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Biomedical subjects

Kazuhiro Nagata

Publications and source records attributed to Kazuhiro Nagata.

At least 37 records · Page 2Linked to original sources
PubMed2005

Microsomal prostaglandin E synthase (mPGES)-1, mPGES-2 and cytosolic PGES expression in human gastritis and gastric ulcer tissue.

Recently, three different prostaglandin E2 synthases have been identified: microsomal prostaglandin E synthase (mPGES)-1, cytosolic PGES (cPGES), and mPGES-2; however, their role and connection to cyclooxygenase (COX)-2 in the gastric ulcer repair process remain unknown. Therefore, we examined mPGES-1, cPGES, and mPGES-2 expression and localization in the stomach in vitro and in vivo. Tissues were obtained from Helicobacter pylori (H. pylori)-infected patients and consisted of surgical resections of gastric ulcers, or biopsies of gastric ulcers or gastritis. mPGES-1 mRNA and protein expression levels were examined by real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. mPGES-1, cPGES, and mPGES-2 localization were analyzed immunohistochemically. Induction of PGES expression in response to interleukin (IL)-1beta was examined in vitro in the cultured human gastric fibroblast line Hs262.St. Real-time PCR analysis of mPGES-1 mRNA expression in biopsy samples showed significantly higher expression levels in open than in closed gastric ulcer tissue. Western blot analysis showed mPGES-1 protein expression limited to open ulcer tissue, while mPGES-2 and cPGES immunoreactivities were seen in both open and closed ulcer tissue. Immunohistochemical analysis showed strong mPGES-1 expression in fibroblasts and macrophages of the ulcer bed, paralleling COX-2 expression. cPGES and mPGES-2 expression levels were seen in both fibroblasts of the ulcer bed and in epithelial cells. Furthermore, stronger cPGES and mPGES-2 immunoreactivities were seen in scattered mast cell-like cells and neuroendocrine-like cells, respectively. Induction of mPGES-1 expression in response to IL-1beta was seen in cultured gastric fibroblasts in vitro, and double immunostaining showed mPGES-1 coexpression with COX-2 in fibroblasts of the ulcer bed in vivo. In conclusion, mPGES-1, cPGES, and mPGES-2 are all expressed in gastric ulcer tissue, but only mPGES-1 parallels COX-2 expression in mesenchymal and inflammatory cells of the ulcer bed, suggesting a key role for this enzyme in the ulcer repair process.

Adult↗
PubMed2005

Influence of depletion of alveolar macrophages on apoptosis in Candida-induced acute lung injury.

Apoptosis plays an important role in acute lung injury (ALI), and alveolar macrophages (AMs) are known to secrete proinflammatory cytokines and promote alveolar inflammation. The authors have previously reported that AMs can be depleted by inhalation of 1 mM 2-chloroadenosine (2-CA). In this study, the authors evaluated the effect of AM depletion by 2-CA inhalation on apoptosis in Candida-induced ALI. The results of in situ terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling (TUNEL) and immunohistochemical studies and measurement of cytokine levels and caspase 3 activities in lung homogenates indicated that the Fas-FasL system and apoptosis of alveolar epithelial cells are suppressed by depletion of AMs by 2-CA inhalation.

2-Chloroadenosine↗
PubMed2005

The maintenance of the endoplasmic reticulum network is regulated by p47, a cofactor of p97, through phosphorylation by cdc2 kinase.

The endoplasmic reticulum (ER) has a characteristic complex polygonal structure with hallmark three-way junctions in many types of cells. To investigate the mechanisms responsible for maintaining the ER network, we established ER disassembly and reassembly assays in semi-intact Chinese hamster ovary (CHO) cells that constitutively expressed heat shock protein-47 fused to the green fluorescent protein (GFP-HSP47) as an ER marker (the cells are referred to as CHO-HSP cells). Using these assays, we found that maintenance of the ER network required cytosol and adenosine triphosphate/guanosine 5'-triphosphate (ATP/GTP) hydrolysis, but not actin filaments or microtubules. We also showed that the ER network was disrupted upon addition of either N-ethylmaleimide-treated cytosol after washing semi-intact cells with high salt solution or mitotic cytosol in nocodazole-treated semi-intact CHO-HSP cells. The disrupted ER network induced by mitotic cytosol was reformed by the addition of interphase cytosol. In addition, we found that p47, a cofactor of p97, was essential for the maintenance of the ER network, and that phosphorylation of p47 by cdc2 kinase resulted in ER network disruption by mitotic cytosol. Taken together, these results imply that the maintenance of the ER network requires a membrane fusion process mediated by p97/p47, and that cell cycle-dependent morphological changes of the ER network are regulated through phosphorylation/dephosphorylation of p47.

Adenosine Triphosphatases↗
PubMed2005

NSF/SNAPs and p97/p47/VCIP135 are sequentially required for cell cycle-dependent reformation of the ER network.

The endoplasmic reticulum (ER) has a characteristic polygonal structure with hallmark three-way junctions. In a previous paper, we reconstituted the disruption of the pre-existing ER network using mitotic cytosol from HeLa cells in streptolysin O (SLO)-permeabilized CHO-HSP cells (stably expressing GFP-HSP47). In addition, we found that interphase cytosol induced reformation of the disrupted ER network into a continuous network structure. Here, we show that the reformation of the ER network is accomplished through two sequential fusion reactions. The first process is mediated by NSF/alpha and gamma-SNAPs, and involves the generation of typical membranous intermediate structures that connect the disrupted ER tubules. A subsequent fusion is mediated by p97/p47/VCIP135, which has been shown to be required for homotypic fusion events in Golgi cisternae regrowth after mitosis. In addition, we also found that both fusion processes involve the t-SNARE, syntaxin 18.

Adenosine Triphosphatases↗
PubMed2005

Gene expression in human keloids is altered from dermal to chondrocytic and osteogenic lineage.

Keloids are a dermal fibrotic disease whose etiology remains totally unknown and for which there is no successful treatment. Here, we employed cDNA microarray analysis to examine gene expression in keloid lesions and control skin. We found that 32 genes among the 9000 tested were strongly up-regulated in keloid lesions, of which 21 were confirmed by Northern blotting. These included at least seven chondrocyte/osteoblast marker genes, and RT-PCR analysis revealed that transcription factors specific for these genes, SOX9 and CBFA1, were induced. Immunostaining and in situ hybridization further supported that these markers are expressed in keloid lesions. Intriguingly, scleraxis, a transcription factor known as a marker of tendons and ligaments, was also induced in keloid fibroblasts. We propose that reprogramming of gene expression or disordered differentiation from a dermal pattern to that of a chondrocytic/osteogenic lineage, probably closer to that of tendon/ligament lineage, may be involved in the etiology of keloids.

Adolescent↗
PubMed2005

Double-cycle, high-dose ifosfamide, carboplatin, and etoposide followed by peripheral blood stem-cell transplantation for small cell lung cancer.

PURPOSES: To determine the tolerability and feasibility of double-cycle, high-dose chemotherapy followed by peripheral blood stem-cell transplantation (PBSCT) after conventional chemotherapy or chemoradiotherapy for small cell lung cancer (SCLC). PATIENTS AND METHODS: Patients with previously untreated SCLC received two cycles of cisplatin, 80 mg/m2, and etoposide, 300 mg/m2 (cisplatin-etoposide [PE]). Later, they were administered high-dose etoposide, 1,500 mg/m2, followed by granulocyte colony-stimulating factor for collection of peripheral blood stem cells. After two additional cycles of PE, the patients received high-dose ifosfamide, 10 g/m2, carboplatin, 1,200 mg/m2, and etoposide, 1,000 mg/m2 (ifosfamide-carboplatin-etoposide [ICE]) followed by PBSCT twice at 3-month to 4-month intervals. Patients with limited disease (LD) concurrently received 50 Gy of irradiation with the last two cycles of PE. RESULTS: Eighteen patients, including 11 patients with LD, were enrolled. Fifteen patients could receive high-dose ICE followed by PBSCT twice, and 3 patients could receive it once. The median number of CD34+ cells collected was 13.11 x 10(6)/kg. The median numbers of days to neutrophil counts > or = 500/microL and platelet counts > or = 50,000/microL were 10 days and 14.5 days after the first PBSCT, and 10 days and 15 days after the second PBSCT, respectively. Grade 3 diarrhea occurred in one cycle, and grade 3 renal toxicity occurred in two cycles. The overall response rate was 100%, with an 83.3% rate of complete or near-complete response. The 2-year and 5-year survival rates were 72% and 55% in patients with LD and 43% and 0% in patients with extensive disease, respectively. CONCLUSION: Double-cycle, high-dose ICE therapy followed by PBSCT is tolerable and feasible even after conventional chemotherapy or chemoradiotherapy in patients with SCLC.

Adult↗
PubMed2005

Proinsulin lacking the A7-B7 disulfide bond, Ins2Akita, tends to aggregate due to the exposed hydrophobic surface.

A single mutation (C96Y) in the Ins2 gene, which disrupts the A7-B7 disulfide bond, causes the diabetic phenotype in Akita mice. We biochemically analyzed the conformation of wild-type and Akita mutant recombinant proinsulins. Gel filtration chromatography and dynamic light scattering revealed that the apparent size of the mutant proinsulin molecules was significantly larger than that of wild-type proinsulin, even in the absence of intermolecular disulfide bonds. Titration with a hydrophobic probe, 1-anilinonaphthalene-8-sulfonate, demonstrated that the mutant proinsulin was more hydrophobic than the wild type. In addition, circular dichroism studies revealed that the conformation of the mutant proinsulin was less stable than the wild type, which is consistent with the observation that hydrophobic residues are exposed on the surface of the proinsulin molecules. Studies with antiserum against the C-peptide of proinsulin indicated that the mutant proinsulin had an immunoreactivity that was at least one-tenth weaker than wild-type proinsulin, suggesting that the C-peptide of mutant proinsulin is buried inside the aggregate of the proinsulin molecule. These findings indicate that increased hydrophobicity of mutant proinsulin facilitates aggregate formation, providing a clue to the dominant negative effect in the Akita mouse.

Anilino Naphthalenesulfonates↗
PubMed2005

[Spinal cord metastases in lung cancer: a clinical review of four cases].

We report 4 cases of spinal cord metastases of lung cancer detected by MRI. Histologically, 3 of the 4 cases were small cell carcinoma and the other was adenocarcinoma. All 3 cases of small cell carcinoma had neoplastic meningitis. MRI taken in these cases showed the multiple nodules in the cauda equina, which were seeded from brain metastases. One of them had intramedullary spinal cord metastases, which appeared as enlargement of the spinal cord or nodules in the spinal cord on MRI. Leg paralysis and incontinence progressed in all cases. The other case of adenocarcinoma had epidural spinal cord compression due to spinal metastasis. In this case irradiation and corticosteroids relieved her leg and back pain. Spinal cord metastases should be considered as a differential diagnosis in patients with numbness, pain or weakness in the extremities.

Adenocarcinoma↗
PubMed2005

[A case of sarcoidosis with diffuse pulmonary arteriovenous malformations].

A 27-year-old woman was admitted because of right dry eye and blurred vision. She was given a diagnosis of uveitis due to sarcoidosis. Chest X-ray film showed bilateral hilar lymphadenopathy and multiple nodules in both lungs. The nodules were considered to be associated with sarcoidosis. However chest CT demonstrated that the nodules were well-defined and connected with two vessels. We diagnosed diffuse pulmonary arteriovenous malformations (AVMs) by 3D-CT. In three of sixteen AVMs, the feeding vessels were more than 3 mm wide in diameter on pulmonary arteriography, and those were embolized by platinum coils. The embolotherapy of diffuse AVMs could not ameliorate the shunt fraction sufficiently, but should reduce the possibility of the catastrophic complication of brain abscess.

Adult↗
PubMed2004

CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum.

Unfolded and malfolded client proteins impose a stress on the endoplasmic reticulum (ER), which contributes to cell death in pathophysiological conditions. The transcription factor C/EBP homologous protein (CHOP) is activated by ER stress, and CHOP deletion protects against its lethal consequences. We find that CHOP directly activates GADD34, which promotes ER client protein biosynthesis by dephosphorylating phospho-Ser 51 of the alpha-subunit of translation initiation factor 2 (eIF2alpha) in stressed cells. Thus, impaired GADD34 expression reduces client protein load and ER stress in CHOP(-/-) cells exposed to perturbations that impair ER function. CHOP(-/-) and GADD34 mutant cells accumulate less high molecular weight protein complexes in their stressed ER than wild-type cells. Furthermore, mice lacking GADD34-directed eIF2alpha dephosphorylation, like CHOP(-/-) mice, are resistant to renal toxicity of the ER stress-inducing drug tunicamycin. CHOP also activates ERO1alpha, which encodes an ER oxidase. Consequently, the ER of stressed CHOP(-/-) cells is relatively hypo-oxidizing. Pharmacological and genetic manipulations that promote a hypo-oxidizing ER reduce abnormal high molecular weight protein complexes in the stressed ER and protect from the lethal consequences of ER stress. CHOP deletion thus protects cells from ER stress by decreasing ER client protein load and changing redox conditions within the organelle.

Analysis of Variance↗
PubMed2004

Accumulation of type IV collagen in dilated ER leads to apoptosis in Hsp47-knockout mouse embryos via induction of CHOP.

Hsp47 is an endoplasmic reticulum (ER)-resident molecular chaperone that is specific for collagen. In Hsp47(-/-) mouse embryos at 9.5 days postcoitus (dpc), immunostaining indicated the absence of type IV collagen, but not of laminin and nidogen-1, in the basement membrane (BM). Electron immunomicroscopy revealed accumulation of type IV collagen in dilated ERs, but not in the BM of Hsp47(-/-) embryos, whereas it was only present in the BM in Hsp47(+/+) embryos. The BM structures stained with anti-laminin and anti-nidogen-1 antibody became disrupted in Hsp47(-/-) embryos at 10.5 dpc. Thus, in the absence of type IV collagen in the BM owing to the lack of Hsp47, the structure of the BM cannot be maintained during the dramatic morphological changes that take place around 10.5 dpc. Type IV collagen is therefore indispensable for the maintenance of BM structures during the late-stage development of mouse embryos, although not essential for the initial formation of the BM. Just before the death of Hsp47(-/-) embryos, DNA fragmentation typical of apoptosis was observed at 10.5 dpc together with significantly upregulated CHOP mRNA expression. ER stress caused by the accumulation of misfolded collagen may have induced apoptosis in Hsp47-knockout embryos through the upregulation of CHOP.

Animals↗
PubMed2004

Insufficient folding of type IV collagen and formation of abnormal basement membrane-like structure in embryoid bodies derived from Hsp47-null embryonic stem cells.

Hsp47 is a molecular chaperone that specifically recognizes procollagen in the endoplasmic reticulum. Hsp47-null mouse embryos produce immature type I collagen and form discontinuous basement membranes. We established Hsp47-/- embryonic stem cell lines and examined formation of basement membrane and production of type IV collagen in embryoid bodies, a model for postimplantation egg-cylinder stage embryos. The visceral endodermal cell layers surrounding Hsp47-/- embryoid bodies were often disorganized, a result that suggested abnormal function of the basement membrane under the visceral endoderm. Rate of type IV collagen secretion by Hsp47-/- cells was fourfold lower than that of Hsp47+/+ cells. Furthermore, type IV collagen secreted from Hsp47-/- cells was much more sensitive to protease digestion than was type IV collagen secreted from Hsp47+/+ cells, which suggested insufficient or incorrect triple helix formation in type IV collagen in the absence of Hsp47. These results indicate for the first time that Hsp47 is required for the molecular maturation of type IV collagen and suggest that misfolded type IV collagen causes abnormal morphology of embryoid bodies.

Animals↗
PubMed2004

Characterization of multiple members of the HSP70 family in platyfish culture cells: molecular evolution of stress protein HSP70 in vertebrates.

A shift from 28 to 37 degrees C in the incubation temperature of a culture of the platyfish fibroblast cell line, EHS cells (platyfish fibroblast cell line), induced a set of stress proteins. A two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis showed that the cells expressed three genetically distinct forms of heat-shock protein 70 (HSP70) family proteins: heat-inducible forms of HSP70, the constitutively expressed heat-shock cognate protein 70 (HSC70) and its phosphorylated isoform, and the glucose-regulated protein 78 (GRP78). Three different clones encoding two major isoforms of heat-inducible HSP70, platyfish HSP70-1 and HSP70-2, and of the HSC70 were isolated from a platyfish cDNA library. We compared the deduced amino acid sequences of the platyfish HSP70 and HSC70 proteins with those of other vertebrates. Phylogenetic analysis showed that vertebrate HSP70 could be classified into four cluster groups: (a) fish HSP70, with two isoforms of heat-inducible HSP70 in fish, fish HSP70-1 and HSP70-2; (b) the mammalian testis-specific HSP70-related protein HST70; (c) the mammalian heat-inducible HSP70B'; and (d) the mammalian major histocompatibility complex (MHC)-linked HSP70, including the MHC-linked heat-inducible HSP70 and the testis-specific HSP70-related protein. These findings suggest that vertebrate HSP70 was derived from a single ancestral HSP70 gene during vertebrate evolution and that multiple copies of heat-inducible HSP70 were probably evolved during genetic divergence in fish and higher vertebrates.

Amino Acid Sequence↗
PubMed2004

Advanced glycation end products increase collagen-specific chaperone protein in mouse diabetic nephropathy.

Advanced glycation end products (AGEs) appear to contribute to the diabetic complications. This study reports the inhibitory effect of OPB-9195 (OPB), an inhibitor of AGEs formation, and the role of a collagen-specific molecular chaperone, a 47-kDa heat shock protein (HSP47) in diabetic nephropathy. Transgenic mice carrying nitric-oxide synthase cDNA fused with insulin promoter (iNOSTg) leads to diabetes mellitus. The iNOSTg mice at 6 months of age represented diffuse glomerulosclerosis, and the expression of HSP47 was markedly increased in the mesangial area in parallel with increased expression of types I and IV collagens. OPB treatment ameliorated glomerulosclerosis in the iNOSTg mice associated with the decreased expression of HSP47 and types I and IV collagens. The expression of transforming growth factor-beta (TGF-beta) was increased in glomeruli of iNOSTg mice and decreased after treatment with OPB. To confirm these mechanisms, cultured mesangial cells were stimulated with AGEs. AGEs significantly increased the expression of HSP47, type IV collagen, and TGF-beta mRNA. Neutralizing antibody for TGF-beta inhibited the overexpression of both HSP47 and type IV collagen in vitro. In conclusion, AGEs increase the expression of HSP47 in association with collagens, both in vivo and in vitro. The processes may be mediated by TGF-beta.

Animals↗
PubMed2004

The endoplasmic reticulum stress response is stimulated through the continuous activation of transcription factors ATF6 and XBP1 in Ins2+/Akita pancreatic beta cells.

The dominant C96Y mutation of one of the two murine insulin genes, Ins2, causes diabetes mellitus in 'Akita' mice. Here we established pancreatic islet beta cell lines from heterozygous mice (Ins2+/Akita). Western blot analysis of endoplasmic reticulum (ER) molecular chaperones indicated that Grp78, Grp94 and Orp150 are significantly increased in Ins2+/Akita cells compared with wild-type (Ins2+/+) cells. Reporter gene assays using the human GRP78 promoter with or without the ER stress response element (ERSE) showed that Ins2+/Akita cells exhibit significantly stronger ERSE-dependent transcriptional activity than Ins2+/+ cells. Transient over-expression of the Ins2 C96Y mutant in wild-type beta cells induces a stronger ERSE-dependent stress response than does wild-type Ins2 over-expression. The ERSE-binding transcription factor ATF6 is strongly activated in Ins2+/Akita cells. The activity of a reporter containing the specific binding sequence of another ERSE-binding transcription factor, XBP1, is also enhanced in Ins2+/Akita cells. Levels of active forms of XBP1 mRNA and protein are both markedly elevated in Ins2+/Akita cells. These results indicate that this cell line is subject to continuous ER stress and that the Ins2 C96Y mutation induces the expression of ER chaperones through the activation of ATF6 and XBP1.

Activating Transcription Factor 6↗
PubMed2004

[Six cases of thymic carcinoma: a clinical review].

Thymic carcinoma is rarer than thymoma and carries a very poor prognosis. No standard treatment has yet been established. Chemotherapy and radiation therapy are usually given to patients in whom surgery is not indicated. We clinically review six patients with thymic carcinoma (three men and three women) who were admitted to our hospital from 1992 through 2002. Their ages ranged from 56 to 81 years (median 70.5). Four patients had chest pain, two were unable to swallow, and in one, who was asymptomatic, the tumor was detected by chance. Histologically, five cases were squamous cell carcinomas, and one was a lymphoepithelioma-like carcinoma. Four of the six patients had distant metastasis at diagnosis. Only one patient was able to undergo surgery, two received radiation therapy alone, and three were administered chemoradiation. The response to the chemoradiation was progression of the disease in two patients and no change in one. The response to radiation therapy was partial response in one patient and no change in one. In summary, chest pain is the most common symptom of thymic carcinoma. A symptomatic case suggests a poor prognosis resulting from the clinical behavior of thymic carcinoma, such as early distant metastasis or direct invasion of surrounding organs. Histologically, squamous cell carcinoma is the most common, and thymic carcinoma appears to be generally resistant to chemotherapy and radiotherapy.

Aged↗
PubMed2004

[Severe asthma complicated with large mucoid impaction: successful removal with balloon catheter].

A 28-year-old woman was hospitalized for renal transplantation. She suffered an asthma attack after transplantation. Once this attack had ended after medical treatment, she again experienced dyspnea after the 11th treatment day. A chest CT showed mucoid impaction in the left main bronchus, atelectasis in the left lower lobe, and pneumomediastinum. A huge mucoid impaction was removed with a balloon catheter under mechanical ventilation without exacerbation of the mediastinal emphysema. Mucoid impaction should be taken into account as a cause of dyspnea in severe asthma if the maximum dose of beta-stimulant is not effective.

Adult↗