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Biomedical subjects

K Ikuta

Publications and source records attributed to K Ikuta.

At least 325 records · Page 18Linked to original sources
PubMed1987

Establishment and characterization of a T-lymphoblastoid cell line MDCC-MTB1 derived from chick lymphocytes infected in vitro with Marek's disease virus serotype 1.

Continuously proliferating T-lymphoblastoid cells, named MDCC-MTB1, were obtained by infection of chick embryo lymphocytes with Marek's disease virus serotype I (MDV1) in culture and subsequent cultivation in the presence of human interleukin 2 (IL-2). The MTB1 cells have now been growing well for at least 4 months with a doubling time of about 10 hr, irrespective of the presence of IL-2. The MTB1 cells show lymphoblastoid morphology, and carry T-lymphocyte marker surface antigens and a karyotype of female chick origin with several abnormal chromosomes. Southern blot hybridization showed that they contain about 10 virus genome equivalents/cell of almost the whole MDV genome. Infectious virus could not be rescued from MTB1 cells by co-cultivation of these with chick embryo fibroblasts. In addition, no virus particles were found in thin-sectioned MTB1 cells by electron microscopy. An immunofluorescence test with monoclonal antibody (MAb) to MDV-specific phosphorylated proteins showed that MTB1 cells expressed the MDV-specific antigen in the cytoplasm only after the cells had been treated with 5-iodo-2-deoxyuridine for 48 hr at 41 degrees C. MTB1 cells can form colonies in 0.33% soft agar, and can be transplanted into chicks by i.p. injection. Thus, continuously growing lymphoblastoid cells were obtained by in vitro infection of chick embryo lymphocytes with oncogenic MDV1 and cultivation of the cells in the presence of human IL-2 during the transformation step. These cells appear to show a similar phenotype to an MD lymphoma-derived cell line.

Animals↗
PubMed1987

Effects of sex steroids on the smoltification of masu salmon, Oncorhynchus masou.

Testosterone (T; 25-micrograms/g diet), estradiol-17 beta (E2; 2.5-, 25-micrograms/g diet), and 5 alpha-dihydrotestosterone (DHT; 2.5-, 25-micrograms/g diet) were administered to yearling masu salmon from February 3 through May 8. T and E2 inhibited the natural smoltification in spring and stimulated gonadotropin (GtH) accumulation in the pituitary, but DHT had no effect. T (25-micrograms/g diet) was administered to underyearling masu salmon from September 30 through December 14. At the end of the T treatment, fish body color darkened and pituitary GtH content increased in the T-treated group. On May 8 in the next spring, however, plasma T levels and pituitary GtH contents already decreased and smoltification occurred in both control and T-treated groups. On October 29, underyearling masu salmon reared under the short daylength (8 L:16 D) for 4 months were switched to a long daylength (16 L:8 D) and simultaneously administered with T (25-micrograms/g diet) or 11-ketotestosterone (11-KT; 25-micrograms/g diet). On December 17, the proportion of smolts was 52.8% in the control, whereas T and 11-KT administration inhibited smoltification under the artificially increased daylength. Pituitary GtH content significantly increased both in the T-treated and 11-KT-treated groups, though it was much lower in the latter. These results indicate that the springtime smoltification which is induced by increasing daylength is inhibited by biologically active sex steroids such as E2, T, and 11-KT.

Animals↗
PubMed1987

Human lymphocyte Fc receptor for IgE: sequence homology of its cloned cDNA with animal lectins.

We have purified the human lymphocyte Fc receptor specific for IgE (Fc epsilon receptor) and its soluble form by using the anti-Fc epsilon receptor monoclonal antibody H107. Using an oligonucleotide probe corresponding to the partial amino acid sequence of the soluble Fc epsilon receptor related to IgE binding factor, we cloned, sequenced, and expressed a cDNA for the receptor. The Fc epsilon receptor has 321 amino acid residues with no NH2-terminal signal sequence. The receptor was separated into two domains by a putative 24-amino acid residue transmembrane region located near the NH2-terminal end. The Fc epsilon receptor showed a marked homology with animal lectins including human and rat asialoglycoprotein receptors, chicken hepatic lectin, and rat mannose binding proteins.

Amino Acid Sequence↗
PubMed1987

Monoclonal antibodies to cowpox virus: polypeptide analysis of several major antigens.

Monoclonal antibodies directed against major antigens induced by cowpox virus (CPV) were produced. The specificities of these antibodies were established by immunoprecipitation, immunoblotting and several serological analyses, and from the cross-reactivities of these antibodies with cells infected with various other poxviruses, ectromelia virus (EV), vaccinia virus and Shope fibroma virus. The antibodies defined included ones reacting with each of the known major antigens of poxviruses, i.e. the common antigen of all poxviruses (probably NP antigen), the Orthopoxvirus-specific antigen (probably LS antigen), the haemagglutinin, the cell surface antigen, the common A-type inclusions in CPV and EV, and the antigen involved in neutralization.

Animals↗
PubMed1987

Differences in the pI heterogeneity of virion and intracellular Moloney murine leukaemia virus p30s.

At least three different p30 forms which vary in isoelectric point (pI) were previously shown by two-dimensional (2D) gel electrophoresis to be present in purified virions obtained from several strains of murine leukaemia virus (MLV). This heterogeneity which had been identified by Coomassie Brilliant Blue staining has been further characterized by immunological techniques. Using as substrates two Moloney (M) MLV chronically infected cell lines (MJD-54 and clone 2 cells), we found that (i) all p30s had the antigenicity of M-MLV p30, when analysed by immunoblotting of virion proteins with anti-p30 sera, and (ii) when cells were labelled with [35S]methionine, a 14C-amino acid mixture, or [14C]serine and lysates of purified virions were immunoprecipitated with goat anti-p30 sera, four p30 spots (pI 6.0, 6.1, 6.3 and 6.6) could be clearly identified. These results strongly support the viral origin of the heterogeneous p30 spots. We next examined infected cell lysates in an attempt to pinpoint the molecular basis of this heterogeneity. When we immunoprecipitated p30s from labelled cell lysates utilizing goat anti-p30 sera it was observed that in contrast to the four virion p30s, there were only three intracellular p30s (pI 6.0, 6.3 and 6.6), there was a threefold greater amount of the intracellular compared to the virion form of p30 with pI 6.0, tryptic peptide maps of both virion and intracellular p30s labelled either with [35S]methionine or 125I showed basically similar patterns with only slight differences in intensity among certain peptides for the p30s with pI 6.1, 6.3 and 6.6, and the intracellular p30 with pI 6.0 had a peptide that was not present in any of the other p30s. These results suggest that due to some as yet uncharacterized modification(s) of p30 and/or some structural differences between different p30s, a heterogeneity in pI exists. This may be important for assembly of the virion capsid. However, it is also possible that the p30 heterogeneity reflects the presence of multiple M-MLV proviruses within each of the infected cell clones.

Antibodies, Viral↗
PubMed1987

Analysis of Marek's disease virus serotype 1-specific phosphorylated polypeptides in virus-infected cells and Marek's disease lymphoblastoid cells.

By use of monoclonal antibodies, a virus-specific cytoplasmic antigen related to phosphorylated polypeptides specific to serotype 1 of Marek's disease virus (MDV)-related viruses (MDV1) has been identified in all MD tumour cell lines examined, as well as in infected cells and in tumour lesions of chickens with MD. At least two phosphorylated polypeptides with mol. wt. 39,000 (39K) to 36K and 24K (pp39/36 and pp24, respectively) were identified in the MD tumour cell line H10 cultured at 33 degrees C by immunoprecipitation with monoclonal antibody M21 which reacts with virus-specific phosphorylated polypeptides. These polypeptides were not detected in cells infected with MDV-related viruses of serotype 2 or 3. Immunoblot analysis indicated that these two polypeptides contained a serotype 1-specific epitope recognized with M21. An additional 41K polypeptide appeared in different virus strains of serotype 1. These polypeptides were found to contain phosphorylated serine but no detectable phosphorylated tyrosine or phosphorylated threonine. Cell fractionation indicated that the two phosphorylated polypeptides were mainly associated with smooth and rough endoplasmic reticulum fractions of cells infected with MDV1. Furthermore, the mRNA coding for pp39/36 could be separated from that coding for pp24 on a sucrose density gradient. These results suggest that pp24 and pp39/36 are translated from distinct mRNAs and encoded from overlapping genes or separate regions with partial DNA homology in the MDV1 genome.

Animals↗
PubMed1987

Methylation of Marek's disease virus DNA in chicken T-lymphoblastoid cell lines.

Methylation of Marek's disease virus serotype 1 (MDV1) DNA in both productively and latently infected cells was examined by restriction endonuclease analyses with the isoschizomeric pair HpaII and MspI. The latent MDV1 DNA in T-lymphoblastoid cell lines established from chicken T-lymphomas was considerably methylated, whereas methylation of the virus DNA sequences was not detected in productively infected cells.

5-Methylcytosine↗
PubMed1987

Amplification of human immuno-deficiency virus production from a virus-producing cell line in culture at high cell density.

The amount of infectious virus released into the culture fluid from human immunodeficiency virus (HIV)-producing MOLT-4 cells (MOLT-4/HIV HTLV-III) was measured as a function of the culture conditions and cell density. After 3 days, the daily yield of infectious virus per cell in the fluid of non-growing cells cultured at high cell density was about 15 times higher than the accumulated yield from growing cells cultured at low cell density. SDS-PAGE analysis also showed a similar difference in the amounts of major gag protein p24.

Cell Line↗
PubMed1987

Suppression by neurotropin of production of human immuno-deficiency virus by the virus-producing MOLT-4 cell line.

The effect of neurotropin on human immuno-deficiency virus (HIV) production in a virus-producing MOLT-4 (MOLT-4/HTLV-III) cell line was studied. Culture of the cells for 4 days in the presence of 100 micrograms/ml of neurotropin resulted in a 72% decrease in the number of infectious virus particles released into the culture fluid. A pulse-labeling experiment to analyze the synthesis of HIV proteins suggested that neurotropin may affect the budding process of the virus.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Selective killing of human immunodeficiency virus-infected and -producing cells by liposomes containing diphtheria toxin fragment A.

Liposomes containing fragment A of diphtheria toxin killed human immunodeficiency virus (HIV)-producing MOLT-4 or TALL-1 cells, and HTLV-I-carrying MT-4 cells infected with HIV, but did not kill these cells when they were not infected with HIV. This killing was not affected by the presence of HIV antibodies. These results show that liposomes containing fragment A of diphtheria toxin selectively killed cells expressing HIV antigens or producing the virus in vitro.

Acquired Immunodeficiency Syndrome↗
PubMed1986

Inhibition of cleavage of Moloney murine leukemia virus gag and env coded precursor polyproteins by cerulenin.

Cerulenin, an inhibitor of de novo fatty acid (and cholesterol) biosynthesis, has been shown to significantly decrease (greater than 75%) the amount of Moloney murine leukemia virus (MMuLV) released into the culture medium of chronically infected mouse fibroblasts (I. Katoh, Y. Yoshinaka, and R.B. Luftig, 1986, Virus Res., in press). In order to clarify the mechanism by which this decrease in virus production occurs, we analyzed the kinetics of gag and env coded protein synthesis in M-MuLV infected, cerulenin-treated cells by immunoprecipitation with monospecific antisera to p30, p12, p10, gp70, and p15(E). We found that in pulse (15 min-2 hr)-chase (0-4 hr) experiments the cleavage of not only Pr65gag to p30 and other gag coded proteins but Pr80env to gp70 and Pr15(E) as well, was greatly reduced by cerulenin treatment. Further, since the total amount of label in the Pr65gag and Pr80env bands remained about the same or was slightly decreased in 2-hr pulsed, cerulenin-treated cells, this suggests that cerulenin decreases virus production, in part, by inhibiting the cleavage of both precursor gag and env coded polyproteins during virus assembly and budding at the cell membrane. We also observed that at longer chase periods (4 hr), the effect of cerulenin could be partially overriden in that minor amounts of cleaved gag and env coded polyproteins were produced and assembled into virion particles. However, these particles contained abnormally large amounts of the uncleaved precursor Pr65gag, suggesting that maturation was incomplete. The above results suggest two independent, but not exclusive, possible mechanisms of cerulenin action to block M-MuLV production, viz. cerulenin decreases the pool of fatty acids, thereby inhibiting fatty acid acylation of Pr65gag, as well as Pr80env, and thus preventing the interaction between gag (the p15 antigenic determinant on Pr65gag) and env [the p15(E) antigenic determinant of Pr15(E)] coded gene products at the cell membrane needed for efficient virus assembly (M. Satake and R. B. Luftig, 1983, Virology 124, 259-273), and cerulenin inhibits one or more proteolytic enzymes responsible for the cleavage of Pr65gag and Pr80env.

Antifungal Agents↗
PubMed1986

The effect of cerulenin on the synthesis of the precursor gag polyprotein in defective murine leukemia and sarcoma virus producing cell lines.

The effect of cerulenin, an inhibitor of de novo fatty acid (and cholesterol) biosynthesis, on the synthesis of the precursor gag polyprotein, Pr65gag in a defective murine leukemia virus (334C) producing murine cell line (3JE) and a defective murine sarcoma virus (Gazdar) producing hamster cell line (HTG-2) was examined. In contrast to Moloney murine leukemia virus (M-MuLV) producing cell lines (MJD-54, clone 2) the amount of the Pr65gag remaining in the presence of cerulenin (20 micrograms/ml) was greatly reduced in both defective virus-infected cells. This effect appears specific for the Pr65gag polyprotein, since the env precursor polyprotein Pr80env was normally synthesized and remained undegraded in cerulenin-treated 3JE-infected cells. Thin-section electron micrographs showed an increased accumulation of virion particles in vesicles of treated HTG-2 cells.

Antifungal Agents↗
PubMed1986

Expression and rearrangement of the alpha, beta, and gamma chain genes of the T cell receptor in cloned murine large granular lymphocyte lines. No correlation with the cytotoxic spectrum.

Using cloned murine large granular lymphocyte (LGL) lines, the expression and the rearrangement of the alpha, beta, and gamma chain genes of the T cell receptor (TCR) were analyzed. Morphological, phenotypical, as well as functional studies indicated that the LGL lines were identical to normal, endogenous NK cells. Northern blot hybridization analysis indicated that the full-length transcripts of all the alpha, beta, and gamma chain genes were expressed in most of the LGL lines, including two lines derived from athymic nude mice. In one line, SPB, however, no transcript of the gamma chain gene was detected, whereas the alpha and beta chain genes were clearly expressed. In every LGL line studied, all of the alpha, beta, and gamma chain genes were rearranged. Conforming to the results of Northern blot hybridization study, the gamma chain gene of the SPB line was aberrantly rearranged, whereas those of all the other lines were productively rearranged. The results clearly revealed that NK cells represented a population of lymphocytes genetically committed to the T cell lineage. It was also suggested that the expression and rearrangement of the TCR genes of NK cells might occur in a thymus-independent fashion. An SPB line without expression of the gamma chain gene could exhibit NK activity indistinguishable from other NK lines. Furthermore, the rearrangement patterns of the beta chain gene did not correlate with the specificity of the cytotoxic activity. These results strongly suggested that the cytotoxic activity in NK cells was not directly mediated by TCR on them. We particularly noted that the beta chain gene of most independently established LGL lines showed identical patterns of rearrangement, indicating that they used the same V beta and J beta gene segments. The significance of the restricted pattern of rearrangement of the beta chain gene in LGL lines, as well as the possible functional roles of TCR on NK cells, was discussed.

Animals↗
PubMed1986

Chromosomal sites for Marek's disease virus DNA in two chicken lymphoblastoid cell lines MDCC-MSB1 and MDCC-RP1.

In situ hybridization of viral DNA fragments to metaphase chromosomes of two chicken T-lymphoblastoid cell lines (MDCC) demonstrated that Marek's disease virus DNA was located in the short arm of chromosome 2 of MDCC-MSB1 and MDCC-RP1 cells and in the short arm of chromosome 4 of MDCC-MSB1 cells. The MDV DNA sequences in these chromosomes are not in the vicinity of oncogenes or integrated retroviruses that have been previously identified in chicken chromosomes.

Animals↗