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Biomedical subjects

K Haraguchi

Publications and source records attributed to K Haraguchi.

At least 109 records · Page 6Linked to original sources
PubMed1995

Influence of five methylsulphonyl PCB congeners on frequency of micronucleated cells in cultured human lymphocytes by cytokinesis block method.

The lungs and blood of Yusho patients and healthy Japanese people have already been contaminated with methylsulphonyl polychlorinated biphenyls (MSF-PCBs) at relatively high concentration. Therefore, we should give due attention to their biological and toxicological effects to man. In this study, in order to mainly evaluate non-S-dependent genotoxicity of five MSF-PCB congeners, namely, 3-MSF-4, 5, 3', 4'-tetrachlorobiphenyl (TCB), 3-MSF-4, 5, 2', 3'-TCB, 3-MSF-2, 5, 2', 4', 5'-pentachlorobiphenyl (PenCB), 4-MSF-2, 5, 2', 3', 4'-PenCB and 4-MSF-2, 5, 2', 3', 5', 6'-hexachlorobiphenyl (HCB). We have examined their effects on the induction of micronucleated cells, which has been frequently used to estimate the dose of ionizing radiation and truly radiomimetic, non-S-dependent, clastogens, in cultured human lymphocytes in the absence or presence of 2, 3, 4, 7, 8-pentachlorodibenzofuran (PenCDF), 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) or 3, 4, 5, 3', 4'-pentachlorobiphenyl (Co-PenCB). The following results were obtained. 1) 4 x 10(-5) M7, 8-benzoflavone (ANF) significantly enhanced the frequency of micronucleated cells and all of the five MSF-PCB congeners failed to induce the formation of micronucleated cells at doses of 5.2 to 9.6 ppm, which were about 35,000 times higher than the concentrations in the lungs and adipose tissue of healthy Japanese people.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1994

Structure of the rat thyroid transcription factor-1 (TTF-1) gene.

TTF-1 is a homeodomain-containing thyroid transcription factor which activates the genes of thyroid specific protein, thyroglobulin, thyroid peroxidase and thyrotropin receptor. We have cloned the TTF-1 gene from rat liver genomic library, and the exon/intron organization and the structure of the 5' flanking region were determined. The clone contained the 5.2 kbp upstream sequence from translation initiation site, and we found that the gene has a single intron in the coding sequence. We found in the 5' flanking region the TTF-1 binding consensus sequence, CTCAAGC, at -175 to -169, which overlaps the consensus sequence of CAAT box, DNase I foot print analysis has revealed that the region is protected by nuclear extract from thyroid cells but not by the extract from the liver, suggesting that expression of the TTF-1 gene is autoregulated by TTF-1.

Animals↗
PubMed1994

Disruption of the first extracellular loop of thyrotropin receptor prevents ligand binding.

In order to understand the function of the first extracellular loop of the human thyrotropin receptor (hTSHR), each of two peptides of nine amino acids was inserted into the first extracellular loop of hTSHR. hTSHR cDNA was subcloned into the eukaryotic expression vector, pRc/CMV (hTSHR/pRc/CMV). B-hTSHR/pRc/CMV, a mutant hTSHR cDNA which encodes a hydrophilic peptide insert (AGTTRRVAI) and C-hTSHR/pRc/CMV which encodes a hydrophobic peptide insert (ATVLVVPMI) between +486 Ileu and +487 Asp of hTSHR were transfected into Chinese hamster ovary cells to generate the B-1 and C-6 cell lines, respectively. Neither thyrotropin (TSH) nor thyroid stimulating antibody (TSAb) stimulated cAMP production by B-1 or C-6 cells. An 125I-TSH binding assay showed that neither cell line bound TSH. Our data demonstrated that these mutations impaired both TSH binding and cAMP production. This evidence suggests that the first extracellular loop of hTSHR may have a crucial role in the TSH- and TSAb-dependent signal transduction.

Animals↗
PubMed1994

Desensitization and internalization of a thyrotrophin receptor lacking the cytoplasmic carboxy-terminal region.

To understand the functional significance of the carboxy-terminal half of the intracellular region of the human TSH receptor (hTSHR), a mutant hTSHR lacking amino acids from the carboxy-terminal to His726 was constructed. Wild type hTSHR cDNA and truncated hTSHR cDNA were subcloned into a eukaryotic expression vector, pRc/CMV, and transfected into Chinese hamster ovary cells to obtain cell lines which stably expressed hTSHRs at high levels. This allowed us to observe highly efficient coupling of hTSHR and adenylyl cyclase as well as desensitization and internalization of hTSHR. Despite the differences in potential phosphorylation sites and internalization signals, dose-dependent stimulation of adenylyl cyclase by TSH, TSH-dependent desensitization and the rate of hTSHR internalization were similar for wild type and truncated hTSHRs. We conclude that the carboxy-terminal half of the intracellular region of hTSHR does not have a major functional role in TSH-dependent signal transduction.

Amino Acid Sequence↗
PubMed1994

[The structure and function of the extracellular domain of human thyrotropin receptor].

Since the molecular cloning of human TSH receptor (TSHR), much efforts have been focused on determining the TSH or TSAb binding sites. Despite many conflicting data, there seems to be a consensus that TSH binds to the various parts of the extracellular domain of TSHR discontinuously. However, following questions are not yet answered 1) Is the binding to extracellular domain of TSHR sufficient to the signal transduction? 2) Is there any contribution of the extracellular loops to TSH binding? Using TSHR mutated at the first or the second extracellular loops, we propose that the extracellular loops are important for TSH binding and signal transduction.

Amino Acid Sequence↗
PubMed1993

A taxonomic review of the genus Microbispora by analysis of ribosomal protein AT-L30.

We analyzed the ribosomal AT-L30 proteins from 13 type strains of species belonging to the genera Microbispora and Actinomadura. The electrophoretic mobilities of the AT-L30 preparations from Microbispora strains, as determined by two-dimensional polyacrylamide gel electrophoresis, revealed that the members of the genus Microbispora are phylogenetically homogeneous. The results of partial amino acid sequencing of AT-L30 preparations from several representative Microbispora strains supported the separation of the genus Microbispora from other related genera. The amino acid sequences of the AT-L30 proteins from strains of species belonging to the genus Actinomadura sensu stricto displayed a diversity that exemplified the low levels of amino acid sequence homology within the genus. This diversity was considered to be a characteristic typical of the genus Actinomadura.

Amino Acid Sequence↗
PubMed1993

Thyrotrophin-dependent desensitization by Chinese hamster ovary cells that express the recombinant human thyrotrophin receptor.

To determine whether thyrotrophin (TSH)-induced desensitization requires a thyroid-specific factor(s), the human TSH (hTSH) receptor was expressed in Chinese hamster ovary cells. The first incubation of the cells with TSH decreased the subsequent response of adenosine 3',5'-cyclic monophosphate to freshly added TSH in the second incubation. This homologous desensitization was observed as early as after 3 h of the first incubation. The lowest dose of TSH that elicited desensitization was 0.1 nmol/l. The desensitization was not overcome by adding higher doses of TSH in the second incubation. A 125I-labelled TSH-binding study revealed a decrease in the number of high-affinity binding sites but not in that of low-affinity binding sites. The data suggest that TSH-induced desensitization in hTSH receptor-transfected cells is caused, at least in part, by a decrease in the number of TSH receptors on the cell surface. The evidence demonstrates, contrary to an earlier report, that a thyroid-specific factor(s) is not required for hTSH receptor desensitization.

Animals↗
PubMed1993

Stereo-controlled C-C bond formation at the anomeric position of uracil nucleosides.

Stereoselective synthesis of 1'-carbon-substituted uracil nucleosides has been achieved through face-selective bromo-pivaloyloxylation of a 1',2'-unsaturated derivative and successive SnCl4-promoted nucleophilic substitution with organosilicon reagents. This constitutes the first example of C-C bond formation at the anomeric position of nucleosides.

Carbon↗
PubMed1993

[Change of PCDF concentrations in the blood of Yusho patients in recent ten years].

The blood of 16 Yusho patients sampled from 1982 (14 years after the incident) to 1992 were analyzed for congeners of polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) by high resolution gas chromatography-mass spectrometry and gas chromatography with electron capture detection, respectively. Biological half-lives of PCDFs and PCBs were estimated by following the change of blood concentrations of the Yusho patients in the equation of 1-compartment model. The biological half-lives of 2, 3, 4, 7, 8-penta-CDF and 1, 2, 3, 4, 7, 8-hexa-CDF in 8 individuals varied from 6.0 to 76 years with median 11.7 years and from 4.8 to 26.5 years with median 7.0 years, respectively. The half-lives of 1, 2, 3, 4, 6, 7, 8-hepta-CDF in 4 individuals varied from 2.1 to 15 years. Concentrations of PCBs in 13 individuals were estimated to be slowly decreased and increased with several years half-life in about half-and-half numbers.

Aged↗
PubMed1993

[PCB methylsulfone accumulated in muscle of mink dosed with PCB (Clophen A50)].

The structural requirements of the parent PCBs for the formation of the retained PCB methylsulfones (MeSO2-CBs) were investigated in mink (Mustela vison) exposed to PCB (Clophen A50). Muscle was analyzed for the MeSO2-CBs, which were determined by comparison of synthesized reference compounds. Major unmetabolized CBs such as 2, 4, 5, 2', 4'-pentaCB, 2, 4, 5, 2', 3', 4'-hexaCB, 2, 4, 5, 2', 4', 5'-hexaCB and 2, 3, 4, 5, 2', 4', 5'-heptaCB were determined in high concentrations in the mink muscle. All these PCBs are substituted in the 2, 4, 5-positions of at least one of the phenyl rings. On the other hand, CBs with free meta/para-positions were readily metabolized. Thus, at least 25 MeSO2-CBs were detected at concentration of 16 micrograms/g in extracted lipids from the muscle, corresponding to one tenth of PCB levels. Most of the methyl sulfone metabolites were 3- and 4-MeSO2-CB isomers of PCBs known to be rapidly metabolized, e.g. 2, 4, 2', 5'-tetraCB, 2, 3, 6, 4'-tetraCB, 2, 5, 3', 5'-pentaCB, 2, 4, 5, 2', 5'-pentaCB, 2, 3, 4, 2', 5'-pentaCB, 2, 3, 6, 3', 4'-pentaCB, 2, 3, 6, 2', 4', 5'-hexaCB and 2, 3, 6, 2', 3', 4'-hexaCB. 3-MeSO2-2, 5, 2', 5'-tetraCB and 3-MeSO2-2, 5, 6, 2', 5'-pentaCB were also found to be retained in the muscle, but their isomeric 4-MeSO2-CBs were not detected. Both the 3- and 4-MeSO2-2, 5, 6, 2', 4', 5'-hexaCB isomers were identified in the muscle extracts while no MeSO2-CB metabolite originating from 2, 3, 2', 4', 5'-pentaCB were detected in the muscle. These results show the PCBs with at least one phenyl ring with 2, 5-dichloro- and 2, 3, 6-trichloro-substitution are strongly favored and may be considered a criterion for the formation of MeSO2-CBs accumulated in mink muscle. These observations are in accordance with MeSO2-CBs detected also in other mammals.

Animals↗
PubMed1993

[Accumulation of hydroxylated PCB metabolites in blood].

In order to investigate some potential accumulate properties of hydroxylated PCB metabolites, blood and tissues such as livers, kidneys, lungs and adipose tissue from rats exposed to a commercial PCB (Aroclor 1254) were analyzed for hydroxylated PCBs (OH-PCBs). A large number of OH-PCBs were detected in the liver 1 day after exposure among which almost 13 have been identified in the blood. OH-PCBs were retained selectively in the blood probably due to binding between the OH-PCBs and a protein in serum. One major OH-PCB determined in rat blood was 4-OH-2', 3, 3', 4', 5-pentaCB formed from 2, 3, 3', 4, 4'-pentaCB after NIH shift. Blood of a grey seal from the Baltic also contained at least 8 OH-PCB congeners with 2 major compounds determined as 4-OH-2', 3, 3', 4', 5-pentaCB and 4-OH-2, 3, 3', 4', 5, 5'-hexaCB. Thus, 4-OH-2', 3, 3', 4', 5-pentaCB is the most retainable OH-PCB congener in blood from rats and seal. Human serum have also been found to contain a few number of OH-PCBs at concentration of 0.6 ng/g serum which corresponds to one fourth of PCB level.

Animals↗
PubMed1992

Single subunit structure of the human thyrotropin receptor.

We have produced rabbit antibody against a synthetic peptide corresponding to N-terminal region of the extracellular domain of human thyrotropin receptor (hTSH-R) (N peptide, aminoacid residues 29-57). Western blot analysis revealed that N-peptide antibody recognized recombinant hTSH-R stably expressing in CHO-K1 cells as a mol. wt. about 104 kDa regardless in the presence or absence of disulfide-reducing agent. The band was not detected in untransfected CHO-K1 cells and no band was also stained by the antibody absorbed with N-peptide. In a reducing condition, the antibody also bound the rat receptor from FRTL5 cells as the same molecular size (104 kDa). These results clearly indicate that TSH-R is composed of a single subunit and that two subunit model for the TSH-R may reflect artifactual proteolytic cleavage of the receptor during membrane preparation.

Amino Acid Sequence↗
PubMed1992

Beta 2-adrenergic receptor mRNA is overexpressed in neoplastic human thyroid tissues.

The expression of beta 2-adrenergic receptor (AR) mRNA was investigated in normal and neoplastic human thyroid tissues. A combination of techniques for reverse transcribing mRNA into cDNA and the incorporation of 32P-gamma ATP into the polymerase chain reaction (PCR)-generated fragments allowed us to detect beta 2-AR mRNA in surgically excised thyroid specimens. The levels of beta 2-AR cDNA generated by PCR in thyroid adenomas and cancers were 3.3 and 6.9 times, respectively, as high as that of normal thyroid tissues. These findings suggest that the level of beta 2-AR mRNA is correlated with the extent of differentiation in neoplastic tissues. The present study provides new insights into the relationships between the AR-adenylate cyclase system and the regulation of the growth and differentiation in neoplastic human thyroid tissues.

Base Sequence↗
PubMed1991

Mouse submandibular gland prorenin-converting enzyme is a member of glandular kallikrein family.

Mouse submandibular gland prorenin-converting enzyme (PRECE) consists of the two polypeptide chains of 17 and 10 kDa and cleaves mouse Ren-2 prorenin at a dibasic site to yield mature renin. Western blot analysis using an antiserum against this enzyme gave rise to multiple bands in mouse submandibular glands, suggesting that PRECE is a member of a protease family. Partial amino acid sequence analysis of purified PRECE and cloning and sequence analyses of its cDNA indicated that it is identical to the mGK-13 gene product, epidermal growth factor-binding protein type B, which is a member of the glandular kallikrein family and is involved in maturation of epidermal growth factor. Conditioned medium from Chinese hamster ovary cells transfected with an expression plasmid for PRECE had prorenin converting activity. These results indicate that PRECE is involved in the maturation of two bioactive polypeptides expressed in mouse submandibular glands, Ren-2 renin and epidermal growth factor.

Amino Acid Sequence↗
PubMed1991

Thyrotropin receptor non-mediated thyroid stimulating immunoglobulin in Graves' disease.

There exists a consensus that hyperthyroid Graves' disease is caused by thyrotropin receptor (TSH-R) autoantibodies. To test the possibility that the TSH-R is the sole antigen for thyroid stimulating antibodies (TSAb), we compared bioactivities of Graves' IgGs between non-thyroid mammalian cells transfected with human TSH-R cDNA and the reference thyroid bioassay. A Graves' IgG with TSH-binding inhibitor immunoglobulin (TBII) activity (89%) markedly stimulated cAMP formation in both CHO-K1 cells transfected with TSH-R cDNA (340 microU/ml of TSH equivalent) and rat thyroid cells, FRTL-5, (410 microU/ml of TSH equivalent). In contrast, a TBII negative (-1.5%) IgG from another patient with Graves' disease showed a strong thyroid stimulating activity (87 microU/ml of TSH equivalent) when FRTL-5 cells were used for the assay. But no stimulating activity was observed in this IgG when CHO-K1 cells transfected with TSH-R cDNA were used, suggesting a possible existence of TSH-R non-mediated thyroid stimulating immunoglobulin in some cases of Graves' disease.

Animals↗
PubMed1991

Carbachol-activated muscarinic (M1 and M3) receptors transfected into Chinese hamster ovary cells inhibit trafficking of endosomes.

We examined the effects of isoproterenol and carbachol on fluid-phase endocytosis by Chinese hamster ovary (CHO) cells transfected with beta-adrenergic, M1, or M3 cholinergic receptors. Isoproterenol increased cAMP production and carbachol increased intracellular Ca, indicating successful expression of the receptor genes and coupling to typical signal transduction pathways. Carbachol inhibited the uptake of horseradish peroxidase (HRP) or Lucifer yellow (markers of fluid-phase endocytosis) in both M1- and M3-containing cells but not in wild-type cells, whereas isoproterenol did not affect pinocytosis in cells transfected with beta-adrenergic receptors. Carbachol inhibited the transit of HRP from an exchangeable pool to a nonexchangeable pool by a latent process requiring minimally 5 min of incubation. During the latent period, only one peak of low-density HRP-containing vesicles was found on Percoll gradients; after 5 min, HRP appeared in both high- and low-density vesicles. Carbachol-treated cells contained less HRP in the high-density fraction enriched in lysosomal markers. Early endosomes from CHO cells labeled for 5 min with HRP underwent fusion to make a more dense population of vesicles in the presence of ATP and KCl at 37 degrees C but not at 4 degrees C. The fused material contained increased levels of G proteins as detected either by ADP ribosylation with appropriate toxins or by immunoblotting with specific antibodies. These findings suggest that GTP binding proteins are internalized in endocytic vesicles and enter into a complex trafficking process involving fusion with other vesicular compartments. Trafficking of endosomes to these compartments is inhibited by activated M1 and M3 muscarinic receptors in CHO cells.

Animals↗