Biomedical subjects

J Miyazaki

Publications and source records attributed to J Miyazaki.

At least 109 records · Page 6Linked to original sources

PubMed1998

Distribution and origin of the intraepithelial nerve fibres in the feline pharyngeal mucosa.

Sensory inputs from the pharynx play an important role in initiation of the swallowing reflex and in feedback control of motor activities. Using an immunohistochemical technique and denervation procedures, we examined the distribution and origin of the intraepithelial nerve fibres in the feline pharyngeal mucosa to clarify the role of the afferent nerve in swallowing. The posterior pillar was very densely innervated, and the posterior and lateral walls of the mesopharynx had a moderate nerve density. In contrast, the base of the tongue, the vallecula, the pharyngeal surface of the epiglottis, and the pyriform sinus had only a few nerve fibres. The epithelium of the rostral and caudal portions of the pharyngeal mucosa were innervated by the glossopharyngeal nerve and superior laryngeal nerve, respectively, with a borderline at the middle level of the epiglottis. A portion of the intraepithelial nerve fibres in the lateral and posterior walls of the mesopharynx originated from the pharyngeal branch of the vagus nerve. It is hypothesized that the intraepithelial nerve fibres that exist in densely innervated areas are related to the initiation of the swallowing reflex induced by stimulation of the pharyngeal mucosa.

Afferent Pathways↗

PubMed1998

Distribution of NADPH-diaphorase activity in the feline laryngeal mucosa.

We studied nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity in the feline laryngeal mucosa using a histochemical technique in an effort to clarify the role of nitric oxide (NO) in the larynx. Many NADPH-diaphorase-positive nerve fibres were distributed around the blood vessels and the laryngeal glands. The majority of neuronal cells in the intralaryngeal ganglia were NADPH-diaphorase-positive. It is likely that NADPH-diaphorase-positive nerve fibres around the blood vessels and glands in the laryngeal mucosa originate from the intralaryngeal ganglia, and that NO regulates circulation and secretion in the larynx.

Animals↗

PubMed1998

Mutation P291fsinsC in the transcription factor hepatocyte nuclear factor-1alpha is dominant negative.

The type 3 form of maturity-onset diabetes of the young (MODY3) results from mutations in the gene encoding the transcription factor, hepatocyte nuclear factor-1alpha (HNF-1alpha). The mechanism by which mutations in only one allele of the HNF-1alpha gene impair pancreatic beta-cell function is unclear. The functional form of HNF-1alpha is a dimer--either a homodimer or a heterodimer with the structurally related protein HNF-1beta--that binds to and activates transcription of the genes whose expression it regulates. HNF-1alpha is composed of three functional domains: an amino-terminal dimerization domain (amino acids 1-32), a DNA-binding domain with POU-like and homeodomain-like motifs (amino acids 150-280), and a COOH-terminal transactivation domain (amino acids 281-631). Because the dimerization domain is intact in many of the mutant forms of HNF-1alpha found in MODY subjects, these mutant proteins may impair pancreatic beta-cell function by forming nonproductive dimers with wild-type protein, thereby inhibiting its activity; that is, they are dominant-negative mutations. This hypothesis was tested by comparing the functional properties of the frameshift mutation P291fsinsC, the most common mutation identified to date in MODY3 patients, and wild-type HNF-1alpha. P291fsinsC-HNF-1alpha showed no transcriptional transactivation activity in HeLa cells, which lack endogenous HNF-1alpha. Overexpression of P291fsinsC-HNF-1alpha in MIN6 cells, a mouse beta-cell line, resulted in an approximately 40% inhibition of the endogenous HNF-1alpha activity in a dosage-dependent manner. Furthermore, heterodimer formation between wild-type and P291fsinsC mutant proteins were observed by electrophoretic mobility shift assay. These data suggest that the P291fsinsC mutation in HNF-1alpha functions as a dominant-negative mutation. However, other mutations, such as those in the promoter region and dimerization domain, may represent loss of function mutations. Thus mutations in the HNF-1alpha gene may lead to beta-cell dysfunction by two different mechanisms.

Animals↗

PubMed1998

Creation and characterization of a mitochondrial DNA-depleted pancreatic beta-cell line: impaired insulin secretion induced by glucose, leucine, and sulfonylureas.

It has been proposed that mitochondrial oxidative phosphorylation in pancreatic beta-cells plays an important role in insulin secretion. To examine the impact of mitochondrial dysfunction on insulin secretion, we created a MIN6 cell line that depleted mitochondrial DNA (mtDNA) by treatment with ethidium bromide (EtBr), and studied the response of the cell line to various secretagogues. MIN6 cells cultured with 0.5 microg/ml EtBr for over 2 months (termed MIN6 deltamt cells) revealed a marked (>90%) decrease in mtDNA content and a lack of mRNAs encoded by mtDNA. MIN6 deltamt cells showed the defects of cytochrome c oxidase activity, glucose- and leucine-induced increase in cellular ATP content, and respiratory chain-driven ATP synthesis, suggesting that MIN6 deltamt cells lost oxidative phosphorylation activity due to the selective disruption of the subunits of respiratory chain enzymes encoded by mtDNA. MIN6 deltamt cells also showed a decrease in glucose utilization, suggesting the impairment of the glycolytic pathway as well. After stimulation with glucose and leucine, MIN6 deltamt cells showed no response in insulin secretion or intracellular free Ca2+ concentration ([Ca2+]i). On the other hand, arginine stimulated insulin secretion and an increase in [Ca2+]i in MIN6 deltamt cells as in MIN6 cells. Glibenclamide also stimulated insulin secretion and an increase in [Ca2+]i in both types of cells, but the responses of MIN6 deltamt cells were significantly lower than those of MIN6 cells. These results suggest the importance of ATP production in insulin secretion and an increase in [Ca2+]i, both induced by glucose and leucine. Moreover, mitochondrial function turns out to be not essential but important for the activation of sulfonylurea-induced insulin secretion.

Adenosine Triphosphate↗

PubMed1997

Role of cytosolic phospholipase A2 in allergic response and parturition.

Phospholipase A2 (PLA2) comprises a superfamily of enzymes that hydrolyse the ester bond of phospholipids at the sn-2 position. Among the members of this superfamily, cytosolic PLA2 has attracted attention because it preferentially hydrolyses arachidonoyl phospholipids and is activated by submicromolar concentrations of Ca2+ ions and by phosphorylation by mitogen-activated protein kinases (MAP kinases). Here we investigate the function of cytosolic PLA2 in vivo by using homologous recombination to generate mice deficient in this enzyme. These mice showed a marked decrease in their production of eicosanoids and platelet-activating factor in peritoneal macrophages. Their ovalbumin-induced anaphylactic responses were significantly reduced, as was their bronchial reactivity to methacholine. Female mutant mice failed to deliver offspring, but these could be rescued by administration of a progesterone-receptor antagonist to the mother at term. Considered together with previous findings, our results indicate that cytosolic PLA2 plays a non-redundant role in allergic responses and reproductive physiology.

Anaphylaxis↗

PubMed1997

Suppression of pulmonary metastasis in murine B16 melanoma cells by transfection of a sialidase cDNA.

A cytosolic sialidase cDNA was transfected into a highly metastatic and invasive cell line, B16-BL6, derived from the murine B16 melanoma. Stable transfection of a cytosolic sialidase expression vector yielded 4 transfectants with high content of the exogenous sialidase protein as well as enzyme activity. These transfectants exhibited markedly decreased experimental pulmonary metastasis, invasiveness in collagen gels and cell motility on colloidal gold-coated glass plates but no change in cell attachment to fibronectin, collagen type VI or laminin. To cast light on the underlying mechanisms, cellular constituents of the transfectants were analyzed. Sialidase over-expression did not lead to any significant changes in cell surface carbohydrates or intracellular glycoproteins, as revealed by lectin flow cytometry and lectin blotting, respectively. Thin layer chromatography of intracellular glycolipids, however, revealed decreased ganglioside GM3 and increased lactosylceramide as major changes.

Animals↗

PubMed1997

Suppression of transcription factor PDX-1/IPF1/STF-1/IDX-1 causes no decrease in insulin mRNA in MIN6 cells.

The insulin gene transcription factor PDX-1/IPF1/STF-1/ IDX-1 plays a key role in directing beta cell-specific gene expressions. Recently, impairment of PDX-1 expression or activity has been observed in beta cell-derived HIT cells cultured under high glucose concentrations, and this has been suggested as a possible cause of the decrease in insulin gene transcription. To investigate the pathophysiological significance of PDX-1 as a determinant of the rate of insulin gene transcription, we suppressed its expression in beta cell-derived MIN6 cells using an antisense oligodeoxynucleotide (ODN) and searched for possible changes in the beta cell-specific gene expression. Treatment of MIN6 cells with an 18-mer phosphorothioate ODN complementary to a sequence starting at the translation initiation codon of PDX-1 caused a potent, concentration-dependent reduction in PDX-1 expression; addition of 2 microM antisense ODN could reduce PDX-1 expression to 14+/-4% of the control. There was also a decrease in its DNA binding to the insulin gene A element. Despite such suppression of PDX-1, Northern blot analysis revealed no decrease in the amount of insulin mRNA in the MIN6 cells. Similarly, no changes were detected in the transcription of the glucokinase or islet amyloid polypeptide gene, for which PDX-1 was shown to function as a transcription factor. Thus, our findings dispute the physiological significance of PDX-1 in determining the rate of insulin gene transcription. This means that other components constituting the transcription-controlling machinery need to be evaluated in order to understand the molecular basis of impaired insulin biosynthesis such as that observed due to glucose toxicity.

Binding Sites↗

PubMed1997

Intranasal immunization of a DNA vaccine with IL-12- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-expressing plasmids in liposomes induces strong mucosal and cell-mediated immune responses against HIV-1 antigens.

A DNA vaccine constructed with the CMV promoter conjugated to env gp160 and rev genes has been shown to induce an effective Th1-type immune response when inoculated via an intramuscular route. In the present study, we obtained high levels of both humoral and cell-mediated immune activity by intranasal administration of this DNA vaccine. The production of mucosal IgA Ab in feces and vaginal fluid was stimulated significantly by intranasal DNA administration. This route of administration resulted in a significant level of HIV-1-neutralizing Abs in feces and serum. Cytokine assays revealed that intranasal administration of this DNA vaccine induces a Th2-type immune response. Interestingly, cationic liposomes greatly enhanced these activities. Abs against HIV-1 were present for at least 10 mo. Coadministration of the DNA vaccine with IL-12- and granulocyte/macrophage-CSF-expressing plasmids induced high levels of HIV-specific CTLs and an increase in delayed type hypersensitivity when administered by the intranasal route. These results clearly demonstrate that intranasal administration of this DNA vaccine with liposomes, together with IL-12- and/or granulocyte/macrophage-CSF-expressing plasmids, induces a strong level of anti-HIV-1 immune response.

Administration, Intranasal↗

PubMed1997

Complete rescue of lethal albino c14CoS mice by null mutation of 4-hydroxyphenylpyruvate dioxygenase and induction of apoptosis of hepatocytes in these mice by in vivo retrieval of the tyrosine catabolic pathway.

Hereditary tyrosinemia 1 (HT1) is characterized by progressive liver damage, from infancy, and by a high risk for hepatocellular carcinoma. HT1 is due to mutations in the fumarylacetoacetate hydrolase gene Fah, encoding the last enzyme in the tyrosine catabolic pathway. Lethal albino deletion c14CoS mice and mice with target-disrupted Fah are models for HT1, but they die in the perinatal period, albeit with a different phenotype from that seen in HT1 in humans. We first asked whether homozygous null mutation of the 4-hydroxyphenylpyruvate dioxygenase gene Hpd could rescue the homozygous c14CoS mice (c14CoS/c14CoS or Fah-/-). The double mutant Fah-/- Hpd-/- mice appeared normal, at least until age 18 months, and there was no evidence of liver disease, findings that facilitated examination of the effect of Fah-/- on mature and unmodified hepatocytes in vivo. The hepatocytes of Fah-/- undergo rapid apoptosis, and acute death follows. Essentially the same phenomena were observed when Fah-/- Hpd-/- mice were administered homogentisate intraperitoneally. These changes in liver pathology in Fah-/- Hpd-/- mice after the administration of homogentisate were associated with massive urinary excretion of succinylacetone. These results suggest that accumulation of fumarylacetoacetate, maleylacetoacetate, or succinylacetone seems to trigger the endogenous process of apoptosis in hepatocytes that lack fumarylacetoacetate hydrolase activity. This apoptosis may be related to the development of hepatocellular carcinomas seen in HT1 patients and pharmaceutically treated fumarylacetoacetate hydrolase-deficient mice.

4-Hydroxyphenylpyruvate Dioxygenase↗

PubMed1997

Murine interleukin 4 transgenic heart allograft survival prolonged with down-regulation of the Th1 cytokine mRNA in grafts.

BACKGROUND: Data supporting the differential activation of T helper (Th) 2 cells in transplantation acceptance/tolerance in rodents have been presented by several investigators. However, the differential activation of Th2 cells may be simply the result of allograft acceptance/tolerance induction instead of a contribution to the maintenance of grafts. METHODS: Therefore, we established interleukin (IL)-4 transgenic mice under the control of a cardiac alpha-myosin heavy chain promotor and transplanted IL-4-expressing heart allografts into unmodified recipients to determine the actual contribution of the Th2 bias to allograft acceptance. RESULTS: Among 16 newborn C57BL/6J (B6) mice, three were positive for the IL-4 transgene. Serum IL-4 levels of transgenic versus control B6xC3H F1 mice were not statistically different. Reverse-transcriptase polymerase chain reaction showed that the transgenic B6xC3H F1 mice expressed IL-4 mRNA in the heart and in the lung, whereas control mice did not express IL-4 in any organ. IL-4 mRNA expression in the transgenic but not in the control heart was also confirmed by RNAse protection assay and fluorescence in situ hybridization. The survival of IL-4 transgenic B6xC3H heart grafts heterotopically placed in C3H recipients was prolonged compared with that of nontransgenic grafts (19.0+/-9.1 vs. 6.8+/-2.2 days, P=0.003). Interferon-gamma mRNA expression in IL-4 transgenic heart grafts on the fifth posttransplant day as assessed by Northern blotting was suppressed compared with that in control grafts. Reverse-transcriptase polymerase chain reaction analysis showed that IL-2 mRNA was suppressed in the IL-4 transgenic grafts compared with that in control grafts, while IL-4 mRNA was observed only in IL-4 transgenic grafts. IL-10 mRNA was detected at similar levels in both transgenic and control grafts. CONCLUSIONS: A Th2 bias may contribute to allograft acceptance in part by inducing the down-regulation of Th1-cytokine mRNAs, but it may not be sufficient to induce indefinite graft survival.

Animals↗

PubMed1997

Developmentally ordered V-J recombination in mouse T cell receptor gamma locus is not perturbed by targeted deletion of the Vgamma4 gene.

Mouse TCR gamma genes in the gamma1 cluster are arranged in the order of Vgamma5, Vgamma2, Vgamma4, Vgamma3, Jgamma1, and Cgamma1 on the chromosome. During thymic ontogeny, each Vgamma gene recombines with the Jgamma1 gene in the order of proximity to Jgamma1. To explore the mechanism of the ordered recombination, we generated Vgamma4-deficient mice by gene targeting and the Cre/loxP system, by deleting the 4.8-kb DNA region between 3' of the Vgamma2 and 3' of the Vgamma4. In semiquantitative PCR analysis, Vgamma2-Jgamma1 recombination was detected frequently in adult thymus, while Vgamma3-Jgamma1 recombination preferentially occurred in fetal thymus of the mutant mice. There was no difference in the frequency of V-J recombinations between control and mutant mice. Southern blot analysis also revealed that recombination of the Vgamma2 gene occurred as frequently as in control mice. In addition, there was no difference in the levels of germline transcripts of Vgamma2 and Vgamma3 genes between control and mutant mice. Therefore, regulation of the Vgamma-Jgamma recombination was not affected by deletion of the Vgamma4 gene. These results suggest that the ordered recombination is controlled by regulatory elements near each Vgamma gene.

Age Factors↗

PubMed1997

Intramuscular injection of expression plasmid DNA is an effective means of long-term systemic delivery of interleukin-5.

It has been demonstrated that intramuscularly injected expression plasmid DNA is taken up by myofibers and subsequently expresses exogenous genes. In the present study, we assessed intramuscular DNA injection as a means of systemically delivering interleukin-5 (IL-5). We constructed an IL-5 expression plasmid, pCAGGS-IL-5, containing murine IL-5 cDNA under the control of the CAG promoter. The soleus muscle of mice was pretreated with bupivacaine. Two days later, mice were injected with pCAGGS-IL-5 or a control pCAGGS plasmid DNA at the same site. At 2 weeks after DNA injection, eosinophils had increased from 2-3% to 8-29% of peripheral white blood cells in 9 of 10 mice injected with pCAGGS-IL-5, while eosinophils never exceeded 3% in control mice injected with pCAGGS. IL-5 mRNA was present in the muscle area injected with pCAGGS-IL-5. IL-5 was also detectable by ELISA in the sera of most mice injected with pCAGGS-IL-5, but in none of the control mice. These results demonstrate that intramuscular plasmid injection serves as a useful method of systemically delivering cytokines by combining the strong CAG promoter and bupivacaine pretreatment.

Animals↗

PubMed1997

Enhancement of cell-mediated immunity against HIV-1 induced by coinnoculation of plasmid-encoded HIV-1 antigen with plasmid expressing IL-12.

Previous investigations have demonstrated that CTL may play an important role in suppressing the disease progression of HIV infection. In this study, we inoculated mice with IL-12 expression plasmid together with plasmid-encoding HIV-1 envelope to enhance CTL activity by activating a Th1-type response. The results of delayed-type hypersensitivity using the footpad swelling response and of CTL activity clearly showed that HIV-1-specific cell-mediated immunity was enhanced by inoculation of the IL-12 expression plasmid. Quantitation of cytokine in the sera of IL-12-inoculated mice revealed that IFN-gamma significantly increased. The enhanced cell-mediated immunity responses were abrogated by combined administration of the IL-12 expression plasmid and neutralizing anti-IFN-gamma Ab. Together, these results suggest that enhanced virus-specific cell-mediated immunity occurred via an endogenously produced IFN-gamma by inoculation of IL-12 expression plasmid.

AIDS Vaccines↗

PubMed1997

Bronchial hyperreactivity, increased endotoxin lethality and melanocytic tumorigenesis in transgenic mice overexpressing platelet-activating factor receptor.

Although platelet-activating factor (PAF) has been shown to exert pleiotropic effects on isolated cells or tissues, controversy still exists as to whether it plays significant pathophysiological roles in vivo. To answer this question, we established transgenic mice over-expressing a guinea-pig PAF receptor (PAFR). The transgenic mice showed a bronchial hyperreactivity to methacholine and an increased mortality when exposed to bacterial endotoxin. An aberrant melanogenesis and proliferative abnormalities in the skin were also observed in the transgenic mice, some of which spontaneously bore melanocytic tumors in the dermis after aging. Thus, PAFR transgenic mice proved to be a useful model for studying the basic pathophysiology of bronchial asthma and endotoxin-induced death, and screening of therapeutics for these disorders. Furthermore, our findings provide new insights regarding the role of PAF in the morphogenesis of dermal tissues as well as the mitogenic activity of PAF and PAFR in vivo.

Animals↗

PubMed1997

In vivo correction with recombinant adenovirus of 4-hydroxyphenylpyruvic acid dioxygenase deficiencies in strain III mice.

Tyrosinemia type 3, caused by a genetic deficiency of 4-hydroxyphenylpyruvic acid dioxygenase (HPD) in tyrosine catabolism, is characterized by convulsion, ataxia, and mental retardation. The III mouse is a model of tyrosinemia type 3. HPD activity and protein are defective in the liver and its blood tyrosine levels are elevated, the range being between 1,100 and 1,656 microM. We constructed a recombinant adenoviral vector bearing the human HPD cDNA (AdexCAGhHPD), which is expressed under the control of a potent CAG promoter. III mice were injected with 1.0 x 10(8) to 1.0 x 10(9) pfu of AdexCAGhHPD through the tail vein. When 3.0 x 10(8) - 1.0 x 10(9) pfu were injected, blood tyrosine levels decreased within 3 hr, reached a normal range (under 300 microM), and remained at a low level for 2-6 weeks. Hepatic HPD activities also increased as early as 3 hr after the injection of 5.0 x 10(8) pfu, reached the levels comparable to the control mice in 3-7 days, and then decreased, and correlated well to blood tyrosine. Hepatic HPD expression was confirmed by Northern blot and immunoblot analyses. Histology revealed no difference (gross or microscopic) between the liver injected with AdexCAGhHPD and the control. No significant changes in blood tyrosine levels were noted after the second injection of 5.0 x 10(8) pfu of AdexCAGhHPD. Thus, the intravenous administration of the adenoviral vector bearing a foreign gene seems suitable for transient, early gene transfer into the liver.

4-Hydroxyphenylpyruvate Dioxygenase↗

PubMed1997

Expression of human islet amyloid polypeptide/amylin impairs insulin secretion in mouse pancreatic beta cells.

Non-insulin-dependent diabetes mellitus (NIDDM) is associated histopathologically with islet amyloid deposits of which a major component is islet amyloid polypeptide (IAPP)/amylin. We examined whether endogenous IAPP controls insulin secretion via a local effect within pancreatic islets and whether overexpression of this peptide contributes to pancreatic beta-cell dysfunction in this disease. Transgenic mice expressing human IAPP in pancreatic beta cell were used in this study. Human IAPP expression did not influence the mouse proinsulin mRNA level and insulin content. Glucose-induced insulin secretion was decreased in the isolated pancreatic islets of transgenic mice. MIN6, a glucose-responsive pancreatic beta-cell line, was transfected with human IAPP cDNA by a lipofectin method. Human IAPP expression was confirmed by RNA blot and immunohistochemical analysis. In two transfectants expressing the largest amount of human IAPP, insulin secretion was increased in response to glucose stimulation; however, the magnitude of the insulin response in cells transfected with human IAPP was smaller than in control clones. Insulin content was not influenced by the expression. We conclude that endogenous IAPP inhibits insulin secretion via an autocrine effect within pancreatic islets, and that the impaired insulin secretion in this disease may be partly caused by overexpression of IAPP.

Amyloid↗

PubMed1997

Myeloid differentiation is impaired in transgenic mice with targeted expression of a dominant negative form of retinoid X receptor beta.

To investigate the in vivo function of retinoid X receptor (RXR) on myelopoiesis, We generated transgenic (Tg) mice with targeted expression of a dominant negative form of RXR beta in myeloid cells. In these Tg mice the transgene is expected to suppress the function of hetero dimeric receptors composed of RXR and its counterparts, such as retinoic acid receptor. Out of 12 mice analysed, one Tg mouse exhibited a severe maturation arrest at the promyelocytic stage. Three other Tg mice showed a mild inhibition of myeloid differentiation, which was further augmented when mice were treated with 5-fluorouracil (5-FU). Furthermore, four Tg mice showed impaired myeloid differentiation in response to the treatment by 5-FU on granulocyte-colony stimulating factor (G-CSF), although they exhibited apparently normal myelopoiesis in the untreated state. The phenotype of Tg mice observed after G-CSF treatment correlated with the expression level of the transgene, although the correlation was not found in untreated mice. These results indicated that myeloid differentiation is perturbed in the Tg mice by the dominant negative effect of the transgenic RXR, indicating that RXR plays a role in myelopoiesis.

Animals↗

PubMed1997

Encapsulation of mammalian cells into synthetic polymer membranes using least toxic solvents.

Immunoisolation, that is, enclosure of cells within a semipermeable membrane to protect them from immunological rejection, may enable the transplantation of cells without use of immunosuppressive drugs. Therefore, in addition to naturally-occurring ionic polymers, several synthetic nonionic polymers which can form dense and strong membranes in water have been studied as materials for immunoisolation. However, such nonionic polymers are required to be soluble in organic solvents which are mostly cytotoxic. In this report we describe enclosure of insulin-releasing cells into water-insoluble poly(2-hydroxyethyl methacrylate) and poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) membranes using X-ray contrast medium as a solvent without use of any special apparatus. The contrast medium employed in our study is iopamidol aqueous solution. Insulin release was observed for 1 months when insulin-releasing cells were encapsulated into these membranes. The permeability of five solutes through the membranes prepared from the iopamidol aqueous solution was also studied to determine their potential immunoisolative efficacy.

Animals↗