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Biomedical subjects

J Miyazaki

Publications and source records attributed to J Miyazaki.

At least 91 records · Page 5Linked to original sources
PubMed1999

Enrichment and efficient screening of ES cells containing a targeted mutation: the use of DT-A gene with the polyadenylation signal as a negative selection maker.

Gene targeting in embryonic stem (ES) cells via homologous recombination can occur at very low frequency. In order to enrich homologous recombinants before screening, a negative selection marker, such as thymidine kinase (TK) and diphtheria toxin A fragment (DT-A), has been commonly used. In this study, we developed a negative selection marker using DT-A gene with polyadenylation signal and it was designated DT-ApA. To determine the difference in targeting efficiency of the negative selections, we constructed three different targeting vectors for each negative selection (first, TK at the 3' end, second, TK at both the 5' and 3' ends < 2 X TK >, and third, DT-ApA at the 5' end of the homologous sequences). Gene targeting experiments using these constructs clearly showed that negative selection using DT-ApA was more efficient than that using TK for homologous recombination and that negative selection using DT-ApA was as efficient as that using 2 X TK. Considering the fact that the use of DT-ApA is more convenient for construction of targeting vectors than that of 2 X TK, DT-ApA is an efficient negative selection marker. In addition, we examined long and accurate PCR (LA-PCR) for screening gene targeted clones. The use of LA-PCR with genomic DNAs from ES cell clones facilitated simple detection of homologous recombinants, suggesting that the screening with LA-PCR is compatible with the use of longer homologous sequences of both arms in vector design. Our results indicate that the use of DT-ApA for negative selection together with the application of LA-PCR for screening ensures efficient and time-saving screening for homologous recombinants.

Animals↗
PubMed1999

Suppression of airway inflammation by theophylline in adult bronchial asthma.

BACKGROUND: Chronic continuous airway inflammation caused by eosinophils has been noted to play critical roles in the pathophysiology of bronchial asthma, in addition to reversible obstruction and hypersensitivity of the respiratory tract. Therefore, suppression of chronic airway inflammation has become more important in asthma treatment. Although theophylline has been a conventionally used bronchodilator, it has been recently reported to have concurrent anti-inflammatory effects. OBJECTIVE: Accordingly, we studied the effects of a slow-release theophylline preparation, Theolong, on airway inflammation. METHODS: Administration of Theolong 400 mg/day to 24 patients with mild or moderate asthma and measuring eosinophil cationic protein (ECP), a marker of airway inflammation, and eosinophils in sputum and peripheral blood at 4 and 8 weeks. RESULTS: As a result, sputum ECP, serum ECP and sputum eosinophil count (%) were significantly lowered after 4 and 8 weeks. CONCLUSION: Thus, in the theophylline-administered group, slow-release theophylline, Theolong, was effective in treating asthma, with anti-inflammatory effects on inflammatory cells besides its bronchodilator action.

Administration, Oral↗
PubMed1999

[A case of primitive neuroectodermal tumor of the kidney].

A primitive neuroectodermal tumor (PNET) is a solid tumor originating from the neural crest. This tumor is known to occur in the central nervous system and soft tissue, but recently determined to also invade the kidney. Although primary renal PNET is very rare, we encountered a case of primary renal PNET with multiple lung metastasis. This case was a 35-year-old man with a chief complaint of macroscopic hematuria, which he noticed in January 1996. He was hospitalized because of right renal tumor detected with ultrasonography. On CT scan, a low-density solid tumor with a maximum diameter of 8 cm was visualized in the right kidney. Low-signal and high-signal tumorous lesions were demonstrated in the same region on T 1-weighted and T 2-weighted MR images, respectively. Then right radical nephrectomy was performed on Feb. 5, 1996. On histopathological observation, the tumor was composed of small tumor cells with solid growth. Immunohistochemical staining revealed that the tumor was positive for CD 99. Thus our final diagnosis was primary right renal PNET. Although 2 metastatic lesions with a diameter of about 1 cm were observed in the right lower lung before the time of surgery, pulmonary metastatic lesions markedly increased in number 3 months after surgery. Thus we initiated CAP chemotherapy with cyclophosphamide, doxorubicin, and CDDP. After 3 cycles, pulmonary metastatic lesions disappeared at CT scan, indicating complete remission. However, a tumor with a maximum diameter of about 10 cm recurred in the retroperitoneum 7 months after complete remission. Although 2 cycles of CAV/PE therapy with cyclophosphamide, doxorubicin, vincristine, CDDP, and VP-16 were performed following resection of the retroperitoneal tumor, he showed no response and decreased 24 months after surgery.

Adult↗
PubMed1998

Pancreatic beta cell-specific expression of thioredoxin, an antioxidative and antiapoptotic protein, prevents autoimmune and streptozotocin-induced diabetes.

The cytotoxicity of reactive oxygen intermediates (ROIs) has been implicated in the destruction of pancreatic beta cells in insulin-dependent diabetes mellitus (IDDM). Thioredoxin (TRX), a redox (reduction/oxidation)-active protein, has recently been shown to protect cells from oxidative stress and apoptosis. To elucidate the roles of oxidative stress in the development of autoimmune diabetes in vivo, we produced nonobese diabetic transgenic mice that overexpress TRX in their pancreatic beta cells. In these transgenic mice, the incidence of diabetes was markedly reduced, whereas the development of insulitis was not prevented. Moreover, induction of diabetes by streptozotocin, an ROI-generating agent, was also attenuated by TRX overexpression in beta cells. This is the first direct demonstration that an antioxidative and antiapoptotic protein protects beta cells in vivo against both autoimmune and drug-induced diabetes. Our results strongly suggest that oxidative stress plays an essential role in the destruction of beta cells by infiltrating inflammatory cells in IDDM.

Animals↗
PubMed1998

A composite CMV-IE enhancer/beta-actin promoter is ubiquitously expressed in mouse cutaneous epithelium.

In most existing transgenic mouse models developed for the study of specific genes in the skin, the goal has been to target transgene expression to defined populations of cells in the cutaneous epithelium. Keratin promoters have been especially useful for this purpose. In some instances, however, it may be desirable to express a transgene in all the cells of the cutaneous epithelium. Since no ubiquitously expressed promoter sequences had previously been identified, we used lacZ reporter transgenes to test two enhancer/promoter sequences for ubiquitous expression in the skin of adult transgenic mice. We find that a CMV enhancer/CMV promoter is not active in most cell types in the skin, whereas a CMV enhancer/modified beta-actin promoter sequence is active in the suprabasal and basal cells of the epidermis as well as in the epithelial cells of the hair follicles, sebaceous glands, and the dermal papillae.

Actins↗
PubMed1998

Lysosomal glycogen storage disease with normal acid maltase with early fatal outcome.

In a male infant who had cardiomyopathy, generalized muscle weakness and increased serum creatine kinase levels, his muscle biopsy revealed myopathic changes with tiny intracytoplasmic vacuoles containing PAS-positive material and high acid phosphatase activity, but had normal acid maltase activity biochemically. These findings were consistent with those seen in lysosomal glycogen storage disease with normal acid maltase (Danon disease). Sarcolemmal indentations commonly seen in this disease were missing, but a complement membrane attack complex, C5b-9 was positive along the surface membrane of the muscle fibers as seen in X-linked vacuolar myopathy. The patient was on a respirator and died at 27 months of age from pneumonia and hypertrophic cardiomyopathy. Lysosomal glycogen storage disease with normal acid maltase may be manifested at birth with marked skeletal and cardiac involvement leading to death in early infancy.

Biomarkers↗
PubMed1998

Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice.

ATP-sensitive K+ (KATP) channels regulate many cellular functions by linking cell metabolism to membrane potential. We have generated KATP channel-deficient mice by genetic disruption of Kir6.2, which forms the K+ ion-selective pore of the channel. The homozygous mice (Kir6.2(-/-)) lack KATP channel activity. Although the resting membrane potential and basal intracellular calcium concentrations ([Ca2+]i) of pancreatic beta cells in Kir6.2(-/-) are significantly higher than those in control mice (Kir6.2(+/+)), neither glucose at high concentrations nor the sulfonylurea tolbutamide elicits a rise in [Ca2+]i, and no significant insulin secretion in response to either glucose or tolbutamide is found in Kir6.2(-/-), as assessed by perifusion and batch incubation of pancreatic islets. Despite the defect in glucose-induced insulin secretion, Kir6.2(-/-) show only mild impairment in glucose tolerance. The glucose-lowering effect of insulin, as assessed by an insulin tolerance test, is increased significantly in Kir6.2(-/-), which could protect Kir6.2(-/-) from developing hyperglycemia. Our data indicate that the KATP channel in pancreatic beta cells is a key regulator of both glucose- and sulfonylurea-induced insulin secretion and suggest also that the KATP channel in skeletal muscle might be involved in insulin action.

Animals↗
PubMed1998

Systemic delivery of interleukin 10 by intramuscular injection of expression plasmid DNA prevents autoimmune diabetes in nonobese diabetic mice.

We previously demonstrated that intramuscular plasmid injection serves as a useful method of long-term systemic delivery of cytokines. In the present study, we assess intramuscular DNA injection as a means of systemically delivering interleukin 10 (IL-10), a cytokine with immunosuppressive properties, and preventing the progression of autoimmune diabetes in the nonobese diabetic (NOD) mouse, an excellent model for human insulin-dependent diabetes mellitus (IDDM). We injected IL-10 expression plasmid (pCAGGS-IL10) or a control pCAGGS plasmid into the muscles of NOD mice twice at 3 and 5 weeks of age. IL-10 was detectable by ELISA in the sera of mice injected with pCAGGS-IL10 for more than 2 weeks after the injection. Although the severity of insulitis at 13 weeks of age was not improved by the intramuscular injection of pCAGGS-IL10, the incidence of diabetes was markedly reduced in NOD mice injected with pCAGGS-IL10 as compared with those injected with pCAGGS or as compared with nontreated NOD mice. These results show that the progression of autoimmune diseases in mice can effectively be suppressed by intramuscular DNA injection, and suggest that this method is potentially applicable to the treatment of human autoimmune diseases.

Animals↗
PubMed1998

Abrogation of autoimmune diabetes in nonobese diabetic mice and protection against effector lymphocytes by transgenic paracrine TGF-beta1.

Paracrine effect of transforming growth factor-beta1 (TGF-beta1) on autoimmune insulitis and diabetes was studied by transgenic production of the active form of porcine TGF-beta1 (pTGF-beta1) in pancreatic islet (islet) alpha cells in nonobese diabetic (NOD) mice under the control of rat glucagon promoter (RGP) (NOD-RGP-TGF-beta1). None of 27 NOD-RGP-TGF- beta1 mice developed diabetes by 45 wk of age, in contrast to 40 and 71% in male and female nontransgenic mice, respectively. None of the NOD-RGP-TGF-beta1 mice developed diabetes after cyclophosphamide (CY) administration. Adoptive transfer of splenocytes of NOD-RGP-TGF-beta1 mice to neonatal NOD mice did not transfer diabetes after CY administration. Adoptive transfer of three types of diabetogenic lymphocytes to NOD-RGP-TGF-beta1 and nontransgenic mice after CY administration led to the lower incidence of diabetes in NOD-RGP-TGF-beta1 mice versus that in nontransgenic mice: 29 vs. 77% for diabetogenic splenocytes, 25 vs. 75% for islet beta cell-specific Th1 clone cells, and 0 vs. 50% for islet beta cell-specific CD8(+) clone cells, respectively. Based on these, it is concluded that autoimmune diabetes in NOD mice is not a systemic disease and it can be completely prevented by the paracrine TGF-beta1 in the islet compartment through protection against CD4(+) and CD8(+) effector lymphocytes.

Adoptive Transfer↗
PubMed1998

Rapid activation and nuclear translocation of mitogen-activated protein kinases in response to physiological concentration of glucose in the MIN6 pancreatic beta cell line.

MIN6 is one of the few pancreatic beta cell lines that respond to physiological concentrations of glucose by secreting insulin, and little is known about the triggered molecular mechanisms. We report below that the response to glucose in the MIN6 cells includes an activation of the p42 and p44 mitogen-activated protein (MAP) kinases (ERK2 and ERK1). This activation also occurred with the antidiabetic sulfonylurea glibenclamide and kainate, a specific agonist of a subtype of the ionotropic glutamate receptors, which depolarize the cytoplasmic membrane. The requirement for a calcium entry through the L-type voltage-gated channels and other characteristics of the regulation of the MAP kinase activity, such as the effect of the elevation of the cAMP concentration by forskolin, were similar to those of the secretion of insulin. However, the activation of the MAP kinases is not required for the secretion of insulin, inasmuch as this effect of glucose was not abolished when the MAP kinases were prevented from activation by PD098059, an inhibitor of the MAP kinase kinase. However, as the MAP kinases were translocated into the nucleus, they might be implicated in the calcium-dependent transcriptional response of the cells to glucose and thus regulate the expression of the insulin gene.

Animals↗
PubMed1998

Impaired anaphylactic responses with intact sensitivity to endotoxin in mice lacking a platelet-activating factor receptor.

Platelet-activating factor (PAF) is a potent phospholipid mediator with diverse biological activities in addition to its well-known ability to stimulate platelet aggregation. Pharmacologic studies had suggested a role for PAF in pregnancy, neuronal cell migration, anaphylaxis, and endotoxic shock. Here we show that disruption of the PAF receptor gene in mice caused a marked reduction in systemic anaphylactic symptoms. Unexpectedly, however, the PAF receptor-deficient mice developed normally, were fertile, and remained sensitive to bacterial endotoxin. These mutant mice clearly show that PAF plays a dominant role in eliciting anaphylaxis, but that it is not essential for reproduction, brain development, or endotoxic shock.

Anaphylaxis↗
PubMed1998

Reduced T helper 1 responses in IL-12 p40 transgenic mice.

To investigate the antagonistic effect of IL-12 p40 on IL-12 activity in vivo, we generated transgenic (Tg) mice in which p40 gene was regulated by a liver-specific promoter. Three Tg mouse lines were generated, and they expressed the p40 transgene predominantly in liver. Serum p40 level was extremely high, and it consisted of mainly monomer and homodimer and also of higher m.w. complexes. These Tg mice did not show any apparent phenotypic difference from control littermates in lymphoid cells. Enhancement of NK cell lytic activity in spleen by administration of rIL-12 to these mice was greatly diminished. Ag induced cytokine production was impaired: decreased production of IFN-gamma and increased production of IL-4 and IL-10. Delayed-type hypersensitivity response was also significantly reduced. Moreover, these Tg mice showed increased susceptibility to the infection with an intracellular pathogen, blood-stage Plasmodium berghei XAT, which is an irradiation-induced attenuated substrain of P. berghei NK65, presumably due to the decreased IFN-gamma production. These results suggest that p40 functions as an IL-12 antagonist in vivo, and that Th1 responses in p40 Tg mice are significantly reduced. Thus, these Tg mice could be a useful model to evaluate the inhibitory effect of p40 on IL-12-mediated various immune responses in vivo.

Animals↗
PubMed1998

A T cell activation antigen, Ly6C, induced on CD4+ Th1 cells mediates an inhibitory signal for secretion of IL-2 and proliferation in peripheral immune responses.

A T cell activation antigen, Ly6C, is considered to be involved in the autoimmunity of some autoimmune-prone mice; however, the function of Ly6C remains largely unknown. We prepared a rat anti-mouse Ly6C monoclonal antibody (mAb) (S14) that inhibits the proliferation of peripheral T cells stimulated with anti-CD3 mAb in vitro. S14 mAb, the specificity of which is confirmed by a cDNA transfectant, recognizes Ly6C antigen preferentially expressed on a part of CD8+ T cells in peripheral lymphoid organs. The immunohistochemical analysis demonstrates that Ly6C appears on CD8+ T cells in the conventional T cell-associated area of BALB/c but not of nonobese diabetic (NOD) mice, confirming the absence of Ly6C+ T cells in NOD mice. Addition of soluble S14 mAb to the culture does not influence the proliferation of T cells in vitro; however, the S14 mAb coated on the plate clearly inhibits the proliferation and IL-2 production of anti-CD3-stimulated peripheral T cells. The T cells are arrested at the transitional stage from G0/G1 to S+G2/M phases, but they are not induced to undergo apoptotic changes in vitro. This inhibitory signal provided through the Ly6C molecule inhibited IL-2 secretion in a subpopulation of the activated CD4+ T cells. Ly6C is expressed on T cell clones of both Th1 and Th2 cells, but the cytokine secretion from Th1 clones is preferentially inhibited. These results suggest that Ly6C mediates an inhibitory signal for secretion of cytokines from Th1 CD4+ T cells, potentially causing the inhibition of immune response in peripheral lymphoid tissues.

Animals↗
PubMed1998

Skeletal muscle regeneration induced by chorio-allantoic grafting.

To examine whether the expression pattern of fast-muscle type troponin-T (TnT) isoforms was fixed in cell lineage, breast muscle pieces (pectoralis major) from chick embryos and young and adult chickens were grafted on to chorio-allantoic membrane of 9-day-old chick embryos and cultured until the host embryos hatched out. Muscle fibre formation of the grafts was investigated by histological and immunohistochemical methods with anti-fast-muscle type and anti-slow-muscle type TnT sera, and the expression of fast-muscle type TnT in the grafts from chick embryos and young chickens was studied by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional SDS-PAGE, and immunoblotting. In the chorio-allantoic grafting, the breast muscle initially degenerated forming pyknotic nuclei and hyaline cytoplasm. The surviving cells, which were supposed to be satellite cells, regenerated new muscle fibres of the same type as those of the grafted muscle in respect of TnT isoform expression. Therefore, we considered that the ability to express specific isoforms of TnT was fixed in the satellite cells, and that chorio-allantoic grafting was a useful technique for studying muscle differentiation.

Allantois↗
PubMed1998

Gene transfer into muscle by electroporation in vivo.

Among the nonviral techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive, and safe. Applications of this method have been limited by the relatively low expression levels of the transferred gene. We investigated the applicability of in vivo electroporation for gene transfer into muscle, using plasmid DNA expressing interleukin-5 (IL-5) as the vector. The tibialis anterior muscles of mice were injected with the plasmid DNA, and then a pair of electrode needles were inserted into the DNA injection site to deliver electric pulses. Five days later, the serum IL-5 levels were assayed. Mice that did not receive electroporation had serum levels of 0.2 ng/ml. Electroporation enhanced the levels to over 20 ng/ml. Histochemical analysis of muscles injected with a lacZ expression plasmid showed that in vivo electroporation increased both the number of muscle fibers taking up plasmid DNA and the copy number of plasmids introduced into the cells. These results demonstrate that gene transfer into muscle by electroporation in vivo is more efficient than simple intramuscular DNA injection.

Animals↗
PubMed1998

Membrane immunoisolation of a diffusion chamber for bioartificial pancreas.

Immunoisolation is a potentially important approach to transplanting islets without any immunosuppressive therapy. The concept of immunoisolation is outlined in systems in which the transplanted tissue is separated from the immune system of the host by an artificial barrier. We previously described a diffusion chamber as a bioartificial endocrine pancreas (Bio-AEP), which was constructed by placing pancreatic endocrine cells, trapped in a mixed matrix, in the center of a ring holder sandwiched between nucleopore membranes, which were shielded by silicone. This experiment was designed to evaluate a suitable pore size for the nucleopore membrane to ensure immunoisolation during xenoimplantation of the Bio-AEP in vitro and in vivo. A nucleopore membrane of pore size 0.1 microm or 0.2 microm was employed as the semipermeable membrane which provided a mechanical barrier between the endocrine pancreas graft and the host immune system. The protective effect of the Bio-AEP from humoral immunity was determined in vitro, using sensitized sheep erythrocytes (EAs). A complement protein did not destroy the cell membranes of the EAs in the diffusion chamber containing the mixed matrix with the nucleopore membrane of 0.1 microm pore size. In an in vivo experiment, 6 streptozotocin (STZ) induced diabetic rats were implanted with Bio-AEPs constructed with nucleopore membranes of pore size 0.1 microm and containing MIN6 cells in the mixed matrix. In the STZ diabetic rats with Bio-AEPs, a return to normoglycemia was observed up to 50 weeks after implantation without the use of any immunosuppressant. Also, the body weights of the rats gradually increased. During the observation, when the Bio-AEPs were removed from the STZ diabetic rats, the blood glucose immediately returned to preimplantation levels, and the body weights of the rats also decreased. The membranes of the Bio-AEPs removed from the STZ diabetic rats showed a very thin layer of fibroblastic cells on the outer surfaces. The results indicated that the Bio-AEP, in which pancreatic endocrine cells were trapped in a mixed matrix and with a 0.1 microm pore size membrane, should be useful for xenoimplantation into diabetic animals and may open a new field in the therapy of human diabetics.

Animals↗