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Biomedical subjects

T Uchida

Publications and source records attributed to T Uchida.

At least 1,081 records · Page 60Linked to original sources
PubMed1985

Binding properties of diphtheria toxin to cells are altered by mutation in the fragment A domain.

CRM197, CRM176, and CRM228 are products of single or multiple missense mutations in the diphtheria toxin gene. CRM197 differs from wild-type toxin in 1 amino acid residue of the fragment A region, and also CRM176 and CRM228 have amino acid substitution(s) in fragment A. We compared the binding properties of CRM197 to toxin-sensitive Vero cells with those of diphtheria toxin and other CRMs. Nicked CRM197 is about 50 times more effective than intact CRM197 in inhibiting the action of diphtheria toxin on sensitive cells, as shown by inhibition of diphtheria toxin cytotoxicity or inhibition of binding of 125I-diphtheria toxin. The binding of native toxin or other CRMs was not significantly affected by nicking. Moreover, the binding of CRM197 to cells was unaffected by ATP, although ATP clearly inhibits binding of diphtheria toxin, CRM176, and CRM228. Two kinds of hybrid protein were formed using fragment B of CRM197: one with fragment A of diphtheria toxin and one with fragment A of CRM228. ATP inhibited the binding of these hybrid proteins. Furthermore, the affinities of these hybrid proteins for diphtheria toxin-sensitive cells were the same as that of native toxin. Thus, it was concluded that the altered binding properties of CRM197 were due to alteration of fragment A and what the interaction of diphtheria toxin with ATP involves both fragments. The results also suggest that fragment A plays a role in diphtheria toxin-receptor interaction.

Adenosine Triphosphate↗
PubMed1985

Molecular cloning of a full-length cDNA encoding the hemagglutinin-neuraminidase glycoprotein of Sendai virus.

We cloned a full-length complementary DNA for the hemagglutinin-neuraminidase (HN) mRNA of Sendai virus (HVJ) using a synthetic 27-mer as a probe. Nucleotide sequence analysis showed that there is a long open reading frame on the mRNA that encodes a protein of 575 amino acids. The deduced amino acid sequence indicated that only one hydrophobic region sufficiently long to anchor the protein in the membrane and located near the N-terminus (amino acids 35-60). It is suggested that HN protein is oriented with its N-terminus inside the membrane.

Amino Acid Sequence↗
PubMed1985

Microinjection of macromolecules into leukemic cells by cell fusion technique: search for intracellular growth-suppressive factors.

To investigate the intracellular molecular events during leukemic cell proliferation, we have examined the method of ghost-mediated microinjection of macromolecules into leukemic cell line cells (HL-60). Samples were packed into red cell ghosts. Microinjection was performed by the fusion of ghosts and HL-60 cells using the hemagglutinating virus of Japan (HVJ). Fusion rate was about 80-90%, when determined by the injection of FITC-labeled globulins (IgG) or diphtheria toxin fragment A into HL-60 cells. When the nuclear protein extract from normal granulocytes was injected into HL-60 cells, their growth was significantly suppressed. The injection of the nuclear protein extract from HL-60 itself into HL-60 cells did not inhibit their growth. This finding suggests that leukemic cells may be deficient in intracellular regulatory factors which have suppressive activity on cell growth.

Animals↗
PubMed1985

Localization of hepatitis B surface and core antigens in human hepatocellular carcinoma by immunoperoxidase methods. Replication of complete virions of carcinoma cells.

The localization of hepatitis B surface antigen (HBsAg) and core antigen (HBcAg) was investigated by an indirect immunoperoxidase method in formalin-fixed, paraffin-embedded liver specimens obtained from 95 Japanese patients with hepatocellular carcinomas. Routine and immune electron microscopic examinations were done in one case. The correlation between expression of hepatitis B virus antigens in the tissue and serum hepatitis B e antigen (HBeAg)/antibody to HBeAg (anti-HBe) status was examined. Hepatitis B surface antigen was detected in the cytoplasm of noncarcinomatous hepatocytes in 28 (29.5%) cases and of carcinoma cells in 11 (11.6%) cases. Hepatitis B core antigen was stained in noncarcinomatous hepatocytes in 13 (13.7%) cases and in carcinoma cells in 4 (4.2%) cases. Hepatitis B core antigen was present mainly in the nuclei, and all HBcAg-positive cases were positive for HBsAg. The routine electron microscopic examination revealed many round particles, 25 to 30 nm in diameter both in the nuclei and in the cytoplasm, and larger particles, 40 to 45 nm in diameter in the cytoplasm of carcinoma cells. Both types of particles had reaction products of HBcAg by immunoelectron microscopic study. Therefore, it was confirmed that the former were cores and the latter were Dane particles. There was a tendency that HBeAg-seropositive cases showed localization of HBcAg in the noncarcinomatous tissue. Among four cases with positive HBcAg in carcinoma cells, two were positive for HBeAg, one was positive for anti-HBe, and the other was negative both for HBeAg and anti-HBe in the sera. The data suggested occasional production of complete hepatitis B viruses of carcinoma cells in anti-HBe-positive as well as in HBeAg-positive hepatocellular carcinomas.

Adult↗
PubMed1985

Some properties of IgG against diphtheria toxin synthesized in Xenopus oocytes containing mRNA from hybridoma.

When a mixture of mRNA from hybridoma producing IgG, anti-diphtheria toxin antibody, and mRNA from rat liver was injected into Xenopus oocytes, most of the IgG synthesized in the oocytes was not secreted into the medium and remained in the rough endoplasmic reticulum fraction. In contrast, rat serum albumin was rapidly secreted. The glycosylation of IgG in the oocytes was of a high-mannose type, while that of IgG secreted very slowly into the medium was of a complex type. The IgG in the membrane fraction and in the medium could both bind to diphtheria toxin.

Animals↗
PubMed1985

Karyometric analysis of liver cell dysplasia and hepatocellular carcinoma. Evidence against precancerous nature of liver cell dysplasia.

Karyometric analysis was performed with respect to anisonucleosis, nuclear deformity and DNA content in cases of liver cell dysplasia (LCD) and hepatocellular carcinoma (HCC). Presence or absence of iron deposition in foci of LCD and HCC was also evaluated in siderotic livers. All LCDs showed marked anisonucleosis and marked increases in DNA content but slight increases in nuclear deformity. A tendency was noted in which the nuclear deformity was increased as the nuclei became larger. In contrast, HCCs showed wide ranges of anisonucleosis, nuclear deformity and DNA content. Hepatocellular carcinomas having marked anisonucleosis similar to that of LCD showed markedly increased nuclear deformity. However, unlike LCD, this increase was independent of their nuclear size. In the siderotic livers iron deposition was noted in the foci of LCD but not in the foci of HCC. These findings do not support the notion of LCD being precancerous.

Carcinoma, Hepatocellular↗
PubMed1985

Introduction of macromolecules into hemopoietic stem cells with an erythrocyte-ghost-mediated system.

We have developed a method of introduction of macromolecules into normal human hemopoietic stem cells. The erythrocyte ghosts were loaded with diphtheria toxin fragment A (molecular weight = 22,000 daltons), which exerts cytotoxicity only in the intracellular space. Granulocyte-macrophage colonies of human bone marrow cells incubated with the above ghosts in the presence of Sendai virus decreased in number to about 10% of the control. This means that the cell fusion and the subsequent introduction of the fragment A into granulocyte-macrophage progenitors occurred at a high incidence (about 90%). This method will be useful to study intracellular events during the proliferation and differentiation of hemopoietic stem cells.

Cell Fusion↗
PubMed1985

Functional analysis of macrophage hybridomas. II. Antibody-independent tumor cytotoxicity and its dissociation from IL-1 production and Ia expression.

Cloned macrophage hybridoma cells derived from fusions between splenic adherent cells of CKB mice and the drug-marked P388D1 cell line were studied to further understand macrophage-mediated tumoricidal activity at the clonal level. Most of the macrophage hybridomas were specifically activated by a combination of lymphokine and lipopolysaccharide (LPS), but were not activated by either agent alone. There is no apparent correlation between macrophage-mediated tumor cytotoxicity and the ability of LPS to induce interleukin 1 (IL-1) secretion or of concanavalin A (Con A) supernatants containing interferon-gamma to increase H-2 and Ia expression. Thus, one hybridoma clone which was unable to kill tumor cells was still sensitive to LPS and lymphokine activation by the latter criterion.

Animals↗
PubMed1985

Inhibition of RNA nucleo-cytoplasmic translocation by anti-nucleus antibody.

Anti-nucleus antibody was raised by immunizing rabbits with rat liver nuclei, and purified by affinity-column chromatography. When purified anti-nucleus IgG molecules were introduced into FL cells by the erythrocyte-ghost fusion method with HVJ (Sendai virus), release of RNA from the nucleus into the cytoplasm was inhibited in the presence of alpha-amanitin, but nuclear accumulation of 125I-labeled non-histone chromosomal protein from the cytoplasm was not inhibited. These findings suggest that the nucleo-cytoplasmic transport mechanisms of RNA and nuclear proteins are different. The molecular weight of the antigen of this antibody was determined to be about 55K by the immunoblotting technique.

Amanitins↗
PubMed1985

Direct measurement of phagolysosomal esterase activity.

A new method of directly measuring esterase activity within phagolysosomes has been developed. Decanoyl fluorescein- binding microspheres were prepared and phagocytosed by human peripheral neutrophils. Within phagolysosomes lysosomal esterase hydrolyzed decanoyl fluorescein on the microspheres, causing the conversion of decanoyl fluorescein- binding microspheres (non-fluorescent) into fluorescein- binding microspheres (fluorescent). The activity of phagolysosomal esterase in intact neutrophils was assayed by the measurement of the fluorescence intensity without rupturing cells. By use of a flow cytometer, esterase activity within phagolysosomes in single cells was measured.

Esterases↗
PubMed1985

Direct measurement of phagosomal reactive oxygen by luminol-binding microspheres.

A new method, utilizing microsphere-bound luminol, which makes possible the direct measurement of highly reactive oxygen within phagosomes, was studied. When Freund's complete adjuvant-elicited mouse peritoneal macrophages and luminol-binding microspheres were mixed, the microspheres were engulfed in macrophages and enclosed in phagosomes, where chemiluminescence (CL) was generated, showing the generation of highly reactive oxygen. The reactive oxygen could be quantitatively assayed by measuring the intensity of CL. The addition of cytochalasin B inhibited the CL. CL production by the thioglycollate-elicited macrophages was found to be only a ninth of that by Freund's complete adjuvant-elicited macrophages, though the phagocytic activities were almost equivalent in both cases.

Animals↗
PubMed1985

Met-enkephalin-Arg6-Gly7-Leu8 in large-cored vesicles of splanchnic nerve terminals innervating guinea pig adrenal chromaffin cells.

Met-enkephalin-Arg6-Gly7-Leu8 (Met-Enk-Arg-Gly-Leu)-like immunoreactivity in the guinea pig adrenal medulla was investigated by the peroxidase-antiperoxidase method for light microscopy and by the protein A-gold method for electron microscopy. Because of a distinct Met-Enk-Arg-Gly-Leu-like immunoreactivity in the splanchnic nerve terminals innervating the chromaffin cell, their distribution and structure were clearly demonstrated. With electron microscopy, specific colloidal gold particles representing Met-Enk-Arg-Gly-Leu were localized in large-cored vesicles.

Adrenal Medulla↗
PubMed1985

A case of adult T-cell leukemia with metastatic calcification.

We report a case of adult T-cell leukemia with increased uptake in both lungs which was detected by a bone scan using 99mTc-MDP. This finding is thought to have been caused by the metastatic calcification which is associated with ectopic parathormone production.

Adult↗
PubMed1985

Elevated spontaneous mutation rate in SV40-transformed Werner syndrome fibroblast cell lines.

Spontaneous mutation rates of the cells from patients with Werner syndrome were examined, and we found that the spontaneous mutation rates at the hypoxanthine-guanine phosphoribosyltransferase locus in SV40-transformed Werner syndrome cell lines were markedly elevated, compared to those in SV40-transformed normal control cell lines. Our results suggest that Werner syndrome is a mutation mutant.

Adult↗
PubMed1985

Characterization of diphtheria-toxin-resistant mutants lacking receptor function or containing nonribosylatable elongation factor 2.

Stable mutants resistant to diphtheria toxin (DT) were isolated from Chinese hamster ovary cells (CHO-K1) by single-step mutations with various mutagens. All the mutants were classified into two major groups as reported by other workers (4-6): toxin-entry mutants (DTrI) and translational mutants (DTRII) at the level of elongation factor 2 (EF-2). These mutants were further characterized by directly measuring the specific uptake of [125I]DT and the content of nonribosylatable EF-2 by two-dimensional gel analysis. DTrI mutants, which showed no cross-resistance to Pseudomonas exotoxin A (PA), had no ability to associate with [125I]DT and contained only ADP-ribosylatable EF-2, like wild-type cells. DTRIIb mutants maintained about 50% of the normal level of cellular protein synthesis in the presence of DT, and two-dimensional gel analysis directly showed that they contained equivalent amounts of ADP-ribosylatable and nonribosylatable EF-2 molecules. Fully toxin-resistant cells, named KEE1 (DTRIIa), were isolated from a DTRIIb mutant (KE1) by two-step mutation. KEE1 cells showed full resistance to DT and PA, the normal level of association with [125I]DT, and produced only nonribosylatable EF-2. Biochemical analysis of somatic cell hybrids indicated that the DT-resistant character of class II behaved codominantly. These results strongly supported the hypothesis that two copies of the gene for EF-2 are functional in CHO-K1 cells.

ADP Ribose Transferases↗
PubMed1985

Functional analysis of macrophage hybridomas. III. Inhibition of lymphocyte blastogenesis and tumor proliferation.

Four cloned macrophage hybridoma cells prepared by fusion of splenic adherent cells with a P388D1 macrophage tumor markedly inhibited the growth of lymphocytes and tumor cells. Macrophage clones 5, 8, 63, and 64 are strong inhibitors of B-cell blastogenesis, T-cell blastogenesis, and tumor proliferation, while the P388D1, tumor line and clones 13, 59, and 67 demonstrated little inhibitory activity in all three systems. The inhibitory effect of macrophage hybridomas can be detected within 8 hr, although greater inhibition was noted following longer incubation. The correlations among these three assay systems suggest that similar mechanisms may be involved. The data indicate that the inhibition of cell proliferation was not due to cell lysis. Furthermore, the inhibition of lymphocyte proliferation in Con A-stimulated cultures was not accompanied by inhibition of lymphokine production in the same cultures. Neither prostaglandins nor hydrogen peroxide appear to be primarily responsible for growth inhibition. The inhibitory properties of these macrophage hybridoma lines represent a stable phenotype and provide a homogeneous source of cells for further analysis of the phenomenon.

Animals↗
PubMed1985

Localization of hepatitis B surface antigen in the human parotid gland.

Localization of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) in the human parotid gland and liver was studied by an immunohistochemical method in four cases with seropositive HBsAg. Neither HBsAg nor HBcAg could be detected in the parenchymal cells of the parotid gland in any of the cases. However, one of four cases, which had the highest titer of serum HBsAg, showed HBsAg immunoreactivity in the vascular wall and luminal fluid of the parotid gland. In liver, HBsAg was detected in three and HBcAg in two of the four cases, respectively. HBsAg was localized in the cytoplasm of hepatocytes, while HBcAg was localized mainly in their nuclei. The detection of HBsAg in the parotid gland and liver was correlated with the serum titer of the antigen. The results indicate that the HB virus does not have an affinity for or a replicate in the parotid gland. Also, it is suggested that the HBsAg found in saliva is derived from HBsAg circulating through the oral mucosa by capillary leakage and not from secretion into the mouth by the salivary gland.

Adolescent↗