal hepatocytes that form normal lobular structures. As the human SAP promoter can direct adult liver-specific expression of heterologous genes, it is assumed that Tag gene expression in the transgenic animals is increased sharply after birth. Interestingly, a similar level of Tag expression was observed in both the cytoplasm and the nucleus. Our results suggest that spatial differences in Tag expression result in the generation of two types of HCC in transgenic mice and that the ST mouse can serve as a model for human hepatocarcinogenesis.

Alanine Transaminase↗
PubMed1991

(BALB/c x C57BL/6)F1 mice are tolerant to undetectable mixed haplotype A beta dA alpha b and mixed isotype A beta dE alpha d self class II molecules.

The existence of mixed haplotype A beta dA alpha b and mixed isotype A beta dE alpha d molecules was demonstrated in A beta d gene introduced C57BL/6 (B6) transgenic and in A beta dE alpha d double gene introduced B6 transgenic mice, respectively. Using alloreactive and antigen reactive T-cell clones, these mixed class II molecules were shown to function as mixed lymphocyte culture reaction (MLR) stimulating determinants and restriction elements for antigen recognition by T cells. The amounts of expression of these mixed class II molecules were sufficient for the stimulation of alloreactive T cells in primary MLR culture. (BALB/c x B6)F1 (CBF1) mice were shown not to express functional mixed haplotype and mixed isotype class II molecules using alloreactive and antigen reactive T-cell clones. Interestingly, however, CBF1 mice did not respond to such mixed haplotype and mixed isotype class II molecules. These results show that CBF1 mice do not respond to undetectably-expressed class II molecule and suggest that they are tolerant to undetectable self class II molecules. The possible interpretations for this are discussed.

Animals↗
PubMed1991

Identification and distribution of tropomyosin isoforms in chicken digestive canal.

Two-dimensional gel electrophoresis of eight digestive organ extracts from 60-day-old chicks was performed. Judging from the similarity of their protein maps, the organs were classified into the following four types: 1) esophagus type, 2) proventriculus type, 3) gizzard type, and 4) intestine type. In four representative organs of these types, the distribution of tropomyosin isoforms was examined, and four high- and five low-Mr-type isoforms in addition to alpha and beta isoforms were detected in the embryonic organs. In the adult organs, however, there were three high- and four low-Mr-type isoforms, which were restricted to the mucous membrane, in addition to alpha and beta isoforms. Immunoblot analysis indicated that the high- and low-Mr-type isoforms in the embryo corresponded with those in the adult mucous membrane, but differences in the number and amount of the isoforms were found between the embryo and the adult mucous membrane.

Animals↗
PubMed1991

Production and effect of infectious Dane particles in transgenic mice.

We have demonstrated by immunoelectron microscopy that 42-nm particles with double-shelled structures characteristic of Dane particles are present in the serum of transgenic mice, 1.2HB-BS 10, carrying partly duplicated hepatitis B virus (HBV) genome. Furthermore, these particles were shown to infect primary human fetal hepatocytes as demonstrated by the elevation of HBV surface antigen (HBsAg) in the culture medium. HBV DNA is known to be expressed in a liver- and kidney-specific manner in the adult mouse, so we examined the developmental expression of viral antigens. In the liver, viral antigens (HBsAg and HBV e antigen) began to be expressed before birth and the level of expression showed a sharp rise after birth. On the other hand, in the kidney, viral antigens began to be expressed after birth. Serum levels of viral antigens were roughly proportional to the levels of expression in the liver, suggesting that the liver is the main source for viral antigens in the serum. None of these transgenic mice produced anti-HBs or anti-HBV core response or showed biochemical or pathological change up to at least 24 months of age. All these results suggest that infectious viral particles can be produced in transgenic mice, and that expression and replication of HBV DNA are not toxic in vivo.

Aging↗
PubMed1990

Tissue-specific expression of rat pyruvate kinase L/chloramphenicol acetyltransferase fusion gene in transgenic mice and its regulation by diet and insulin.

We produced transgenic mice carrying about 3 kb of the 5'-flanking sequence of the rat pyruvate kinase L gene linked to the chloramphenicol acetyltransferase (CAT) structural gene. Expression of the transgene was observed only in tissues in which the endogenous L-type pyruvate kinase is expressed. Dietary glucose or insulin induced similar increases in the levels of CAT and L-type pyruvate kinase mRNAs in the liver. However, the fructose-induced level of CAT mRNA was about 3- and 6- fold lower than those of endogenous L-type pyruvate kinase mRNA in the liver and kidney, respectively, confirming our previous finding that stabilization of the transcripts of the pyruvate kinase L gene is an important regulatory step in fructose induction, especially in the kidney. Thus we conclude that all the cis-acting elements responsible for tissue-specific expression of the L-type pyruvate kinase and its stimulation by dietary components and insulin are localized in the sequence from about nucleotide -3000 to +37 in the pyruvate kinase L gene.

Animals↗
PubMed1990

Direct evidence for the contribution of the unique I-ANOD to the development of insulitis in non-obese diabetic mice.

Insulin-dependent diabetes mellitus is characterized by the infiltration of lymphocytes into the islets of Langerhans of the pancreas (insulitis) followed by destruction of insulin-secreting beta-cells leading to overt diabetes. The best model for the disease is the non-obese diabetic (NOD) mouse. Two unusual features of the class II major histocompatibility complex (MHC) of the NOD mouse are the absence of I-E and the presence of unique I-A molecules (I-ANOD), in which aspartic acid at position 57 of the beta-chain is replaced by serine. This feature is also found in the HLA-DQ chain of many Caucasians with insulin-dependent diabetes mellitus. We have previously reported that the expression of I-E prevents the development of insulitis in NOD mouse. Here we report that the expression of I-Ak (A alpha kA beta k) in transgenic NOD mice can also prevent insulitis, and that this protection is seen not only when the I-A beta-chain has aspartic acid as residue 57, but also when this residue is serine. These results show that the single amino-acid substitution at position 57 of the I-A beta-chain from aspartic acid to serine is not sufficient for the development of the disease.

Animals↗
PubMed1990

HBV production in transgenic mice.

We produced transgenic mice by microinjecting a partially duplicated copies of hepatitis B virus (HBV) gene into fertilized eggs of C57BL/6 mice. One mouse was a high producer of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in the serum. All offspring carrying HBV DNA were positive for both antigens in the serum. The HBV DNA was expressed in liver- and kidney-specific manner. The normal process of HBV replication, including the packaging of the pregenome 3.5-kb RNA into a nucleocapsid, the reverse-transcription of the complete minus strand DNA, and the release of Dane particles into the serum before the completion of synthesis of plus strand, occurred in the liver of these transgenic mice. These results suggest that the species specificity of HBV infection is not due to the inability to replicate in nonnatural host but to the lack of receptors or factors needed for virus adsorption and internalization. The founder mouse is now 19 months of age but shows no clinical or pathological change, suggesting that HBV itself is not cytopathic.

Animals↗
PubMed1990

Tissue specificity of tropomyosin from the crayfish, Cambarus clarki.

The molecular heterogeneity and tissue specificity of crustacean tropomyosin were investigated, using muscle and nonmuscle tissues from the crayfish, Cambarus clarki. In muscle, three types of tropomyosin isoforms were found on two-dimensional gel electrophoresis. One of them was specific to cardiac muscle, and the other two were shared by skeletal and visceral muscles. In nonmuscle tissues, four types of isoforms were found on two-dimensional gel electrophoresis and in immunoreplica tests using an antiserum against crayfish skeletal muscle tropomyosin. Two of them were common to the muscle isoforms, but the other two were not detected in muscles. Furthermore, nonmuscle tissues contained several peculiar isoforms, the electrophoretic mobilities of which were considerably higher than those of the other isoforms mentioned above. When tropomyosin was purified from the mid-gut gland, these isoforms with high mobilities were found in the crude tropomyosin preparation. These results showed that the crayfish tropomyosin was heterogeneous and that the isoforms were distributed in a tissue-specific manner, like vertebrate tropomyosin. However, the results did not coincide with those of our previous study on horseshoe crab tropomyosin, which showed molecular heterogeneity but no tissue specificity. In view of the difference in the isoform distributions between the two major groups (Crustacea and Merostomata) of Arthropoda, the significance of the tissue specificity of tropomyosin isoforms was discussed.

Animals↗
PubMed1990

Correlation of tissue-specific methylation with gene inactivity in hepatitis B virus transgenic mice.

We produced transgenic mice using two constructs, HB-GII and 1.2HB-BS, of hepatitis B virus (HBV) DNA. The former has been designed to express mRNAs for HBV surface antigen (HBsAg), and the later to express all mRNAs of HBV. Several lines of the transgenic mice carrying each construct were examined for the tissue-specificity and level of HBV DNA expression, and for the relationship between expression and methylation of the transgenes. Only one out of ten for HB-GII and one out of eight for 1.2HB-BS were high producers of viral antigens. In high producers, transgenes were expressed in the liver and the kidneys. But in low producers, transgenes were usually expressed only in the kidneys. There is a reciprocal relationship between the level of expression and the degree of methylation, that is, the higher the level of expression, the less the degree of methylation. We also observed that the expression of the integrated HBV-DNA was repressed by methylation following its passage through the female germline in one line. Thus, in addition to transacting factors that can control the gene expression positively or negatively, this tissue-specific methylation may also be involved in the regulation of HBV gene expression.

Animals↗
PubMed1990

Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms.

Two cell lines have been established from insulinomas obtained by targeted expression of the simian virus 40 T antigen gene in transgenic mice. These cell lines, designated MIN6 and MIN7, produce insulin and T antigen and have morphological characteristics of pancreatic beta cells. MIN6 cells exhibit glucose-inducible insulin secretion comparable with cultured normal mouse islet cells, whereas MIN7 cells do not. Both cell lines produce liver-type glucose transporter (GT) mRNA at high level. Brain-type GT mRNA is also present at considerable level in MIN7 cells, but is barely detectable in MIN6 cells, suggesting that exclusive expression of the liver-type GT is related to glucose-inducible insulin secretion. MIN6 cells do not express either major histocompatibility (MHC) class I or class II antigens on the cell surface. However, treatment with interferon-gamma induces high levels of MHC class I antigens, and a combination of interferon-gamma and tumor necrosis factor-alpha induces a MHC class II antigen on the cell surface. These results emphasize that the MIN6 cell line retains physiological characteristics of normal beta cells. The MIN6 cell line will be especially useful to analyze the molecular mechanisms by which beta cells regulate insulin secretion in response to extracellular glucose concentrations. We discuss a possible role of GT isoforms in glucose sensing by beta cells.

Animals↗