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Biomedical subjects

Y Hashimoto

Publications and source records attributed to Y Hashimoto.

At least 775 records · Page 43Linked to original sources
PubMed1994

Transition from TCR-beta dimer to TCR-alpha beta-expressing cells by introduction of an alpha-chain in an immature thymocyte cell line.

The molecular basis for the surface expression of TCR-beta chain in the absence of association with TCR-alpha, -gamma, or -delta chain by an immature thymocyte cell line was investigated. The TCR-beta chain expressed by this cell line was not encoded by any unique DNA sequence, nor was it inserted into the membrane via a glycosyl-phosphatidylinositol linkage. Transfection of two other beta-chains derived from mature T cell clones resulted in the surface expression of dimers of the transfected beta-chains in both cases. Immunoprecipitation of the beta-dimer-CD3 complex demonstrated that the association of the beta-dimer with the CD3 complex, especially the CD3 zeta chain, was so weak that they dissociated under the detergent conditions in which the TCR-CD3 complex of mature T cells is kept intact. Transfection of TCR-alpha chain resulted in the expression of a TCR-alpha beta-CD3 complex and the disappearance of beta-dimers. In accordance with the changes in TCR complex components, the association between TCR-alpha beta and CD3 complex became stable and the cells transduced signals more efficiently. The results demonstrate that the expression of TCR-beta as part of an incomplete TCR-CD3 complex is developmentally regulated and the expression of TCR-alpha chain results in normal configuration and function of TCR complex.

Animals↗
PubMed1994

Erythroid differentiation-inducing activity of protoporphyrin IX and its analogs on human leukemia K562 cell line.

Protoporphyrin IX, which had been regarded as ineffective in cell differentiation, induced erythroid differentiation of K562 cell line, and its analogs such as hematoporphyrin IX, mesoporphyrin IX, deuteroporphyrin IX and protoporphyrin IX dimethyl ester were also differentiation inducers. The differentiation induced by these compounds was augmented by a retinoid, as is hemin-induced differentiation.

Benzoates↗
PubMed1994

Formation of alpha-helix 88-98 is essential in the establishment of higher-order structure from reduced lysozyme.

Lys96 and Lys97 in lysozyme are located at the C terminus of alpha-helix 88-98. The positive charges of these residues are supposed to stabilize the helical structure, and these residues are conserved as the basic amino acids among c-type lysozymes. The renaturation rate of reduced mutant lysozyme, where both Lys96 and Lys97 were mutated together to Ala, was slower than that of native lysozyme at pH 8.0 and 37 degrees C by SH-SS interchange reactions. In order to investigate the reason, the peptide fragment 36-105 (where we can obtain information of the interaction between helix 88-98 and Trp62 and Trp63 residues) was prepared. CD spectra were compared between peptide fragment 36-105 and its acetylated form, where the positive charges of Lys96 and Lys97 were eliminated, and it was elucidated that the displacement of positive charges at the C terminus of the helix caused the shift of the advantageous structure of the fragment from alpha-helix to coil. Moreover, we obtained evidence that there was interaction of the helix with Trp62 and/or Trp63, which maintained a thermodynamically stable higher-order structure. Therefore, these results suggest that the formation of alpha-helical structure in 88-98 is a significant factor in the establishment of native structure from reduced lysozyme.

Amino Acid Sequence↗
PubMed1994

Epidermal growth factor-receptor mutant lacking the autophosphorylation sites induces phosphorylation of Shc protein and Shc-Grb2/ASH association and retains mitogenic activity.

Epidermal growth factor (EGF) receptor (EGFR) can induce cell growth and transformation in a ligand-dependent manner. To examine whether the autophosphorylation of EGFR correlates with the capacity of the activated EGFR to induce cell growth and transformation, we truncated the human EGFR just after residue 1011, removing all three major autophosphorylation sites (DEL1011). Further, a point mutation was introduced at another autophosphorylation site, Tyr-992-->Phe (DEL1011+F992). The wild-type and mutant receptors were stably expressed in a NIH 3T3 variant cell line that expresses an extremely low level of endogenous EGFR and does not grow with EGF. As expected, DEL1011 and DEL1011+F992 were found to be severely impaired in EGF-induced autophosphorylation, due to the deletion of the appropriate target tyrosines. However, mutant receptors still could induce EGF-dependent DNA synthesis, morphological transformation, and anchorage-independent growth, although the extent of these was significantly reduced when compared with wild-type EGFR. EGF-induced tyrosine phosphorylation of Ras-GTPase activating protein-associated protein p62 and phospholipase C gamma 1 was dramatically reduced in the cells expressing DEL1011 and DEL1011+F992. On the other hand, tyrosine phosphorylation of Shc, complex formation of Shc-Grb2/Ash, and activation of microtubule-associated protein kinase were still fully induced upon EGF stimulation without binding of Shc or Grb2/Ash to the mutant receptor. Thus, tyrosine phosphorylation of Shc may play a crucial role for activating Ras and generating mitotic signals by the activated EGFR mutant.

3T3 Cells↗
PubMed1994

Delayed allograft rejection by T cell receptor V beta 8.1 transgenic mice peripherally tolerized to Mls-1.

One commonly studied model system for peripheral tolerance is the antigen-specific unresponsiveness of T cells from mice previously inoculated with superantigens such as Mls-1a. In this study, we used a TcR V beta 8.1 transgenic mouse model to investigate whether mice peripherally tolerized to Mls-1a exhibit delayed skin allograft rejection. We report dramatic prolongation of skin allograft survival in V beta 8.1 transgenic but not in non-transgenic mice tolerized to Mls-1a. Peripherally induced unresponsiveness to Mls-1a can, therefore, be considered true tolerance.

Animals↗
PubMed1994

The effects of natural and synthetic retinoids on the differentiation of RCJ C5.18 chondrogenic cells.

RCJ C 5.18 (C 5.18) is a chondrogenic clonal cell line which, under standard culture conditions, develops chondroblastic features including the production of a cartilagenous matrix. Retinoic acid (RA) is known to inhibit the chondrogenic differentiation of C 5.18 cells and this may parallel the teratogenic effects of retinoids in vivo; however, the question as to which of the 3 retinoic acid receptors (RAR alpha, beta, gamma) or the 3 retinoid X receptors (RXR alpha, beta, gamma) mediate this RA-induced inhibition remains unanswered. We tested several retinoids with different receptor binding characteristics. Cartilage formation in C 5.18 cultures was evaluated by counting the number of cartilage nodules formed, and by quantitating the glycosaminoglycan content of the cultures using alcian blue staining. All of the retinoids prevented cartilage formation in a dose-dependent manner. Treatment with the retinoids did not affect cell number, thereby ruling out any toxic effects. RA, which binds to all 3 RARs with similar affinity, produced a 50% inhibition (IC50) of cartilage formation at 4 x 10(-10) M. We also tested Ch55, which also binds to all 3 RARs, but with higher affinity than RA. This compound was approximately 10 times more potent than RA (IC50 2 x 10(-11) M). 9-cis RA, which binds to the 3 RARs with affinities similar to RA and also binds to the 3 RXRs, was less active (IC50 8 x 10(-9) M), suggesting that RXR binding interferes with the inhibitory effect of ligand-activated RARs. 9-cis retinal, for which the binding characteristics are unknown, had the same effect as 9-cis RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Rapid superoxide production by endothelial cells and their injury upon reperfusion.

Cultured human umbilical vein endothelial cells (EC) were exposed to hypoxia for various time periods followed by reoxygenation, and production of both O2- and H2O2 by EC was measured using the cytochrome c and scopoletin methods, respectively. Production of significant amounts of O2- was observed as early as 1 min after reoxygenation, reached maximum at 3 min and seemed to decline thereafter. Production of H2O2 was observed slightly later than that of O2-. Endothelial cell injury during hypoxia/reoxygenation was determined by both the trypan blue dye exclusion and chromium release methods. Significant increase of EC injury was detected after reoxygenation by both methods. This injury was significantly inhibited by allopurinol but not by superoxide dismutase or catalase. These results indicate that the production of reactive oxygen metabolites by EC and EC injury occur very rapidly after reoxygenation, which has not been reported, and these should be taken in consideration for the understanding and treatment of the clinical reperfusion injury.

Allopurinol↗
PubMed1994

Cloning and characterization of the hemA gene for synthesis of delta-aminolevulinic acid in Xanthomonas campestris pv. phaseoli.

The gene from Xanthomonas campestris pv. phaseoli that is involved in the C5 pathway of delta-amino-levulinic acid (ALA) of Escherichia coli. Subcloning of deletion fragments from the initial 2.5-kilobase (kb) chromosomal fragment allowed the isolation of a 1.6-kb fragment that could complement the hemM mutation. Nucleotide sequence analysis of the 1.6-kb DNA fragment revealed an open reading frame that encodes a polypeptide of 426 amino acid residues, and the deduced molecular mass of this polypeptide is 46768 Da. The amino acid sequence shows a high degree of homology of the HemA protein, which is glutamyl-tRNA reductase, to other organisms. Thus, we examined the complementation test of the cloned gene from Xanthomonas with a hemA mutation of E. coli and found that the gene complemented the hemA mutation. These results suggest that the cloned gene is hemA and the gene from Xanthomonas also complements both hemA and hemM mutations, as in the case of the E. coli hemA.

Aldehyde Oxidoreductases↗
PubMed1994

Decrease in lymphokine-activated killer sensitivity of a human renal-cell carcinoma cell line after cytokine treatment.

Recent studies have shown that cytokine treatment of tumor cells alters the sensitivity of these cells to lymphokine-activated killer (LAK) cells, depending on the cell line. In this study, we analyzed the decrease in LAK sensitivity of a human renal-cell carcinoma cell line (SMKT-R-3). The LAK sensitivity of SMKT-R-3 was decreased by treatment with a combination of interferon gamma (IFN gamma) and tumor necrosis factor (TNF). However, the cytokine treatment increased the expression of intercellular adhesion molecule-1 (ICAM-1) on the renal-cell carcinoma cell surface. The conjugate-formation assay also confirmed a slight increase in the binding rate of LAK cells to the renal-cell carcinoma cells. When actinomycin D (a protein synthesis inhibitor) was added to the culture medium prior to treatment with IFN gamma and TNF, the LAK sensitivity of SMKT-R-3 recovered to the level demonstrated by the cells that had not received any cytokine treatment. These results suggest that the effect of cytokines in reducing LAK sensitivity of SMKT-R-3 is mediated by protein synthesis occurring when LAK cells are bound to SMKT-R-3 cells.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1994

Interaction of marine toxin dolastatin 10 with porcine brain tubulin: competitive inhibition of rhizoxin and phomopsin A binding.

Dolastatin 10, a cytostatic peptide containing several unique amino acid subunits, was isolated from the marine shell-less mollusk Dolabella auricularia. It inhibits microtubule assembly at concentrations below 5.0 microM (IC50, 3.0 microM) and causes formation of tubulin aggregates at higher (> 10 microM) concentrations in a somewhat different manner from that caused by vinblastine. Electron microscopical analysis showed irregular aggregates of microtubule proteins in the presence of 10 microM dolastatin 10. Dolastatin 10 inhibited the binding of both radiolabeled rhizoxin and phomopsin A to tubulin with inhibition constants (Ki) of 7 x 10(-8) M and 1 x 10(-7) M, respectively. The results suggest that at least one of the binding sites of dolastatin 10 on tubulin is the rhizoxin binding site.

Animals↗
PubMed1994

Effect of chylomicron remnants on apolipoprotein E synthesis in HepG2 cells.

The effect of chylomicron remnants (CM-R) on apolipoprotein (apo) E synthesis by HepG2 cells was studied. The accumulation rate of apo E in the medium was increased in cells incubated with CM-R (chylo-cells) compared with that in the control cells. However, the abundance of apo E mRNA was not greater in chylo-cells than in control cells. The time course of apo E accumulation in the medium showed that apo E increased in the chylo-cells linearly up to 24 hours, whereas it increased in the control cells linearly only up to 6 hours, when it reached a plateau. The results suggest that the secretion rate of apo E was not affected by incubation with CM-R, but that uptake of the newly secreted apo E by the cells may be depressed by incubation with CM-R. Autoradiography of newly synthesized apo E showed that chylo-cells produced much more "larger" apo E than did the control cells. Two-dimensional electrophoresis of the apo E newly synthesized by the chylo-cells showed that the larger apo E was slightly acidic even after treatment with neuraminidase. Pulse-chase labeling of apo E showed that the larger apo E was secreted by both control and chylo-cells equally and that the larger apo E was converted to regular apo E in the medium more effectively in control cells than in chylo-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoproteins E↗
PubMed1994

Relationship between occurrence of tremor/convulsion and level of beta-carbolines in the brain after administration of beta-carbolines into mice.

Fifteen beta-carboline derivatives, including those found in the South American hallucinogenic plant Banisteriopsis caapi, were injected IP and IVC into mice. Subsequent behavioral changes were observed and the levels of the compounds in brain tissue were determined. It was found that following IP administration, tremors and/or convulsions were induced by beta-carbolines having aliphatic alkyl groups, but not by those with carbonyl and oxo groups substituted at carbon-1 of the C ring. These effects were potentiated by the presence of a methoxy group at carbon-7 of the A ring, and their duration of actions were prolonged by 3,4-dihydro derivatives. When induced, tremors/convulsions correlated with levels of beta-carbolines in the brain. The smaller ED50 values of beta-carbolines that cause tremors/convulsions showed lower levels of beta-carbolines in brain tissue.

Animals↗
PubMed1994

Characterization of a cytotoxic factor produced by rat bladder cancer cell line: its identity to TNF alpha.

Clone C19 derived from a rat bladder cancer cell line BC31ad constitutively released cytotoxic factor RCF. The serum-free C19-conditioned medium was partially purified by DEAE-Sephacel, Sephacryl S-200 and MonoQ-FPLC chromatographies to the specific activity of 6.4 x 10(6) U/mg protein (LM assay). RCF was a 17-kDa protein with pI 4.6-5.0. Anti-RCF serum neutralized the cytotoxic activity of rat and mouse, but not human, tumor necrosis factor-alpha (TNF alpha) as well as RCF. The partial amino acid sequence of RCF was homologous with the sequence 43-58 of rat TNF alpha. RCF and rat TNF alpha showed a similar cytotoxic spectrum against several tumor cells. Furthermore, they induced a marked proliferation of mouse T cells in combination with a suboptimal dose of ConA or anti-CD3 antibody, however, human TNF alpha almost not. Taken together, RCF was substantially regarded as rat TNF alpha.

Amino Acid Sequence↗
PubMed1994

Expression of Thy-1 on rat T cells and its relevance as a maturation marker of T cells in rats.

Expression of Thy-1 and CD3 antigens on thymocytes and the nylon wool-non-adherent (nylon-nonad) fractions of peripheral lymphocytes in F344 rats was studied by flow cytometry using anti-Thy-1 and anti-CD3 antibodies. In terms of the expression and density of Thy-1 and CD3, the thymus and peripheral lymphoid tissues in rats contained different populations. Nearly all thymocytes showed a high density of Thy-1 (Thy-1hi); a small population with a low density of Thy-1 (Thy-1lo) was found in CD3 bright-positive (CD3hi) thymocytes. Three-color analysis with CD4, CD8 and Thy-1 revealed Thy-1lo thymocytes to have mainly CD4 or CD8; Thy-1hi thymocytes also contained CD4 or CD8 single-positive (SP) cells as a minor population. Thus, SP thymocytes bearing CD3 were divided into two populations by evaluation of the density of Thy-1. Of peripheral CD3+ T cells, 15% showed a low density of Thy-1+ and the others were Thy-1-. Both T cells responded to anti-CD3 monoclonal antibody (mAb). However, no Thy-1hi T cells were found in peripheral lymphoid tissues. These results suggested that SP thymocytes in rats develop with the reduction of Thy-1, but not with its loss, and are newly supplied to peripheral lymphoid tissues as the phenotype, Thy-1lo, and are altered to Thy-1- T cells. Analysis of each cell size and disappearance of peripheral Thy-1lo T cells with age supported the above conclusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗