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Biomedical subjects

J Miyazaki

Publications and source records attributed to J Miyazaki.

At least 127 records · Page 7Linked to original sources
PubMed1996

Differences in inducibility of CYP1A1-mRNA by benzimidazole compounds between human and mouse cells: evidences of a human-specific signal transduction pathway for CYP1A1 induction.

Three benzimidazole compounds, omeprazole (OP), thiabendazole (TBZ), and lansoprazole (LP), were compared with respect to the induction of CYP1A1-mRNA in human hepatoma cells, HepG2. OP was the most potent inducer among the three compounds, but LP was found to be a weak inducer. Induction by TBZ was at an intermediate level. None of these compounds induced CYP1A1-mRNA in a mouse hepatoma cell line, Hepa-1. The transient expression of mouse Cyp1a1-CAT gene into HepG2 cells showed that OP treatment of the transfectants induced CAT activity to the same degree as 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment. Therefore, the cellular factors in human cells were able to work on the mouse regulatory element. The expression of human aryl hydrocarbon (Ah) receptor in the mouse Hepa-1 mutant cell line cl-19, which is defective in Ah receptor, did not increase the induction level of CYP1A1-mRNA by OP treatment. When the cultured medium of HepG2 cells in the presence of OP was added to the mouse Hepa-1 cell culture medium, CYP1A1-mRNA was not induced in Hepa-1 cells. It is thus concluded that metabolites of OP in human cells are not the ligands for the human Ah receptor. Therefore, in human cells, but not mouse cells, there must be an OP-sensitive activation factor for the human Ah receptor.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1996

Cloning of chicken slow muscle troponin T and its sequence comparison with that of human.

A full-length cDNA coding for chicken slow muscle troponin T (TnT) was for the first time isolated from a cDNA library of 10-day-old embryos, using an RT-PCR product of chicken slow muscle TnT. It showed about 60% homology for chicken fast and slow muscle TnTs and 75.2% for human slow muscle TnT. The 16 amino acid sequence found in the carboxyl terminus of human slow muscle TnT was absent in the chicken slow muscle TnT. The 5E-5A-7E sequence found in the amino terminal region of chicken slow muscle TnT was partly similar to the counterpart of human slow muscle TnT, but not to those of chicken fast and cardiac muscle TnTs. With this report of chicken slow muscle TnT, cDNA information on chicken TnTs of all three striated muscles was completed following those of human TnTs.

Amino Acid Sequence↗
PubMed1996

Proprotein-processing endoprotease furin decreases regulated secretory pathway-specific proteins in the pancreatic beta cell line MIN6.

Prohormone convertases PC2 and PC3, yeast Kex2-family endoproteases specific to the regulated secretory pathway, cleave proinsulin to insulin in the secretory granules of pancreatic beta cells. The well-differentiated beta cell line MIN6 expresses PC2 and PC3 and another regulated secretory pathway-specific protein chromogranin A. Furin, another yeast Kex2 endoprotease, exists in the trans-Golgi networks of many cell types. The beta cell line RINm5F (a cell line that is less differentiated than the MIN6 cell line) does not express the regulated pathway-specific proteins, but strongly expresses furin. We suspected that furin expression may cause the decrement of regulated secretory pathway-specific proteins. To test this hypothesis, we expressed a furin cDNA with a metallothionein promoter in MIN6 cells. With Zn2+ stimulation of furin expression, the messages of PC2, PC3, and chromogranin A decreased, and the processing of proinsulin to mature insulin became less efficient. The furin-expressing MIN6 cells exhibited less insulin content and weakened insulin secretion in response to a high glucose concentration. The conditioned medium from furin-expressing MIN6 cells also exerted a decrease of PC2 and PC3 expression in unaltered MIN6 cells. Thus, proteins cleaved by furin inside the cells or by truncated furin shed into the culture medium appear to cause decreased PC2 and PC3 expression, insulin content, and glucose-responsive insulin secretion in MIN6 cells.

Amino Acid Sequence↗
PubMed1996

Calcium as a second messenger of the action of transforming growth factor-beta on insulin secretion.

In MIN6 insulinoma cells, transforming growth factor-beta (TGF-beta) induced the oscillatory elevation of the cytoplasmic free calcium concentration, [Ca2+]c, in the presence of 5.5 mM glucose. The increase in [Ca2+]c induced by TGF-beta was totally dependent on calcium entry and attenuated by nifedipine or nickel chloride. In contrast, carbachol elevated [Ca2+]c in the presence of nickel chloride. When the plasma membrane was hyperpolarized by diazoxide, TGF-beta did not raise [Ca2+]c, whereas both carbachol and depolarizing concentration of potassium elevated [Ca2+]c under the same conditions. TGF-beta did not affect either the cellular cyclic AMP or inositol trisphosphate levels. In the presence of 5.5 mM glucose, TGF-beta induced a 3-fold increase in insulin secretion and the effect of TGF-beta was blocked by either nifedipine or nickel chloride. TGF-beta did not stimulate insulin secretion in the presence of 100 microM diazoxide, whereas both carbachol and 40 mM potassium chloride significantly increased insulin secretion. These results suggest that TGF-beta induces the oscillatory elevation of [Ca2+]c in MIN6 cells by stimulating calcium entry via voltage-dependent calcium channels. Calcium is an intracellular messenger of the action of TGF-beta on insulin secretion.

Calcium↗
PubMed1996

Detection of autoantibodies to the pancreatic islet heat shock protein 60 in insulin-dependent diabetes mellitus.

Autoantibodies against heat shock protein (hsp) 60 have been reported to be detected in sera of non-obese diabetic mice, in an experimental model of IDDM. However, there are only a few studies which have examined IDDM patients for antibodies against mammalian hsp60. We produced murine hsp60 derived from pancreatic beta cells which has high homology to human hsp60 and examined antibodies against the hsp60 in IDDM patients using an enzyme-linked immunosorbent assay. We extended the analysis to patients with other immune-mediated diseases and non-insulin-dependent diabetes mellitus (NIDDM). Positive sera for hsp60 antibody were more frequently detected in 13 out of 84 IDDM (15.5%) and 5 out of 25 rheumatoid arthritis patients (20%), when compared to healthy subjects (1/85; 1.2%, P < 0.001 and P < 0.01, respectively). The levels of hsp60 antibodies of IDDM (0.218 +/- 0.227) and rheumatoid arthritis patients (0.259 +/- 0.191) were significantly higher than those of healthy subjects (0.076 +/- 0.131, P < 0.001, P < 0.01, respectively). Patients with slowly progressive IDDM (n = 26), autoimmune thyroid disease (n = 42), or NIDDM (n = 40) had levels of hsp60 antibodies similar to those in healthy subjects. We found no relationship between the levels of hsp60 antibodies and islet cell antibodies (ICA) or antibodies to glutamic acid decarboxylase (GAD65) in IDDM patients. In conclusion, hsp60 antibodies were detected in Japanese IDDM as well as in rheumatoid arthritis patients. Although the positivity was low, the detection of hsp60 antibodies may be helpful for diagnosis of IDDM especially in GAD65 Ab- or JCA-negative Japanese patients.

Autoantibodies↗
PubMed1996

Neuroendocrine cells in the cat laryngeal epithelium.

The structure and distribution of neuroendocrine cells in the feline laryngeal epithelium were examined using immunohistochemical techniques. Neuroendocrine cells were often spindle shaped, with cytoplasmic processes directed towards the lumen and basement membrane. The apical portion of the cells usually reached the laryngeal lumen with microvillous projections. The cytoplasm always contained variable numbers of electrondense cored vesicles. The number of neuroendocrine cells decreased in the following order: subglottis, posterior glottis, supraglottis, anterior glottis. Neuroendocrine cells contained calcitonin gene-related peptide, substance P and/or 5-hydroxytryptamine. They also showed protein gene product 9.5 or neuron-specific enolase immunoreactivity. These observations suggest that neuroendocrine cells play a part in the regulatory function of the cat larynx by releasing various peptides. These substances may contribute to allergic reactions or control mucus secretion by acting via the endocrine or paracrine pathways and/or neurosecretory pathways.

Animals↗
PubMed1996

The significance of E-cadherin in transitional-cell carcinoma of the human urinary bladder.

E-cadherin is a Ca(2+)-dependent intracellular adhesion molecule in the epithelial tissue. The urothelium also expresses E-cadherin molecules. We determined E-cadherin expression in bladder carcinoma immunohistochemically and investigated its relationship with pathological and clinical data. The percentage of cases showing pattern B, less than 50% of which cancer cells expressed the same intensity of E-cadherin as did the normal epithelium, was higher in cases of high-grade or high-stage tumors as compared with those of low grade or low stage. Heterogeneous staining was observed in the same specimens, which suggested changes in the cell phenotype. Immunoblotting demonstrated no evidence of gross alteration of E-cadherin molecules. The decrease in E-cadherin expression was associated with the invasiveness of bladder carcinoma, as has been reported in other carcinomas.

Cadherins↗
PubMed1996

GGAAAT motifs play a major role in transcriptional activity of the human insulin gene in a pancreatic islet beta-cell line MIN6.

The insulin gene is specifically expressed in pancreatic islet beta cells. Various cis-acting DNA elements in the 5'-flanking region of the human insulin gene were examined for their contribution to the transcriptional activity using sensitive human growth hormone (hGH) reporter plasmids. The hGH constructs, having successively deleted human insulin promoter sequences, were transfected to a pancreatic islet beta-cell line MIN6. The deletion of two GGAAAT (GG) motifs, GG2 at -145 to -140 bp and GG1 at -134 to -129 bp, decreased the transcriptional activity to 6.5% of that of the promoter sequence from -156 to +1 bp. The selective mutations in both GG motifs also decreased the transcriptional activity to 5.5%. One-base mutations of GG2 and GG1 decreased the transcriptional activity to 82 and 11%, respectively. The two-base mutations between GG2 and GG1 affected the transcriptional activity more strongly than those just outside the GG motifs. A single set of GG motifs in the upstream of thymidine kinase promoter increased the transcriptional activity to 216% compared to that of thymidine kinase promoter alone in MIN6 cells. With an electrophoretic mobility shift assay (EMSA), a nuclear factor in MIN6 cells was shown to bind the DNA fragments containing two GG motifs. This factor did not bind to another GGAAAT-like sequence at -313 to -305 bp in the human insulin gene. These results suggested that the GG motifs contributed to the cell-specific transcription of the human insulin gene in association with the binding of the sequence-specific nuclear factor.

3T3 Cells↗
PubMed1996

Rectification of diabetic state in C57BL/KsJ-db/db mice by the implantation of pancreatic beta cell line MIN6.

C57BL/KsJ mice carrying homozygous db/db mutation (db/db mice) are characterized by extreme obesity and early onset of hyperglycemia. In an attempt to rectify diabetes of these mice, a pancreatic beta cell line MIN6, which retains glucose-inducible insulin secretion, was transplanted subcutaneously into the back of the mice. Glucose and insulin levels of individual mice were examined biweekly and their weight gain weekly. All mice were sacrificed at 100 days after the transplantation of MIN6 cells. In db/db mice that had received MIN6 cells, blood insulin levels were restored and blood glucose levels were reduced to those of non-diabetic mice, although they remained obese. Glucose tolerance test suggested that transplanted MIN6 cells responded to loaded glucose as beta cells of non-diabetic mice. Immunohistochemical study showed that transplanted MIN6 cells produced insulin. Fatty liver associated with diabetes mellitus observed in db/db mice was not found in the MIN6 cell-transplanted mice. Implication of the results is discussed with reference to potential therapies for severe diabetes.

Animals↗
PubMed1996

Defective B-1 cell development and impaired immunity against Angiostrongylus cantonensis in IL-5R alpha-deficient mice.

We generated interleukin-5 receptor alpha chain (IL-5R alpha)-deficient (IL-5R alpha-/-) mice by gene targeting. The IL-5R alpha-/- mice showed decreased numbers of B-1 cells concomitant with low serum concentrations of IgM and IgG3. They showed no IL-5-induced enhancement of B cell responses to T-independent antigens. The number of alpha beta T cell receptor-positive thymocytes tended to decrease in 3-week-old IL-5R alpha-/- mice, returning to normal by 6 weeks of age. The IL-5R alpha-/- mice produced basal levels of eosinophils, while their bone marrow cells failed to form eosinophilic colonies in response to IL-5. Impaired eosinophilopoiesis in IL-5R alpha-/-mice enhanced the survival of Angiostrongylus cantonensis. These results indicate that IL-5-induced eosinophils serve as potent effector cells in the killing of Angiostrongylus cantonensis in mice.

Angiostrongylus cantonensis↗
PubMed1996

Clonal prevalence of T cells infiltrating into the pancreas of prediabetic non-obese diabetic mice.

The non-obese diabetic (NOD) mouse spontaneously develops T-cell-mediated autoimmune insulitis. We analyzed the clonotypes of T cell infiltrates of the NOD mouse islets using a new method we have developed recently, which consists of RT-PCR amplification of the CDR3 region of the TCR beta chain mRNA and subsequent single-strand conformation polymorphism (SSCP) analysis. NOD mice of 10-32 weeks of age were shown to accumulate oligoclonal T cells in the pancreas. To examine whether each T cell clone stays in a small area of the pancreas or spreads over the whole pancreas, a pancreas was divided into two pieces, which were then subsequently analyzed in a pair by the above PCR-SSCP method. When a pair produces common bands with the same mobility in SSCP gel, they are likely to represent the presence of the same T cell clones between these two parts of the pancreas. Aged mice (24-32 weeks old) with severe insulitis obviously produced more common bands for most of the Vbeta subfamilies than younger mice (10 weeks old) with only periinsulitis. DNA sequencing verified that these common bands have the same TCR junctional sequences, suggesting that they were derived from the same T cell clones. These results suggest that clonal prevalence of T cells infiltrating into the pancreas occurs in the late stage of insulitis development and that a limited number of T cell clones finally predominate over the whole pancreas.

Animals↗
PubMed1996

Preferential requirement of CD3 zeta-mediated signals for development of immature rather than mature thymocytes.

Antigen recognition signals by the TCR are transduced through activation motifs present in the cytoplasmic region of CD3 chains. In vitro analysis has suggested that the CD3zeta chain mediates different signals from other CD3 chains. To analyze the in vivo function of CD3zeta-mediated signals for T cell development, mice expressing a mutant CD3zeta chain lacking all the activation motifs were generated by introducing the transgene into zeta-knockout mice. Mature CD4(+) single-positive (SP) thymocytes in these mice were greater in number than in zeta-deficient mice, and the promoted differentiation was indicated by the changes of CD69 and HSA phenotypes. We found that even in the absence of activation motifs in CD3zeta, these mature cells became functional, being able to induce Ca2+ mobilization and proliferation upon stimulation. On the other hand, CD4(-)CD8(-) double-negative (DN) thymocytes, most of which were arrested at the CD44(-)CD25(+) stage similarly to those in zeta-deficient mice, could not be promoted for differentiation into CD4(+)CD8(+) double-positive thymocytes in these mice in spite of the fact that the expression of the transgene in DN thymocytes was higher than that of zeta in wild-type mice. These results demonstrate the preferential dependence of the promotion of development and/or expansion of DN thymocytes rather than mature thymocytes upon the activation signals through the zeta chain and suggest differential requirements of TCR signaling for mature SP and immature DN thymocyte developments in vivo.

Animals↗
PubMed1996

Muscle-type tropomyosin of sea urchin egg increases the actin-binding of nonmuscle-type tropomyosin.

Tropomyosin isoforms in eggs of several species of sea urchins are classified into two types, muscle-type and nonmuscle-type, based on their antigenicities. Their actin-binding abilities were investigated using muscle-type isoform (32K) and nonmuscle-type isoform (30K), which were purified by the method previously reported and separated by isoelectric focusing from eggs of sea urchin, Strongylocentrotus intermedius. Co-sedimentation assays revealed that 32K could stoichiometrically bind to actin filaments independently of the 30K, but 30K alone bound very poorly. The actin-binding of 30K was, however, considerably increased in the presence of 32K, and the molar ratio of the bound 30K and 32K was approximately 1:1. The increase in the actin-binding of 30K is probably caused by the interaction of 30K with 32K in a head-to-tail manner, as indicated by the higher specific viscosity of the mixture than that of 32K alone.

Actins↗
PubMed1996

Two distinct signaling pathways activated by activin A in glucose-responsive pancreatic beta-cell lines.

Activin A stimulates insulin secretion in pancreatic beta-cells by a calcium-dependent mechanism. The present study was conducted to further characterize the effects of activin A in two glucose-responsive insulinoma cell lines, MIN6 and HIT-T15 cells. In HIT-T15 cells, activin A evoked an increase in cytoplasmic free calcium concentration, stimulated insulin secretion, maintained glucose responsiveness of the cells and inhibited DNA synthesis. However, activin A did not have any effect in MIN6 cells. Measurement of 125I-labeled activin A binding in MIN6 cells revealed that the number of binding sites was markedly reduced, suggesting that the refractoriness was due, at least partly, to the reduced numbers of the activin receptor. Stable transfectants of MIN6 cells that overexpressed the type II activin receptor were then developed. The transfected cells (MIN6-ActR cells) expressed ten times more 125I-labeled activin A-binding sites than parental cells and the apparent Kd was 1.15 nM, which was nearly identical to that in parental cells. Affinity cross-linking in MIN6-ActR cells showed that a 90 kDa type II receptor as well as a 52 kDa protein, presumably follistatin, was markedly labeled with 125I-labeled activin A. Although MIN6-ActR cells expressed significant numbers of activin receptors, activin A did not induce immediate calcium-dependent responses in these cells. In contrast, activin A was capable of inducing long-term effects in MIN6-ActR cells; thus, reduction of the glucose concentration in culture medium from 25 to 5.5 mM for 4 days resulted in a remarkable loss of insulin response to glucose stimulation but this decline in response to glucose was prevented by the addition of activin A during culture. In addition, activin A inhibited DNA synthesis in MIN6-ActR cells. Hence, although activin A did not induce calcium-dependent responses, it evoked some calcium-independent effects in MIN6-ActR cells. Taken together, activin A elicits various effects in beta-cells by both calcium-dependent and -independent mechanisms.

Activin Receptors↗
PubMed1996

Expression of chicken troponin T isoforms in cultured muscle cells.

Cells prepared from chicken skeletal muscles of different developmental stages were cultured to study their troponin T isoform expression, using antisera specific to the fast- and slow-muscle-type isoforms. We found that the cultured myogenic cells from chickens and chick embryos were classified into two types, fast type and fast/slow type in which fast- and slow-muscle-type isoforms were coexpressed. Cells expressing only slow-muscle-type troponin T isoforms could not be found. Most cells prepared from pectoralis major (fast muscle) and gastrocnemius (mixed muscle) of 11-day old embryos belonged to the latter, with only a small fraction belonging to the former. The percentage of fast type cells in those cells prepared from pectoralis major increased along development to over 90% by the 17th day of incubation, while, in the cells prepared from gastrocnemius, it reached a plateau of 30-40% by the 13th day of incubation. All the cells from anterior latissimus dorsi (slow muscle) belonged to the fast/slow type. Ratios of these two types of muscle cells varied depending on their origins and stages. The in vitro expression of troponin T isoforms was different from the in vivo expression, and each muscle seems to be determined differently in the composition of cell types during the developmental course.

Animals↗
PubMed1996

Immune response to heat-shock protein correlates with induction of insulitis in I-E alpha d transgenic NOD mice.

To evaluate the correlation between heat-shock protein (HSP) and insulitis, we compared lymphocyte proliferative response to Mycobacterium leprae HSP65 of NOD mice with that of I-E alpha d transgenic NOD (I-E+NOD) mice, which show no insulitis. We found that splenocytes from 15-week-old NOD mice showed a more marked proliferative response to HSP than did those from age-matched I-E+NOD mice (P < 0.05). We then transferred splenocytes from 12-week-old NOD mice into I-E+NOD mice to induce insulitis in the recipients and examined antibody levels against HSP. By 6 weeks posttransfer, insulitis was successfully transferred to four out of five recipients of NOD splenocytes and antibody levels against HSP were significantly higher in the NOD splenocyte-transferred group than in controls, which showed no insulitis (P < 0.01). These results suggest that immune response to HSP correlates with insulitis in NOD mice. Our results support the assertion that HSP is a useful antigen for investigating the etiology of IDDM.

Animals↗
PubMed1996

Effect of mitochondrial and/or cytosolic glycerol 3-phosphate dehydrogenase overexpression on glucose-stimulated insulin secretion from MIN6 and HIT cells.

The glycerol phosphate shuttle consists of FAD-linked mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) and its cytosolic NAD-linked isoform (cGPDH). Impaired mGPDH activity has recently been suggested to be one of the primary causes of insulin secretory defects in beta-cells. We found that mGPDH and cGPDH activities in MIN6 cells are comparable to those of isolated islets and higher than those in HIT cells by eightfold and threefold, respectively. Therefore, we selected the MIN6 cell line as a beta-cell model with normally regulated insulin secretion and normal shuttle enzyme activities and the HIT cell line as a beta-cell model with impaired insulin secretion and lower activities of these enzymes. The role of these dehydrogenases in glucose-stimulated insulin secretion was addressed by examining the effects of overexpression of mGPDH and/or cGPDH via recombinant adenoviruses in these cells. Infection with recombinant adenovirus with a cDNA encoding the Escherichia coli beta-galactosidase gene resulted in expression of its gene in 90% of MIN6 and HIT cells. Infection with a recombinant adenovirus with mGPDH cDNA (Adex1CAmGPDH) caused 2.1-fold and 5.7-fold increases in dehydrogenase activity as compared with those of control MIN6 and HIT cells, respectively. Infection with a recombinant adenovirus with cGPDH cDNA (Adex1CAcGPDH) caused a more than 50-fold increase in activity in both cell lines. Glycerol phosphate shuttle flux, as estimated by [2-3H]glycerol conversion to [3H]H2O, was increased to 120-130% by infection with Adex1CAmGPDH, but not with Adex1CAcGPDH infection, in both MIN6 and HIT cells. No further increase in flux through the glycerol phosphate shuttle was detected when the cells were infected with Adex1CAmGPDH together with Adex1CAcGPDH. Furthermore, neither [U-14C]glucose oxidation nor the insulin secretory response to glucose was affected in either cell line. Thus, mGPDH abundance in MIN6 and HIT cells is not directly related to their insulin secretory capacity in response to glucose, and reduced expression of mGPDH is not the primary cause of abnormal insulin secretory responses in HIT cells. The present data indicate that the emerging hypothesis pointing to mGPDH deficiency as a possible cause of NIDDM needs to be carefully evaluated.

Adenoviridae↗