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Biomedical subjects

Akira Murakami

Publications and source records attributed to Akira Murakami.

At least 73 records · Page 4Linked to original sources
PubMed2004

Zerumbone, a tropical ginger sesquiterpene, activates phase II drug metabolizing enzymes.

Zerumbone (ZER), a sesquiterpene compound occurring in tropical ginger Zingiber zerumbet Smith, has been implicated as one of the promising chemopreventive agents against colon and skin cancer. In the present study, we investigated the phase II detoxification enzymes induction of ZER using a cultured rat normal liver epithelial cell line. Exposure of RL34 cells to ZER resulted in the significant induction of glutathione S-transferase, while the reduced analogues of ZER (alpha-humulene and 8-hydroxy-alpha-humulene) did not show any inducing effect. Therefore, the electrophilic property, characterized by the reactivity with intracellular nucleophiles including protein sulfhydryls as well as low molecular weight thiols, at the 8-position alpha,beta-unsaturated carbonyl group plays an important role in the induction of phase II enzymes. ZER induced nuclear localization of the transcription factor Nrf2 that binds to antioxidant response element (ARE) of the phase II enzyme genes, suggesting that ZER is a potential activator of the Nrf2/ARE-dependent detoxification pathway. This is consistent with the observation that ZER potentiated the gene expression of several Nrf2/ARE-dependent phase II enzyme genes, including gamma-glutamylcysteine synthetase, glutathione peroxidase, and hemeoxygenase-1. The present study also implied the antioxidant role of this detoxification system activation by ZER in the neutralization of lipid peroxidation in hepatocytes, providing a new insight for cancer prevention.

Animals↗
PubMed2004

Zerumbone, a sesquiterpene in subtropical ginger, suppresses skin tumor initiation and promotion stages in ICR mice.

We recently showed that zerumbone, a sesquiterpene found in subtropical ginger, suppresses colonic tumor marker formation in rats and induces apoptosis in colon cancer cell lines. In our present study, the anti-tumor initiating and promoting activities of zerumbone in mouse skin were evaluated using a conventional 2-stage carcinogenesis model. A single topical pretreatment to mouse skin (2 micromol) 24 hr before application of dimethylbenz[a]anthracene (0.2 micromol) markedly suppressed tumor incidence by 60% and the number of tumors by 80% per mouse. Repeated pretreatment (16 nmol) twice weekly during the post-initiation phase reduced the number of 12-O-tetradecanoylphorbol-13-acetate (TPA, 1.6 nmol)-induced tumors by 83% as well as their diameter by 57%. Multiple reverse transcriptase (RT) PCR experiments revealed that zerumbone (2 micromol) enhanced the mRNA expression level of manganese superoxide dismutase, glutathione peroxidase-1, glutathione S-transferase-P1 and NAD(P)H quinone oxidoreductase in the epidermis, but not that of cytochrome p450 1A1 or 1B1. Further, it diminished TPA-induced cyclooxygenase-2 protein expression and phosphorylation of extracellular signal-regulated kinase 1/2, while pretreatment(s), in either the priming or activation stage or both, reduced double TPA application-induced hydrogen peroxide formation and edema induction by 29% to 86%, respectively. Histologic examination revealed that pretreatment(s) with zerumbone suppressed leukocyte infiltration and reduced proliferating cell nuclear antigen-labeling indices. Together, our results indicate that zerumbone is a promising agent for the prevention of both tumor initiating and promoting processes, through induction of anti-oxidative and phase II drug metabolizing enzymes as well as attenuation of proinflammatory signaling pathways.

Animals↗
PubMed2004

SOX7 and GATA-4 are competitive activators of Fgf-3 transcription.

Fgf-3 is expressed in a dynamic and complex spatiotemporal pattern during mouse development. Previous studies identified GATA-4 as a transcription factor that binds the key regulatory element PS4A of the Fgf-3 promoter and stimulates transcription. Here we show that members of the SOX family of transcription factors also bind PS4A and differentially modulate transcription. At least five SOX genes, Sox2, Sox6, Sox7, Sox13, and Sox17, were expressed in F9 cells, and of these, Sox7 and Sox17 were dramatically induced in parallel with Fgf-3 following differentiation into parietal endoderm-like cells with retinoic acid and dibutyryl cAMP. Complexes could be detected on PS4A with SOX2, SOX7, and SOX17 by using nuclear extracts from differentiated F9 cells. However, only Sox7 expression markedly activated the Fgf-3 promoter in these cells. By contrast, SOX2 was a poor activator of Fgf-3 transcription, and when Sox2 was coexpressed with Gata4, it negatively modulated the strong activation mediated by GATA-4. More detailed analyses showed that SOX7 competes with GATA-4 for PS4A occupancy and to activate the Fgf-3 promoter. In situ hybridization analysis showed that Sox7 is co-expressed with Fgf-3 and Gata4 in the parietal endoderm of E7.5 mouse embryos. In culture, GATA-4-deficient embryonal stem cells were shown to express Fgf-3 upon differentiation into embryoid bodies, although at lower levels than were found in wild type embryonal stem cells. This Fgf-3 expression was virtually abolished when Sox7 expression was suppressed by RNA interference. These results show that SOX7 is a potent activator of Fgf-3 transcription.

Animals↗
PubMed2004

Induction of apoptosis in human myeloid leukemic cells by 1'-acetoxychavicol acetate through a mitochondrial- and Fas-mediated dual mechanism.

PURPOSE: The purpose of this investigation was to determine the antileukemic effects of 1'-acetoxychavicol acetate (ACA) obtained from rhizomes of the commonly used ethno-medicinal plant Languas galanga (Zingiberaceae). EXPERIMENTAL DESIGN: We evaluated the effects of ACA on various myeloid leukemic cells in vitro and in vivo. We further examined the molecular mechanisms of ACA-induced apoptosis in myeloid leukemic cells. RESULTS: Low-dose ACA dramatically inhibited cellular growth of leukemic cells by inducing apoptosis. Because NB4 promyelocytic leukemic cells were most sensitive to ACA, we used NB4 cells for further analyses. Production of reactive oxygen species triggered ACA-induced apoptosis. ACA-induced apoptosis in NB4 cells was in association with the loss of mitochondrial transmembrane potential (DeltaPsim) and activation of caspase-9, suggesting that ACA-induced death signaling is mediated through a mitochondrial oxygen stress pathway. In addition, ACA activated Fas-mediated apoptosis by inducing of casapse-8 activity. Pretreatment with the thiol antioxidant N-acetyl-L-cysteine (NAC) did not inhibit caspase-8 activation, and the antagonistic anti-Fas antibody ZB4 did not block generation of reactive oxygen species, indicating that both pathways were involved independently in ACA-induced apoptosis. Furthermore, ACA had a survival advantage in vivo in a nonobese diabetic/severe combined immunodeficient mice leukemia model without any toxic effects. CONCLUSIONS: We conclude that ACA induces apoptosis in myeloid leukemic cells via independent dual pathways. In addition, ACA has potential as a novel therapeutic agent for the treatment of myeloid leukemia.

Adult↗
PubMed2004

Cancer prevention with food factors: alone and in combination.

Cancer prevention strategies making use of combined agents with distinct molecular mechanisms, rather than individual agents, are considered promising for higher efficacy and lower toxicity. Although there is increasing understanding of the synergistic combinations of synthetic agents, our knowledge regarding such combinations of food factors remains limited. We recently found that free radical generation suppressants from food items in combination with their scavengers at low concentrations exhibited notable synergistic effects in activated leukocytes, whereas combinations of agents with similar modes of action showed additive or antagonistic effects. For example, pound -)-epigallocatechin gallate (EGCG) from green tea has been shown to increase the endotoxin-induced production of proinflammatory mediators such as prostaglandin E(2) and tumor necrosis factor-alpha in RAW264.7 macrophages, whereas the soybean isoflavonoid genistein compensated for these inverse properties of EGCG, leading to marked suppression in combination. The present review briefly highlights the potential effectiveness of combinations of several agents with anti-oxidative and anti-inflammatory properties for cancer preventive strategies.

Anti-Inflammatory Agents↗
PubMed2004

Zerumbone, an anti-inflammatory phytochemical, induces expression of proinflammatory cytokine genes in human colon adenocarcinoma cell lines.

Zerumbone, a sesquiterpene occurring in zingiberaceous plants in Southeast Asian countries, has been shown to have anti-inflammatory effects in several independent experimental studies. We examined its effect on the expression of proinflammatory genes in human colon adenocarcinoma cell lines, Caco-2, Colo320DM, and HT-29, using reverse transcription-polymerase chain reaction (RT-PCR) assays. Surprisingly, zerumbone markedly induced the expression of interleukin (IL)-1alpha, IL-1beta, IL-6, and tumor necrosis factor (TNF)-alpha in each cell line in concentration- and time-dependent manners. Results of a previous pharmacological approach using specific inhibitors of mitogen-activated protein kinases (MAPKs) suggested that the activation of both c-Jun N-terminal kinase and extracellular signal-regulated protein kinase, however, not that of p38 MAPK, may be involved in zerumbone-induced IL-1beta expression pathways in Caco-2 cells. The present results imply that zerumbone increases the production of proinflammatory cytokines in cancerous tissues in the colon and that this biochemical property may cause side-effects.

Adenocarcinoma↗
PubMed2004

Citrus nobiletin inhibits azoxymethane-induced large bowel carcinogenesis in rats.

The inhibitory effects of dietary feeding of citrus nobiletin on azoxymethane (AOM)-induced rat colon carcinogenesis using a long-term bioassay were investigated. Five-week old male F344 rats were initiated with two weekly subcutaneous injections of AOM (20 mg/kg bw) to induce colonic tumors. They were also given the diets containing 0.01% or 0.05% nobiletin for 34 weeks, starting one week after the last dosing of AOM. At the end of the study, the incidence of colonic adenocarcinoma were 67% in the AOM alone group, 55% in the AOM-->0.01% nobiletin group, 35% (p<0.05) in the AOM-->0.05% nobiletin group. Also, nobiletin feeding reduced the cell-proliferation activity, increased the apoptotic index, and decreased the prostaglandin E2 content in colonic adenocarcinoma and/or colonic mucosa. These findings might suggest that citrus nobiletin has chemopreventive ability against AOM-induced rat colon carcinogenesis.

Animals↗
PubMed2004

Regulatory analysis of the mouse Fgf3 gene: control of embryonic expression patterns and dependence upon sonic hedgehog (Shh) signalling.

Fgf3 displays a dynamic and complex expression pattern during mouse embryogenesis. To address the molecular mechanisms underlying Fgf3 expression, we used a transgenic approach to assay genomic regions from the mouse Fgf3 gene for regulatory activity. We identified an enhancer that mediates major components of embryonic expression, governing expression in the midbrain, hindbrain, surface ectoderm, dorsal roots and dorsal root ganglia (DRG), proximal sensory ganglia, and the developing central nervous system (CNS). Deletional analysis of the enhancer further delimited this regulatory activity to a 5.7-kb fragment. We have also revealed sonic hedgehog (Shh) -dependent and Shh-independent aspects of Fgf3 expression through breeding the Fgf3 reporter transgene into Shh mutants. In the absence of Shh signalling, Fgf3 reporter expression is lost in the ventral CNS, DRG, and superior cervical nerves, whereas activation of reporter expression in cranial ganglion cells is Shh independent. Moreover, detailed re-examination of the Shh phenotype revealed that Shh signalling is required for the correct development/maturation of the DRG.

Animals↗
PubMed2004

Compound heterozygous mutations of M1S1 gene in gelatinous droplike corneal dystrophy.

PURPOSE: To report the genetic findings in a Chinese patient diagnosed with gelatinous droplike corneal dystrophy (GDLD). DESIGN: Case report and experimental study. METHODS: Molecular genetic analysis was performed on the DNA extracted from peripheral leukocytes from a Chinese patient with GDLD and his unaffected parents. Fifty healthy, unrelated, Chinese participants were used as control subjects. The M1S1 gene was amplified by polymerase chain reaction and directly sequenced. RESULTS: The patient was clinically diagnosed with GDLD. Direct sequencing of the M1S1 gene revealed heterozygous changes in both alleles, a novel Y184C mutation on one allele and a Q118X mutation on the other that was reported as a founder mutation in the Japanese population. The patient's unaffected parents showed only the heterozygous Q118X or Y184C mutation. The mutation was not detected in the 50 unaffected subjects. CONCLUSIONS: This is the first genetic analysis of a Chinese patient with GDLD. Because the compound heterozygote mutations Q118X and Y184C cosegregated with the phenotype, they are likely the cause of GDLD in this patient.

Adolescent↗
PubMed2004

Microarray analysis identified differentially expressed genes in keratocytes from keratoconus patients.

PURPOSE: To identify differentially expressed genes in human keratoconus keratocyte by cDNA microarray. METHODS: Normal and keratoconic cornea were cultured for keratocytes. RNA was extracted. cDNA probe labeled with fluorescence dye was made from Poly A+ RNA, hybridized with microarray slide, containing 164 human apoptosis genes. Signal intensity was measured. Expression ratio between keratoconus and normal was determined using ImaGene Ver.3.0. Identified genes were further evaluated by RT-PCR and real-time PCR. RESULTS: Five over-expressed and four under-expressed genes were identified. Of these, differential expression of tumor necrosis factor alpha-induced protein 6 (TNFAIP6), human insulin-like growth factor binding protein 5 (IGFBP5), and IGFBP3 were verified and confirmed by RT-PCR. Real-time PCR showed TNFAIP6 increased by 3.3 folds, while IGFBP5, IGFBP3 decreased by 14 and 11 folds respectively. CONCLUSIONS: The identified genes could be important and deserve further investigation. Significant differential expression of TNFAIP6, IGFBP5, and IGFBP3 may indicate an important role of these genes in the mechanism underlying stromal thinning.

Adult↗
PubMed2004

Tear chymase in vernal keratoconjunctivitis.

PURPOSE: To determine the levels of mast cell chymase and tryptase activity in the tears of patients with vernal keratoconjunctivitis (VKC). METHODS: Subjects were 38 VKC patients and 18 healthy controls whose chymase and tryptase activity in tears was measured by enzyme assay. VKC severity was quantified based on the following clinical signs: papillary hypertrophy, conjunctival hyperemia, edema, punctate keratitis, Trantas dots, and mucus production. Of the 38 VKC patients, the degree of disease severity was mild in 13, moderate in 18, and severe in 7. RESULTS: Mean chymase activity and standard deviation in tears was 0.23+/-0.07mU in mild VKC, 0.68+/-0.22mU in moderate VKC, 1.91+/-0.71 mU in severe VKC, and 0.11+/-0.05 mU in healthy controls. The increase in all VKC stages was statistically significant compared to that in healthy control. The degree of chymase activity in tears correlated significantly with VKC severity (r = 0.9245, p < 0.001). High tryptase activity was also detected in the tears of VKC patients, although increased tryptase activity in tears did not correlate with disease severity (r = 0.1999). CONCLUSIONS: Chymase activity in tears may thus be a sensitive marker for determining the severity of VKC.

Adolescent↗
PubMed2004

An enhanced site-specific transcription efficiency of DNA/polypeptide-vector polyplexes.

A novel gene delivery system based on the enhanced release of plasmid DNA from polyplexes in response to intracellular proprotein convertase (PC), furin, that cleaves the carboxyl side of Arg-X-Arg/Lys-Arg, was successfully established. Polypeptides that have this recognition sequence were synthesized by the Fmoc solid phase method and evaluated as gene vectors. The fragmented polypeptides were found to lose the abilities to form polyplexes with plasmid DNA. When the Fur-polypeptide/plasmid DNA polyplexes were treated with furin in vitro, their disassembly and the release of plasmid DNA was observed in agarose gel electrophoresis analysis. The Fur-polypeptide showed higher transgene expression in COS-1 cells, while the control polypeptides which do not have the recognition sites did not. These results indicate that the cationic nature of the polypeptides is not sufficient for gene carrier and this intracellular signal-responsive gene transfer system is the useful strategy to enhance the transcription efficiency of the delivered plasmid DNA.

Amino Acid Sequence↗
PubMed2004

Transcriptome analysis using fluorescence-labeled oligonucleotide.

In our previous studies, the fluorescence intensity at 480 nm of the bispyrene-labeled 2'-O-methyl-oligoribonucleotide (OMUpy2) was drastically enhanced only when it was hybridized with its complementary RNA. In this study, we demonstrated the potential of the OMUpy2 for transcriptome analysis in the cell free system and in the living cell. For the analysis in the cell free system, the micro-chamber was designed and used to evaluate the detection limit of the target RNA. The detection limit of oligo-RNA was approximately 15 fmol. For the analysis of the living cells, C-OMUpy2 was introduced to the cervical carcinoma cells, C4II, and the fluorescence from C-OMUpy2 was monitored. The fluorescence from the cells began to be observed 5 min after the serum stimulation, and the fluorescence was decreased after 20 min. These results seemed to be consistent with the results from the RT-PCR analysis and suggested that OMUpy2 could be a useful tool for the transcriptome analysis.

Cell Line, Tumor↗
PubMed2004

Synthesis and properties of fluorescent-labeled oligonucleotides containing amide-linked dinucleoside with pyrene.

Oligonucleotides containing 2'-pyrene modified uridine (Upy) exhibit the increase of the pyrene monomer emission when hybridize with the complementary RNA. In order to examine the effect of restricted C3'-endoconformation of the Upy residue for the enhancement of the pyrene monomer emission, an oligodeoxyribonucleotide containing amide-linked dinucleoside with a pyrene group at the sugar residue (UapyODN) was synthesized by use of 3'-carboxymethyl-5'-O-dimethoxytrityl-2'-O-(1-pyrenylmethyl)-3'-deoxyuridine. NMR analysis indicated that the furanose ring of the uridine derivative was fixed to C3'-endo conformation. The UapyODN showed increase of fluorescence intensity to 7-fold at 375 nm when hybridized with complementary oligoribonucleotide. This result suggests that the UapyODN can detect the target RNA.

Amides↗
PubMed2004

Dietary supplementation of the citrus antioxidant auraptene inhibits N,N-diethylnitrosamine-induced rat hepatocarcinogenesis.

OBJECTIVES: We have previously reported that an antioxidant, auraptene (AUR), isolated from citrus fruit effectively inhibits chemically induced carcinogenesis in digestive tracts, such as the oral cavity, esophagus and large bowel. In this study, we investigated the modifying effects of dietary supplementation with AUR on N,N-diethylnitrosamine (DEN)-initiated hepatocarcinogenesis in male F344 rats in two different experiments to determine whether the compound exerts a cancer-chemopreventive action in other organs. METHODS: In the first experiment, animals were fed diets containing AUR at dose levels of 100 and 500 ppm for 7 weeks 1 week before, during, and 1 week after the start of liver carcinogenesis induced by DEN (40 ppm in drinking water for 5 weeks) to predict the modulatory effect on hepatocarcinogenesis. After 7 weeks, the numbers of hepatocellular enzyme-altered foci (EAF; cm(2)) which stained positive for the placental form of glutathione S-transferase (GST-P) and transforming growth factor (TGF)-alpha were determined on immunohistochemically stained sections. In the second experiment conducted to confirm the findings, animals subjected to DEN treatment were fed AUR-containing diets (100 and 500 ppm) during either the initiation stage ('initiation' feeding for 7 weeks) or post-initiation phase ('post-initiation' feeding for 25 weeks) of DEN-induced hepatocarcinogenesis. RESULTS: In the first experiment, feeding with AUR at both doses during DEN exposure decreased the mean numbers of GST-P-positive and TGF-alpha-positive EAF/cm(2), and the reduction in the number of TGF-alpha-positive EAF by feeding 500 ppm AUR was statistically significant (p < 0.005). In the second experiment, the 'initiation' feeding with 500 ppm AUR significantly inhibited the incidence (33 vs. 83%, p = 0.000511) and multiplicity (0.67 +/- 1.09 vs. 1.96 +/- 1.85, p < 0.005) of liver cell carcinoma. Also, the 'post-initiation' feeding with AUR at both doses significantly reduced the development of hepatocellular carcinoma (100 ppm: incidence, 15%, p = 0.000006; multiplicity: 0.25 +/- 0.64, p < 0.001; 500 ppm: incidence, 11%, p = 0.000002; multiplicity, 0.26 +/- 0.81, p < 0.001). In addition, AUR feeding reduced cell proliferation and the apoptotic index in liver cell neoplasms. CONCLUSIONS: The results suggest that the citrus antioxidant AUR is a potential chemopreventive agent against DEN-induced hepatocarcinogenesis in rats.

Animals↗
PubMed2004

Variants in optineurin gene and their association with tumor necrosis factor-alpha polymorphisms in Japanese patients with glaucoma.

PURPOSE: To investigate sequence variations in the optineurin (OPTN) gene and their association with TNF-alpha polymorphisms in Japanese patients with glaucoma. METHODS: The OPTN gene was analyzed in blood samples from 629 Japanese subjects. There were 194 patients with primary open-angle glaucoma (POAG), 217 with normal-tension glaucoma (NTG), and 218 with no eye disease (control subjects). The gene was screened for mutations by denaturing high-performance liquid chromatography. Genotyping of three polymorphisms of -308G-->A, -857C-->T, and -863C-->A in the TNF-alpha promoter region was performed. The associations between the genotypes and age, intraocular pressure (IOP), and visual field defects at the time of diagnosis were examined. RESULTS: A possible glaucoma-causing mutation, His26Asp, was identified in 1 of the 411 Japanese patients with glaucoma. A c.412G-->A (Thr34Thr) polymorphism in the OPTN gene was significantly associated with POAG (genotype frequency, P = 0.011; allele frequency, P = 0.003). The frequency of TNF-alpha/-857T and optineurin/412A carriers was significantly higher (P = 0.006) in patients with POAG than in control subjects. Among the patients with POAG who were carriers of TNF-alpha/-857T, the optineurin/412A carriers had significantly worse (P = 0.020) visual field scores than the non-optineurin/412A ones. The frequency of TNF-alpha/-863A and optineurin/603A (or Lys98) carriers was significantly higher in patients with POAG (P = 0.008) or NTG (P = 0.027) than in control subjects. Among the patients with POAG who were carriers of TNF-alpha/-863A, the ones with optineurin/603A (or Lys98) had significantly worse (P = 0.026) visual field scores than did those with non-optineurin/603A (or Lys98). CONCLUSIONS: These findings demonstrated that the OPTN gene is associated with POAG rather than NTG in the Japanese. Statistical analysis showed a possible interaction between polymorphisms in the OPTN and the TNF-alpha genes that would increase the risk for glaucoma.

Adult↗
PubMed2004

Suppressive effects of selected antioxidants on the activated leukocytes-induced mutagenesis in the co-culture assay systems.

We recently established a novel co-culture assay system using activated inflammatory cells and AS52 Chinese hamster ovary cells, and demonstrated that reactive oxygen and nitrogen species (RONS) generated from activated inflammatory leukocytes induce mutations in the gpt recorder gene in AS52 cells. In this study, we examined the inhibitory effects of 19 agents with antioxidative properties on RONS generation in cultured inflammatory cells and on mutagenesis in AS52 cells co-cultured with activated inflammatory cells. The results demonstrate that there is a linear correlation between the ability of these agents to suppress RONS production in activated inflammatory cells and to inhibit mutation in AS52 cells.

Animals↗