Biomedical subjects

Masafumi Tomita

Publications and source records attributed to Masafumi Tomita.

13 recordsLinked to original sources

PubMed2007

Menaquinone-7 regulates gene expression in osteoblastic MC3T3E1 cells.

Previous study has shown that the vitamin K2 analog menaquinone-7 (MK-7) induces expression of the osteoblast-specific genes osteocalcin, osteoprotegerin, receptor activator of NFkappaB, and its ligand. Since MK-7 may also regulate osteoblast cell function, we examined the expression of osteoblast genes regulated by MK-7 administration. Differences between gene expression in control and MK-7-administered MC3T3E1 cells were analyzed using the suppression subtractive hybridization method. After 24 h of MK-7 administration, genes upregulated by MK-7 included tenascin C and BMP2. Genes downregulated by MK-7 administration included biglycan and butyrophilin. Real-time PCR showed a marked increase in tenascin C. When the protein level was examined using Western blot analysis, tenascin C was higher in MK-7-administered cells than in control cells. These results indicated that MK-7 affected the cellular function of osteoblastic MC3T3E1 cells. Considering BMP2 mRNA expression was higher in MK-7-administered cells than in control cells, the effect of MK-7 administration on the signal transduction system was examined. Western blot analysis showed that cells administered MK-7 displayed a higher phosphorylated Smad1 level than control cells. Because MC3T3E1 cells have a nuclear binding receptor for MK-7, this result might indicate an indirect effect of MK-7 through BMP2 production.

Animals↗

PubMed2006

Mouse model of paraquat-poisoned lungs and its gene expression profile.

Paraquat (PQ)-induced pulmonary toxicity is characterized by initial development of pulmonary edema, infiltration of inflammatory cells, and damage to the alveolar epithelium, which may progress to severe fibrosis. However, the exact role of PQ in the progression of the pathogenesis has not been clearly established. To understand the mechanism of PQ in pulmonary toxicity, we developed an animal model of PQ-induced lung injury by intranasal instillation of PQ solution using C57Black/6J mice. Twenty microliters of PQ solution (0.01, 0.01, and 0.04 mg/mouse) was applied through the nares, and the same amount of vehicle was applied in control mice. The pathological progression of lung pathology in our mouse model was very similar to that of patients suffering from PQ poisoning. The lungs of some animals exposed to PQ showed acute fulmination, resulting in death from 5 days post-exposure, but others showed a more protracted injury, resulting in typical pulmonary fibrosis at 3 weeks. Using this PQ-poisoned mouse model, we examined the gene expression at the initial destructive phase (within 5 days) that fibrosis has not completely developed. We prepared RNAs after 6h, 24h, and 5 days and examined the changes of the expression levels for 45 selected genes. The genes showing >2-fold increase at 6h or a time-dependent decrease during this experimental period may be the early markers for the destructive phase. These genes are Mt1, Mt2, Hmox1, Gcl, GR, IL-6, IL-13, Txn1, Fas, FasL, Lpin2, Mmp1a, Mmp12, Sfp-B, Sfp-D, CAT, EC-SOD, GST, and Pltp. On the other hand, the genes involved in the development of fibrosis, such as procollagen, Fn1, Eln, SMA, and Mmp9, Timp1 were significantly increased on day 5, not at 6h nor at 24h, after PQ treatment (the late marker). The genes showing a significant increase (Mmp3 and Mmp8) or decrease (VEGFA) at 24h and 5 days and not at 6h may be also the late markers. These changes in gene expression, which are equalled to functional activities of proteins, will be the targets for future studies focused on the development on PQ-induced pulmonary damage.

Administration, Intranasal↗

PubMed2006

Paraquat-induced gene expression in rat kidney.

Paraquat, one of the most widely used herbicides, is highly toxic to humans and animals. There is much information regarding its toxic effects on the lungs, but less is known about its toxicity in other organs. Paraquat is thought to play pivotal roles in the pathophysiology of acute renal failure and the progression of chronic kidney disease. We investigated the effects of paraquat on gene expression in the kidneys of rats treated with paraquat using a DNA array system, and the gene up-regulation observed was confirmed by quantitative real-time RT-PCR. Rats were sacrificed at 3, 24 h after the first injection (20 mg/kg), and at 3 h after the second injection. Expression of six genes had increased significantly by 3 h after the first injection: metallothionein-1 (MT-1), phosphoenolpyruvate carboxykinase, Na/K-transporting ATPase beta1 subunit, glutamate oxaloacetic transaminase, glutathione-S-transferase, and heme oxygenase-1 (HO-1). The transcription levels of MT-1 and HO-1 showed the biggest increases, but the increases did not continue until 24 h after injection, and the second injection had less effect than the first. Up-regulation of MT-1 and HO-1 mRNA levels was confirmed at the protein level. We observed a paraquat-induced increase of these proteins at 3 h post-injection, whereas this level did not continue until 24 h, as observed in RNA levels. The MT-1 protein in kidneys had been consumed. In addition, the protein level due to the second injection did not increase to the same level as that due to the first injection. These results suggest that protection against paraquat injury is mediated by induction of expression of some genes, and suppression on the induction of MT-1 and HO-1 may explain the injury observed due to paraquat intake. This is the first report of inducible pathways of defense against paraquat-induced oxidative stress in the kidney.

Animals↗

PubMed2006

Involvement of IL-10 and Bcl-2 in resistance against an asbestos-induced apoptosis of T cells.

To analyze the possibility that immunological alteration in asbestos-related diseases (ARDs) such as asbestosis (ASB) and malignant mesothelioma (MM) may affect the progression of cancers, a human adult T cell leukemia virus-immortalized T cell line (MT-2Org) was continuously exposed to 10 mug/ml of chrysotile-B (CB), an asbestos. After at least 8 months of exposure, the rate of apoptosis in the cells became very low and the resultant subline was designated MT-2Rst. The MT-2Rst cells were characterized by (i) enhanced expression of bcl-2, with regain of apoptosis-sensitivity by reduction of bcl-2 by siRNA, (ii) excess IL-10 secretion and expression, and (iii) activation of STAT3 that was inhibited by PP2, a specific inhibitor of Src family kinases. These results suggested that the contact between cells and asbestos may affect the human immune system and trigger a cascade of biological events such as activation of Src family kinases, enhancement of IL-10 expression, STAT3 activation and Bcl-2 overexpression. This speculation was partially confirmed by the detection of elevated bcl-2 expression levels in CD4 + peripheral blood T cells from patients with MM compared with those from patients with ASB or healthy donors. Further studies will be required to verify the role of T cells with enhanced bcl-2 expression in tumor progression induced by asbestos exposure.

Apoptosis↗

PubMed2006

Alterations of Fas and Fas-related molecules in patients with silicosis.

Persons with silicosis have not only respiratory disorders but also autoimmune diseases. To clarify the mechanisms involved in the dysregulation of autoimmunity found in patients with silicosis, we have been focusing on Fas and Fas-related molecules in the Fas-mediated apoptotic pathway, because Fas is one of the most important molecules regulating auto-immunity involving T cells. Our findings showed that patients with silicosis exhibited elevated serum soluble Fas levels, an increased relative expression of the soluble fas and dcr3 genes in peripheral blood mononuclear cells, high levels of other variant messages of the fas transcript, relatively decreased expression of genes encoding several physiological inhibitors (such as survivin and toso), and dominancy of lower-membrane Fas expressers in lymphocytes, which transcribe soluble fas dominantly, compared with soluble fas transcription in healthy donors. These findings are consistent with known features regarding immunological factors, such as serum immunogulobulin G levels and the titer of anti-nuclear autoantibodies in silicosis. In addition, anti-caspase 8 autoantibody and anti-Fas autoantibody were detected in serum specimens from patients with silicosis, and a functional assay showed that anti-Fas antibody stimulated Fas-mediated apoptosis. We hypothesize that there are two subpopulations of silicosis lymphocytes. One is a long-term surviving fraction that includes self-recognizing clones showing lower levels of membrane Fas and inhibition of Fas/Fas ligand binding in extracellular spaces. The other subpopulation exhibits apoptosis caused by silica and silicates, is recruited from bone marrow, shows higher levels of membrane Fas, and is sensitive to anti-Fas autoantibody. Further investigation should be performed to confirm the effects of silica and silicates on the human immune system.

Autoantibodies↗

PubMed2006

Gene expression in rat lungs during early response to paraquat-induced oxidative stress.

Paraquat (PQ) is a well-known pneumotoxicant and provides an established model of oxidative stress. This study focused on the transcriptional response to PQ-driven oxidative stress in rat lungs during an early phase post-injection. Rats were sacrificed at 3 h and 24 h after PQ injection (i.p., 20 mg/kg b.w.), and at 3 h after a second injection (i.p., 20 mg/kg b.w.). The left lungs were rapidly excised and used immediately for RNA preparation. The lung tissues did not show any pathological damage microscopically. Differential expression of RNAs in the lung at 3 h was investigated using a DNA array system. Fifteen genes showed a >1.7-fold change in expression level, which was confirmed by real-time PCR. Five genes related to oxidative stress, TRX, HO-1, GST-Yc, NQO-1, and RL/IF-1, and one gene, CLK3, whose function is unknown, showed a significant increase in their expression due to PQ injection. Two genes, HO-1 and NQO-1, that showed 3- and 2-fold increases at 3-h post-injection, were localized by immunohistochemistry. HO-1 was expressed in the bronchial epithelial cells, some type II cells and macrophages of control lungs, and the cells, especially the bronchial epithelial cells, were strongly stained 3 h following PQ treatment. Immunohistochemical analysis of NQO-1 also showed an increase in positive staining in the bronchial epithelial cells of PQ-treated lung sections. The expression of CYP2C6, 2C7, and 2C12, which are specific to or dominant in female liver, decreased markedly, while the male-specific CYP2C13 and 2C11 showed an increase or no effect. Further investigation is needed to clarify the role of these CYP2C family genes on the early phase of PQ toxicity. These results indicate that the acclimation to oxidative stress is already a highly complex process at the onset of PQ-induced damage and that the genes described herein may prove to be major contributors to the subsequent pulmonary fibrosis.

Animals↗

PubMed2005

Menaquinone-7 regulates the expressions of osteocalcin, OPG, RANKL and RANK in osteoblastic MC3T3E1 cells.

Epidemiological studies show that dietary intake of natto, which contains significant amount of vitamin K(2), reduces the risk of bone formation loss. However, many confounding factors, such as calcium and isoflavone, are found in natto, because it is made from soybeans. In this study, the direct effects of MK-7, a vitamin K(2) analogue, were assessed in osteoblasts. Osteoblastic MC3T3E1 cells were cultured with or without MK-7 for 10 days and the number of cells was calculated. The cell count was not different between MK-7 treated cells and control cells for 1, 2, and 4 days. However, it was significantly suppressed in MK-7 treated cells at 10 days, suggesting that MK-7 suppressed cell proliferation. Real-time PCR analysis showed that mRNAs of osteocalcin (OC), osteoprotegerin (OPG), and the receptor activator of the NFkappaB ligand (RANKL) were induced after MK-7 administration to the culture medium. RANK mRNA expression was also enhanced by MK-7 administration. Immunocytochemical analysis showed that MK-7 increased the protein levels of OC and RANKL. RANK protein was also enhanced, but this induction was suppressed by anti-RANK antibody administration. This suppression was recovered when anti-RANK antibody and MK-7 were administered. These observations suggest that MK-7 may directly affect MC3T3E1 cells and stimulate osteoblastic differentiation, not proliferation.

Animals↗

PubMed2005

Changes in gene expression level for defense system enzymes against oxidative stress and glutathione level in rat administered paraquat.

The herbicide paraquat (PQ) forms reactive oxygen species during enzymatic activation. We examined the effect of PQ on the relative levels of gene expression of antioxidant enzymes and glutathione (GSH) status in lungs of rats exposed to 20 mg/kg PQ. At 16 h after PQ intake, the mRNA expression level of glutathione reductase (GR) showed the greatest increase, and those of catalase (CAT) and manganese-superoxide dismutase (MnSOD) showed more modest increases. In contrast, PQ had little or no effect on the levels of mRNAs for copper/zinc-superoxide dismutase (CuZnSOD) and glutathione peroxidase (GPX). These findings indicate that CAT and MnSOD are coordinated and play a major role in removal of oxidants. On the other hand, PQ caused a significant increase in the GSH level in the lungs, but not in the liver. This increase in the lungs was, at least in part, caused by stimulation of the gamma-glutamylcysteine synthetase gene. However, the expression of GPX mRNA was not stimulated as described above. Because GSH is a substrate for GPX and serves as a scavenger of hydroxyl radicals, the increase in GSH as well as GR expression may be insignificant. This imbalance may be a result of oxidative stress due to PQ.

Animals↗

PubMed2004

Early differential gene expression of rat lung after exposure to paraquat.

Paraquat (PQ), a quaternary nitrogen herbicide, is highly toxic to humans and animals. Acute poisoning and death due to PQ exposure have been reported over the past few decades. Excessive production of oxygen free radicals has been proposed to play an important role in the pulmonary pathology. The aim of the present work was to evaluate the implications for genes that are regulated by oxidative stress at the early stage of PQ exposure in rat lungs. We performed differential display RT-PCR (DD-PCR) on total RNA extracted from rat lungs after injection of 20mg per kg body weight. The experimental DD-PCR conditions, primer length and annealing temperature, were adjusted to improve reproducibility, and 19 differentiated clones were isolated. Sequence analysis followed by conventional RT-PCR and real-time RT-PCR analyses were used to confirm the results. Four clones were finally determined to be significantly affected. These genes were mRNAs for plasma phospholipid transfer protein (PLTP), CL1BA protein, (latrophilin: LPH), and alphaII-spectrin as well as one unknown gene. We demonstrated the distribution of mRNA expression of one gene, LPH, in lung tissues. The present study suggests that 20mg per kg intraperitoneal PQ affects the expression of numerous genes in the lung at 3 h, the onset of pulmonary injury, and that the four genes specified may be major contributors to serious lung injury due to PQ exposure.

Animals↗

PubMed2003

Consecutive administration of paraquat to rats induces enhanced cholesterol peroxidation and lung injury.

It is our hypothesis that as a consequence of increased oxidative stress, rats develop lung injury with increased cholesterol-derived hydroperoxides and oxysterols in lung after consecutive exposure of the rats to paraquat. To test this we administered 10 mg/kg of paraquat i.p. once or seven times (once a day) to Wistar rats. Rats were killed, and lung tissue was collected 24 h after the last paraquat injection. We found that in response to consecutive paraquat doses, there were significant increases in 7alpha- and 7beta-hydroperoxycholest-5-en-3beta-ol (7alpha-OOH and 7beta-OOH; P=0.01) as well as 7alpha- and 7beta-hydroxycholesterol (7alpha-OH and 7beta-OH; P=0.01), and 7-ketocholesterol (7-keto; P=0.03). In addition, pulmonary hemorrhage, thickening of alveolar septum, and inflammatory cell infiltration of macrophages were observed. This is the first report showing enhanced cholesterol peroxidation and lung injury of rats due to consecutive doses of paraquat.

Animals↗

PubMed2002

Paraquat-induced gene expression in rat lung tissues using a differential display reverse transcription-polymerase chain reaction.

Increased formation of reactive oxygen species is a cause of paraquat (PQ)-induced injury and also provides a link between the signaling pathways and transcriptional events that regulate the expression of a large number of genes. However, the molecular mechanisms involved in PQ-induced injury remain unclear. To investigate the changes in gene expression at the onset of PQ injury, we used the differential display-polymerase chain reaction (PCR) method. Rats were treated intraperitoneally with 20 mg/kg PQ, and after 3 h the lungs were immediately excised. Samples of mRNA from normal and treated rats were used to prepare radiolabeled cDNAs, which were electrophoresed. Then the transcription levels were compared. We isolated 26 fragments of cDNA that were potentially affected by PQ, and determined their nucleotide sequences. Six clones of interest were selected and analyzed further. The reverse transcript-PCR based on their sequence information confirmed the differential expression for five clones: four clones were up-regulated and one was down-regulated. We were particularly interested in two genes that had homology with the known gene: TATA box-binding protein-associated factor, RNA polymerase II, B, 150 kDa (TAFIIB), and a candidate gene for lipodystrophy, Lpin2. Both genes were significantly up-regulated within 3 h of PQ intake and the stimulation continued during our 24-h observation period. In addition, up-regulation of Lpin2 was observed in the lungs, but not in the liver and kidneys. In situ hybridization using lung sections showed that the expression of both genes was strongly visualized in Clara cells and in alveolar macrophages. These findings suggest a stimulation of transcription levels and changes in lipid metabolism in Clara cells and in macrophages in the lungs, which result in their playing a crucial role at the onset of PQ-driven pulmonary injury.

Animals↗

PubMed2002

Hypervariable locus of the 3'-flanking region of the neurotensin receptor gene: an effective region for personal identification in forensic practice.

We examined the complex short tandem repeat (STR) locus at the 3'-flanking region of the neurotensin receptor (NTR) gene. The polymorphism of this locus was first reported as a simple tetranucleotide repeat variation by Le et al., but it also offers a surprisingly informative variation, that permits reliable individual identification by two complementary strategies: fluorescent-labelled polymerase chain reaction (PCR)/electrophoresis and direct sequencing of the PCR products. We determined the alleles in 203 Japanese by fluorescent-labelled PCR/electrophoresis. Determination was based on their length with a reliability of +/-1 bp, and the frequency of each allele was very low. Sequencing analysis further grouped these alleles in detail. Sequencing demonstrated that the locus varied by six repetitive units and three insertion/deletion positions of nucleotide fragments. We detected multiple alleles having different structures even in the same allele length. We found structural differences in homozygous alleles having the same base pair size. We also determined that apparently homozygous alleles were heterozygous from sequencing electropherograms showing an overlap of nucleotides or +/-1 bp difference. These results indicate that this locus is structurally hypervariable in addition to having allelic length variations, promising a great advance in individual identification in forensic practice.

Alleles↗

PubMed2002

Oxysterols as indices of oxidative stress in man after paraquat ingestion.

The aim of this study is to evaluate oxidative stress in man after paraquat ingestion by analyzing 7alpha- and 7beta-hydroperoxycholest-5-en-3beta-ol (7alpha- and 7beta-OOH) as well as oxysterols, cholesterol oxidation products, as indices of lipid peroxidation. Lung, kidney, and liver were collected at autopsy from seven patients with paraquat poisoning and seven controls matched for age and sex. We identified for the first time 7-ketocholesterol (7-keto) and 7-hydroxycholesterol (7alpha-OH and 7beta-OH) in human kidney by LC-MS. Next, we quantified 7alpha-OOH and 7beta-OOH by HPLC with postcolumn chemiluminescence as well as oxysterols by HPLC-UV. Both 7alpha-OOH and 7beta-OOH detected in lung and kidney from the controls were as low as the paraquat group. In contrast, we found both 7-keto and 7beta-OH in lung and 7-keto in kidney from the paraquat group were significantly higher than from the controls. This is the first report on accumulated oxysterols in lung and kidney from human paraquat poisoning. It seems to reflect greater oxidative stress in the pathology of paraquat intoxication.

Adult↗