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T Yamaguchi

Publications and source records attributed to T Yamaguchi.

At least 829 records · Page 46Linked to original sources
PubMed1997

In vivo expression of IL-4, IL-5, IL-13 and IFN-gamma mRNAs in peripheral blood mononuclear cells and effect of cyclosporin A in a patient with Kimura's disease.

Semiquantitative reverse transcription-polymerase chain reaction was used to analyse the expression of cytokine mRNAs in freshly isolated peripheral blood mononuclear cells obtained from a patient with Kimura's disease. The patient was treated with cyclosporin A (CsA) after incomplete tumour resection and irradiation of lesions. Levels of interleukin (IL)-4, IL-5 and IL-13 mRNA were elevated and the level of interferon (IFN)-gamma mRNA was within normal limits before treatment. The levels of IL-4, IL-5 and IL-13 mRNA, the number of eosinophils, and the serum level of IgE decreased markedly after surgery and radiation therapy. CsA treatment suppressed these values in a dose-dependent manner, but had a minimal effect on the level of IFN-gamma mRNA. The number of peripheral eosinophils decreased in association with decreases in the levels of IL-4, IL-5 and IL-13 mRNAs during CsA therapy; the serum level of IgE remained low during therapy and did not fluctuate in association with changes in cytokine levels. These results suggest the Th2 cytokines play a part in the development of Kimura's disease and that CsA suppresses the activity of this disease.

Aged↗
PubMed1997

Chiral influences of feedback inhibition with dCTP on murine deoxycytidine kinase.

The inhibitory effects of 4 kinds of 2'-deoxy-L-nucleoside 5'-triphosphates, which are enantiomers of natural dNTPs, on murine deoxycytidine kinase (dCK) were investigated. When ATP was used as the phosphate donor, L-dCTP showed significant inhibitory action noncompetitively and competitively with 2'-deoxycytidine (dCyd) and ATP, respectively. Thus L-dCTP, like dCTP, could serve as a feedback inhibitor for dCK. Recently, it has been demonstrated that human dCK can utilize L-dCyd as a substrate (Verri, A. et al. (1997) Mol. Pharmacol., 51, 132). The present results suggest that dCK is also unable to discriminate the chirality of nucleotides at the phosphate donor binding site of the enzyme.

Adenosine Triphosphate↗
PubMed1997

In vitro interleukin 12 activation of peripheral blood CD3(+)CD56(+) and CD3(+)CD56(-) gammadelta T cells from glioblastoma patients.

Interleukin (IL)-12 has recently been shown to be directly involved in the activation of natural killer and alphabeta T cells via an IL-2-independent pathway. We show here that another type of human cytotoxic cell, gammadelta T cells activated by solid-phase anti-CD3 antibody and expanded using IL-2, obtained, in this case, from the peripheral blood of glioblastoma patients, displays significant tumoricidal activity. In addition, its cytotoxic activity against K562 or Daudi cells or against autologous glioblastoma targets (but not lymphocytes) is significantly enhanced when costimulated with IL-2 and IL-12. To study this synergistic activation by the two interleukins of the patients' gammadelta T cells, we screened the cells for the presence of the IL-2 receptor (IL-2R) and IL-12 receptor (IL-12R) using both flow cytometric analysis and PCR. The patients' gammadelta T cells constitutively expressed the high-affinity IL-2R; when stimulated with IL-12 plus IL-2, the levels of IL-2Ralpha and IL-2Rbeta increased, whereas that of IL-2gamma did not. They also expressed marginal levels of low-affinity IL-12R both immediately after IL-2 expansion and after 24-h incubation, and significantly higher levels after 72-h incubation, consistent with the level of gammadelta T-cell activation. IL-12 alone induced little proliferation of patients' gammadelta T cells in a 24-h assay and none in a 72-h assay; however, it caused a marked inhibition of the IL-2-induced proliferative response in the 72-h assay. The synergistic action of IL-2 and IL-12 was completely abolished by combined pretreatment with anti-IL-2alpha, beta, and gamma mAbs. IL-12-mediated enhancement of gammadelta T cell cytotoxic activity was inhibited by anti-IL-2Rbeta mAb in a dose-dependent manner but not by anti-IL-2Ralpha or anti-IL-2Rgamma mAbs. Thus, the increased expression of the IL-2Rbeta is critical for the synergistic activation of gammadelta T cells by IL-12 plus IL-2; it is also probable that at least the low-affinity IL-12R contributes to the activation of gammadelta T cells mediated by either IL-12 alone or IL-12 plus IL-2. We have, therefore, demonstrated that IL-12 can stimulate the cytotoxic activity of gammadelta T cells from glioblastoma patients, acting via the IL-2Rbeta component of the IL-2R and low-affinity IL-12R. IL-12 activation of patients' gammadelta T cells could possibly be of potential use in the treatment of glioblastoma patients.

Antigens, CD↗
PubMed1996

Low stringency hybridization study of the dopamine D4 receptor revealed D4-like mRNA distribution of the orphan seven-transmembrane receptor, APJ, in human brain.

We recently reported that the density of mRNA for the dopamine D4 receptor was extremely low in human cerebral cortex but unexpectedly higher in the corpus callosum and spinal cord both of which contain substantial white-matter area. Under low stringency conditions, Northern blot analysis using the D4 probe detected cross-hybridized mRNAs having a similar distributional profile to the D4 mRNA in human brain regions, suggesting the mRNA distributional profile is not peculiar to the D4 receptor. Homology screening revealed one of the mRNAs to be an orphan seven-transmembrane receptor, APJ, abundantly expressed in the corpus callosum and spinal cord. In porcine spinal cord the APJ mRNA was detected at a higher level in white-matter rather than in gray-matter area. These data suggest that a group of seven-transmembrane receptors, including the D4 and APJ receptor, is preferentially expressed in the white-matter area probably in non-neuronal glial cells.

Animals↗
PubMed1996

Cloning and characterization of the cDNA encoding human biliverdin-IX alpha reductase.

Biliverdin reductase is classified into two isoforms in substrate specificity; biliverdin-IX alpha reductase and biliverdin-IX beta reductase with a molecular mass of 22 kDa and 34-42 kDa, respectively. We have cloned the cDNA encoding human biliverdin-IX alpha reductase from MOLT4 cDNA library. The cDNA of 1146 bp in nucleotide length contained an entire reading frame coding 296 amino acid residues. The NADH/NADPH binding consensus sequence was found in the amino-terminal region. Comparison between human and rat biliverdin-IX alpha reductases showed 82.8% identity in amino acid sequences and 80.3% identity in the coding nucleotides. The amino acid sequence of human biliverdin-IX alpha reductase showed no significant homology to that of human biliverdin-IX beta reductase. Northern blot analysis of poly(A) RNA from eight different human tissues revealed that the reductase mRNA was abundant in the brain, lung and pancreas but not in the liver. The distribution pattern of biliverdin-IX alpha message was different from that of heme oxygenase activity which is known to be high in the liver and to be low in the heart and lung.

Amino Acid Sequence↗
PubMed1996

YM-50001: a novel, potent and selective dopamine D4 receptor antagonist.

We investigated some in vitro pharmacological properties of a novel human dopamine D2-like receptor antagonist, YM-50001 [(R)-5-chloro-4-cyclopropylacarbonylamino-2-methoxy-N-[1-(3-methox ybenzyl)- 3-pyrrolidinyl]benzamide monooxalate]. Receptor binding studies revealed that YM-50001 had a potent affinity for human D4 receptors (Ki = 5.62 nM). YM-50001 displayed weak or negligible affinity for other neurotransmitter receptors including human D2 and D3 receptors. YM-50001 shifted the dopamine response curve on each human D2-like receptor subtype-mediated low-Km GTPase activity to the right. YM-50001 also exhibited good D4 selectivity with respect to D2-like receptor antagonism in the functional assay. These results indicate that YM-50001 is a novel, potent and selective D4 receptor antagonist.

Benzamides↗
PubMed1996

Inactivation of a novel neuropeptide Y/peptide YY receptor gene in primate species.

Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) belong to a family of structurally related peptides which have numerous functions in both neural and endocrine signaling. By homology screening, we cloned a novel gene sharing the highest homology with the NPY Y1 receptor gene from humans, rabbits, and several other species. This novel gene of rabbit encodes a functional NPY/PYY receptor, designated Y2b, which prefers NPY13-36 rather than [Leu31,Pro34]NPY despite its higher identity with the Y1 receptor. Although, at low levels, mRNA was detected in the tissues and brain regions, including hypothalamus. Further, sequence data revealed that this gene is the orthologue of the recently cloned mouse novel NPY receptor, Y5. However, our study demonstrates that the receptor function of this gene has been inactivated in primates by a frameshift mutation occurring early in primate evolution. This novel NPY receptor represents the first neurotransmitter receptor identified that has universally lost its receptor function in primate species. Interestingly, despite its inactivation in humans, the transcripts were abundantly detected in the heart and skeletal muscle, suggesting a novel function of the human gene.

Amino Acid Sequence↗
PubMed1996

Expression of soluble human beta-globin chains in bacteria and assembly in vitro with alpha-globin chains.

Authentic soluble human beta-globin chains were produced in Escherichia coli using an expression plasmid (pHE2beta) containing full-length cDNAs coding for human beta-globin chain and methionine aminopeptidase. Spectral properties of the purified beta-globin were identical to those of authentic beta-globin. Soluble beta-globin showed low (16 kDa) and high molecular mass (32 kDa) forms that could be separated by gel filtration chromatography. SDS-polyacrylamide gel electrophoresis and electrospray mass spectrometry revealed the 32-kDa species was dimeric beta-globin formed by an intermolecular disulfide bond, while the 16-kDa species was authentic monomeric beta-globin. Monomeric forms of beta-globin, like authentic native beta-globin, formed tetrameric hemoglobin (Hb) A (alpha2beta2) in vitro upon incubation with alpha-globin, while dimeric forms did not. When beta-globin dimers, however, were converted to monomers by incubation with dithiothreitol, the beta-globin chain monomers assembled with alpha-globin and formed hemoglobin tetramers. alpha-Globin was more thermally unstable than beta-globin, while assembled tetramers promoted higher stability. Disulfide-bonded beta-globin dimers showed a slight increase in thermal stability compared with beta-globin; however, dimers were still more unstable than tetrameric Hb A. These results indicate that presence of alpha chains favors assembly with beta-globin, beta-beta dimers cannot bind alpha chains, and that Hb A tetramer formation results in the most thermally stable species.

Biopolymers↗
PubMed1996

Expression studies of delta-globin gene alleles associated with reduced hemoglobin A2 levels in Greek Cypriots.

We previously identified five delta-globin gene alleles associated with reduced hemoglobin (Hb) A2 (Trifillis, P., Ioannou, P., Schwartz, E., and Surrey, S. (1991) Blood 78, 3298-3305). We have now evaluated functional consequences of the changes after expression in COS-1 cells to monitor effects on RNA splicing. In addition, variant Hb A2 tetramers were expressed in yeast to assess effects of amino acid changes on oxygen binding and stability to heat and mechanical agitation. The G --> T change at codon 27 and the A --> G change in IVS-2 both affect RNA splicing, whereas the C --> T change at codon 97 and the AT deletion in IVS-2 have no effect. Oxygen equilibrium curves of the Hb A2 variants expressed in yeast were similar to that of wild type Hb A2. None of the three variant Hb A2 tetramers (Thr --> Ile at codon 4 (Hb deltaT4I), Ala --> Ser at codon 27 (Hb deltaA27S), and Arg --> Cys at codon 116 (Hb deltaR116C)) showed decreased heat stability compared with Hb A2, whereas the Hb deltaT4I variant showed highest instability to mechanical agitation. Co-expression in yeast of alpha-globin chain and the delta-chain variant containing a Leu --> Pro change at codon 141 yielded no identifiable tetramers, suggesting lack of assembly or severe tetramer instability. These studies show the probable cause for decreased Hb A2 for two alleles is due to defective splicing, whereas decreased protein stability, increased tetramer association with red cell membranes, increased interdisulfide bond formation of delta-chains, which inhibits assembly with alpha-chains, and/or reduced assembly is suggested for the other three alleles.

Alleles↗
PubMed1996

Intertrabecular pattern of tumors metastatic to bone.

BACKGROUND: The prevalence of radiologically invisible metastatic tumors to bone is not known. To clarify this, the authors examined histologic sections, radiographs, bone scans, and magnetic resonance (MR) images of autopsied patients with bone metastases. METHODS: Complete slices of the vertebral column, including C3-S1, were obtained from 52 cadavers with metastatic tumors to the spine. Macroscopic and histologic features were compared with postmortem roentgenographs (52 patients), bone scans with 99mTc-labelled methylene diphosphonate (17 patients), and MR images (4 patients). Morphologic patterns were classified into intertrabecular, osteoblastic, osteolytic, mixed, small lesion, and compression fracture. RESULTS: Metastatic lesions were found in 734 of the 1194 vertebral bodies. The lesions were intertrabecular in 255 vertebrae, osteoblastic in 185, osteolytic in 114, mixed in 70, small lesion in 83, and compression fracture in 27. Postmortem radiographs demonstrated lesions in 367 (50%) of the 734 vertebrae, bone scans in 103 (34.7%) of the 297 vertebrae examined, and MR images in all 42 (100%) vertebral bodies imaged. Lesions showing an intertrabecular pattern were detected in only 7.1% of the radiographs and 4.5% of the bone scans. CONCLUSIONS: Radiographs and bone scans often fail to show metastatic lesions, especially those with an intertrabecular pattern. The intertrabecular pattern is a unique histologic form that is difficult to detect on radiographs and bone scans.

Adult↗
PubMed1996

Control of calcium oscillations by phosphorylation of metabotropic glutamate receptors.

Stimulation of two metabotropic glutamate-receptor subtypes, mGluR1 and mGluR5, triggers the release of Ca2+ from intracellular stores through the inositol-(1,4,5) trisphosphate (InsP3) pathway. Here we report that glutamate induces single-peaked intracellular Ca2+ mobilization in mGluR1alpha-transfected cells but elicits Ca2+ oscillations in mGluR5a-transfected cells. The response patterns of the intracellular Ca2+ increase depend upon the identity of a single amino acid, aspartate (at position 854) or threonine (at position 840), located within the G-protein-interacting domains of mGluR1alpha and mGluR5a, respectively. Pharmacological and peptide mapping analyses indicated that phosphorylation of the threonine residue at position 840 of mGluR5a by protein kinase C (PKC) is responsible for the generation of Ca2+ oscillations in mGluR5a-expressing cells. To our knowledge this is the first evidence that PKC phosphorylation of G-protein-coupled receptors is important in producing oscillations in intracellular Ca2+ signalling.

3T3 Cells↗
PubMed1996

Identification of sequence changes responsible for the attenuation of highly virulent infectious bursal disease virus.

The genetic changes responsible for the attenuation of infectious bursal disease virus (IBDV) have not been defined at the molecular level, although passage of the virus in cell culture results in the loss of virulence. To understand the molecular basis of IBDV virulence and attenuation, the IBDV genome segment encoding the precursor polyprotein (NH2-VP2-VP4-VP3-COOH) of a cell culture-adapted OKYMT strain derived from highly virulent OKYM was cloned as cDNA, and the nucleotide sequence was determined. Comparison of the identified nucleotide and deduced amino acid sequences of the attenuated strain with the parental virulent OKYM strain revealed only five amino acid differences: four in the VP2 variable domain and one in the VP3. Two amino acid substitutions at positions 279 (Asp-->Asn) and 284 (Ala-->Thr) in the VP2 variable domain were commonly predicted in another cell culture-adapted strain. These two amino acid changes resulted in reduced hydrophila of this region and deletion of the alpha-helix which might alter the conformation of the virion surface structures. These results may imply that the amino acid residues at position 279 and 284 in VP2 variable domain contribute to virulence of IBDV.

Animals↗
PubMed1996

A photolabile 2',3'-dideoxyuridylate analog bearing an aryl(trifluoromethyl)diazirine moiety: photoaffinity labeling of HIV-1 reverse transcriptase.

In order to develop a photoaffinity labeling reagent for DNA polymerases, including retroviral reverse transcriptase (RT), we utilized 2',3'-dideoxy-E-5-[4-(3-trifluoromethyl-3H-diazirin-3-yl) styryl]UTP (TDSddUTP) as a substrate dTTP analog. Photoaffinity labeling experiments with human immunodeficiency virus type-1 (HIV-1) RT using a radioactive labeling reagent ([gamma-32P]TDSddUTP) and poly(A).oligo(dT) as the template/primer yielded different results depending on the concentration of Mg2+. In the presence of 0.025 mM Mg2+, photoaffinity labeling showed that TDSddUTP bound selectively to the dTTP binding site in the 66 kDa subunit of the p66/p51 heterodimeric enzyme protein when irradiated by near-UV light (365 nm). In the presence of 4 mM Mg2+ or 0.05 mM Mn2+, TDSddUTP was incorporated into the 3'-end of the primer strand due to RT activity and the resulting photolabile primer bound to the 66 kDa subunit of HIV-1 RT on photoirradiation. These results suggest that TDSddUTP could be a useful tool for studying the substrate binding site(s) of DNA polymerases, including HIV-1 RT, which show affinity for this compound.

Affinity Labels↗
PubMed1996

Characterization of YM90K, a selective and potent antagonist of AMPA receptors, in rat cortical mRNA-injected Xenopus oocytes.

The inhibitory potencies of 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride (YM90K), 2-3,dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) and 1-(4-amino-phenyl)-4-methyl-7,8-methyl-endioxyl-5H-2,3-benzodiazep ine (GYKI 52466) at excitatory amino acid receptors were examined in rat cortical mRNA-injected Xenopus oocytes using a two-electrode voltage clamp. Schild analysis of YM90K and NBQX inhibition of kainate currents yielded pA2 values of 6.83 +/- 0.01 and 7.24 +/- 0.01, respectively. GYKI 52466 reduced the maximum kainate response and increased the kainate EC50 in a dose-dependent manner, suggesting that the antagonism of AMPA receptors by GYKI 52466 is mixed competitive and non-competitive for kainate. Schild analysis of YM90K and NBQX inhibition of kainate currents in the presence of 30 microM cyclothiazide yielded pA2 values of 6.62 +/- 0.03 (slope: 1.02 +/- 0.01) and 7.10 +/- 0.02 (slope: 1.00 +/- 0.02), respectively, consistent with competitive antagonism. Cyclothiazide potentiated the AMPA response as well as the kainate response and increased the apparent Hill coefficients in a concentration-dependent manner. The potency of YM90K to inhibit AMPA-induced current could be reduced by increasing the concentration of cyclothiazide. We showed that YM90K is a potent and competitive antagonist for AMPA receptors and the apparent affinity of competitive antagonists was reduced by cyclothiazide. Cyclothiazide can affect the interaction between receptors and both agonists and antagonists, suggesting that it might allosterically alter the affinity of agonists and competitive antagonists for their binding site on the AMPA receptor complex.

Animals↗
PubMed1996

Establishment and characterization of two cultured cell lines derived from malignant rhabdoid tumors of the kidney.

Malignant rhabdoid tumor of the kidney (RTK) is a rare renal sarcoma of childhood. Its histogenesis is unclear, and it is highly resistant to multimodality therapy. To elucidate the origin and the oncogenetic potential of RTK, we investigated the characteristics of 2 newly established RTK cell lines, SWT-1 and SWT-2. Both cell lines were verified to be RTK, since they did not exhibit contact inhibition and exhibited intermediate filaments, a specific marker for RTK. These cells possess the characteristics of mesenchymal cells based on their positive reactions with anti-vimentin and anti-laminin antibodies and their negative reactions with anti-keratin and anti-desmin antibodies. The karyotype of SWT-1 was 46,XX and that of SWT-2 was 46,XX,del(11)(pter-p13::p12-qter). Since 11p13 is the location of the WT-1 tumor-suppressor gene, and del(11p13) is associated with the aniridia-Wilms'-tumor syndrome, these findings link RTK with Wilms' tumor. While SWT-1 was negative for the tumor markers examined, SWT-2 released tissue polypeptide antigen into the culture supernatant. No rearrangement or amplification of the myc and ras oncogenes or of the p53 tumor-suppressor gene were detected. Wild-type RB protein and cyclin A were expressed in both cells. Our data suggest that these 2 cell lines may be useful in identifying the oncogenetic pattern of RTK.

Antineoplastic Agents↗
PubMed1996

Overexpression of bax sensitizes human breast cancer MCF-7 cells to radiation-induced apoptosis.

Resistance to apoptosis plays an important role in tumors that are refractory to chemotherapy and ionizing radiation (IR). bax, which forms a heterodimer with bcl-2 and accelerates apoptosis, is not, or only weakly, expressed in most human breast cancer cells, and weak bax expression is considered to be related to the resistance of breast cancer cells to apoptosis. bax expression vector was introduced to human breast cancer MCF-7 cells, which exhibit weak expression of bax, to demonstrate its role of modulating radiation-induced apoptosis. bax overexpression in MCF-7 cells by stable transfection does not affect viability by itself, but each stable transfectant was more sensitive to IR than the parental MCF-7 cells. The degree of enhancement in radiosensitivity was dependent on the expression level of bax. IR upregulated p53 and p21WAF1 about 5- to 10-fold and downregulated bcl-2 and bcl-XL by 80-90% at 6 hr in both parent and bax stably transfected MCF-7 cells to the same degree. FACS analysis and DNA electrophoresis revealed that this sensitization was due to apoptosis. We suggest that exogenous bax expression might be one of the factors determining cellular radiosensitivity in MCF-7 breast cancer cells and may have therapeutic applications for enhancing radiation sensitivity in breast cancer cells.

Apoptosis↗