Biomedical subjects

Akira Murakami

Publications and source records attributed to Akira Murakami.

At least 19 recordsLinked to original sources

PubMed2007

Zerumbone, a bioactive sesquiterpene, induces G2/M cell cycle arrest and apoptosis in leukemia cells via a Fas- and mitochondria-mediated pathway.

We demonstrated here for the first time that zerumbone (ZER), a natural cyclic sesquiterpene, significantly suppressed the proliferation of promyelocytic leukemia NB4 cells among several leukemia cell lines, but not human umbilical vein endothelial cells (HUVECs), by inducing G2/M cell cycle arrest followed by apoptosis with 10 microM of IC50. Treatment of NB4 cells with growth-suppressive concentrations of ZER resulted in G2/M cell cycle arrest that was associated with a decline of Cyclin B1 protein, but with the phosphorylation of ATM/ Chk1/Chk2. In addition, ZER induced the phosphorylation of Cdc25C at the Thr48 residue and Cdc2 at the Thr14/Tyr15 residues. Furthermore, ZER-induced apoptosis in NB4 cells was initiated by the expression of Fas (CD95)/Fas Ligand (CD95L), concomitant with the activation of caspase-8. ZER was also found to induce the cleavage of Bid, a mediator that is known to connect the Fas/CD95 cell death receptor to the mitochondrial apoptosis pathway. ZER also induced the cleavage of Bax and Mcl-1 proteins, but not Bcl-2 or Bcl-XL. ZER-induced apoptosis took place in association with a loss of the mitochondrial transmembrane potential as well as the activation of caspase-3 and -9, resulting in the degradation of the proteolytic poly (ADP-ribose) polymerase (PARP). ZER also triggered a release of cytochrome c into the cytoplasm. Both antagonistic anti-Fas antibody ZB4 and pan-caspase inhibitor Z-VAD inhibited ZER-induced apoptosis in NB4 cells. Taken together, ZER is an inducer of apoptosis in leukemic cells that specifically triggers the Fas/CD95- and mitochondria-mediated apoptotic signaling pathway.

Antineoplastic Agents↗

PubMed2006

Auraptene decreases the activity of matrix metalloproteinases in dextran sulfate sodium-induced ulcerative colitis in ICR mice.

Previously we reported that auraptene was a potent suppressant for matrix metalloproteinase (MMP)-7 expression in HT-29 human colon cancer cells. In the present study, we examined the effects of auraptene on MMP-2, -7, and -9 expression in colonic mucosa from dextran sulfate sodium (DSS)-induced ulcerative colitis mice. Auraptene remarkably suppressed the DSS-induced gelatinolytic activity of MMP-7 as well as the expression of MMP-2 and -9, suggesting that it might be useful in anti-metastatic therapies via the targeting of MMPs.

Animals↗

PubMed2006

Mutational analysis of RPGR and RP2 genes in Japanese patients with retinitis pigmentosa: identification of four mutations.

PURPOSE: To identify mutations in RPGR and RP2 genes in a series of Japanese retinitis pigmentosa (RP) families and to determine the association between the phenotypic changes in patients/carriers and the mutations. METHODS: A total of 37 unrelated RP families were recruited, three of which were with typical X-linked RP (XLRP), and other 34 families included 29 multiplex families and 5 simplex RP cases with no family history of RP. In addition, At least one RP patient had myopia >-3.0D in these families. RPGR and RP2 genes were comprehensively screened by using the direct polymerase chain reaction-sequencing method. Detailed phenotypes of the families with confirmed mutations were assessed by routine ophthalmic examinations, Goldmann perimetry, electroretinography and color fundus photography. RESULTS: Four mutations in RPGR and RP2 genes were identified. Of the three XLRP families, one had an ORF15 mutation and another had an RP2 mutation. Two ORF15 mutations were found in three of the other 34 RP families, with two families sharing a same mutation, g.ORF15+652-653delAG. All the three ORF15 mutations were first reported in the Japanese population. Affected males showed relatively severe symptoms while female carriers showed a wide spectrum of severity. A tapetal-like reflex was observed in two young females, indicating clinically the carrier status. CONCLUSIONS: We identified three ORF15 mutations and one RP2 mutation in five Japanese RP families. Moderate or severe myopia might be an indicator for the XLRP status in multiplex RP families which pedigree data are insufficient to allow accurate subtyping. It is suggested that mutational analysis of RPGR and RP2 may help to identify the causative mutation in a proportion of multiplex RP patients with myopia.

Adult↗

PubMed2006

Citrus auraptene targets translation of MMP-7 (matrilysin) via ERK1/2-dependent and mTOR-independent mechanism.

Matrix metalloproteinase (MMP)-7 is considered to play essential roles in cancer progression. We examined the efficacy of auraptene, a citrus coumarin derivative, for suppressing MMP-7 expression in the human colorectal adenocarcinoma cell line HT-29. Auraptene remarkably inhibited the production of proMMP-7 protein, without affecting its mRNA expression level. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), showed similar results, suggesting that auraptene suppresses mTOR-dependent proMMP-7 translation. Interestingly, however, auraptene showed no effects on the activation of Akt/mTOR signaling, whereas the phosphorylation levels of 4E binding protein (4EBP)1 and eukaryotic translation initiation factor (eIF)4B were substantially decreased. In addition, auraptene remarkably dephosphorylated constitutively activated extracellular signal-regulated kinase (ERK)1/2. Transfection of ERK1/2 siRNA led to a significant reduction of proMMP-7 protein production as well as of the phosphorylation of eIF4B. These results demonstrate that auraptene targets the translation step for proMMP-7 protein synthesis by disrupting ERK1/2-mediated phosphorylation of 4EBP1 and eIF4B.

Antibiotics, Antineoplastic↗

PubMed2006

Involvement of MAP kinases in lipopolysaccharide-induced histamine production in RAW 264 cells.

Roles of mitogen-activated protein (MAP) kinases in lipopolysaccharide (LPS)-induced production of histamine in the mouse macrophage-like cell line RAW 264 were analyzed. Incubation of RAW 264 cells in the presence of LPS increased histamine levels in the conditioned medium in a concentration- and time-dependent manner. The levels of histidine decarboxylase (HDC) mRNA and the 74-kDa HDC protein were also increased at 4 to 8 h and 8 to 12 h, respectively. LPS elicited the phosphorylation of p44/42 MAP kinase, p38 MAP kinase, and c-Jun N-terminal kinase (JNK). The MAP kinase-Erk kinase 1 inhibitor U0126 (0.1-10 microM) suppressed the LPS-induced phosphorylation of p44/42 MAP kinase, and inhibited the LPS-induced production of histamine and expression of the HDC mRNA and 74-kDa HDC protein in a concentration-dependent manner. The JNK inhibitor SP600125 (3-30 microM) suppressed the LPS-induced phosphorylation of c-Jun, and inhibited the LPS-induced production of histamine and expression of the HDC mRNA and 74-kDa protein in a concentration-dependent manner. Combined treatment with U0126 (0.3 microM) and SP600125 (10 microM) inhibited the LPS-induced production of histamine additively. The p38 MAP kinase inhibitor SB203580 (0.1-10 microM) partially inhibited the LPS-induced production of histamine. These findings suggest that LPS increases histamine production in RAW 264 cells by inducing the expression of the 74-kDa HDC protein, and that the LPS-induced expression of HDC is up-regulated at the transcriptional level by MAP kinases, especially p44 MAP kinase and JNK.

Animals↗

PubMed2006

Polymorphism of beta-adrenergic receptors and susceptibility to open-angle glaucoma.

PURPOSE: The human trabecular meshwork and ciliary body, which express beta-adrenergic receptors (ADRB1 and ADRB2), control aqueous humor dynamics. We investigated associations of ADRB polymorphisms with open-angle glaucoma (OAG), because ADRB gene polymorphisms alter receptor function. METHODS: We studied 240 Japanese controls and 505 Japanese OAG patients including 211 with primary open-angle glaucoma (POAG), and 294 with normal-tension glaucoma (NTG). Associations of four polymorphisms (Ser49Gly and Arg389Gly in the ADRB1 gene; Arg16Gly and Gln27Glu in the ADRB2 gene) were compared between patients and controls. Age, intraocular pressure (IOP), and visual field defects at diagnosis were examined for associations with polymorphisms. RESULTS: The Arg389Gly polymorphism in the ADRB1 gene showed significantly different allele and genotype frequencies in patients with NTG than in controls (p = 0.004 and 0.006, respectively). Other polymorphisms did not show a significant frequency difference. In POAG patients, carriers of Gly16 in the ADRB2 gene were significantly younger at diagnosis than noncarriers (p<0.001). The IOP at diagnosis was significantly higher in OAG patients carrying 27Glu in the ADRB2 gene than in patients without this allele (p<0.001). Clinical characteristics of OAG patients did not differ significantly in relation to other polymorphisms. CONCLUSIONS: Certain polymorphisms of the ADRB1 and ADRB2 genes influence the pathophysiology of OAG in Japanese patients.

Aged↗

PubMed2006

Nobiletin, a citrus flavonoid, suppresses phorbol ester-induced expression of multiple scavenger receptor genes in THP-1 human monocytic cells.

Unregulated uptake of oxidized low-density lipoproteins (ox-LDL) via macrophage scavenger receptors (SRs) such as lectin-like ox-LDL receptor-1 (LOX-1) is a key event in atherosclerosis. In this study, we examined the effects of five selected food phytochemicals on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced LOX-1 mRNA expression in THP-1 human monocyte-like cells. Nobiletin, a citrus polymethoxylated flavone, markedly reduced it in dose- and time-dependent manners. It also suppressed the phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2, c-Jun NH2-terminal kinase (JNK) 1/2, and c-Jun (Ser-63), thereby inhibiting the transcriptional activity of activator protein-1. Further nobiletin attenuated expression of SR-A, SR-PSOX, CD36, and CD68, but not CLA-1, mRNA, leading to the blockade of DiI-acLDL uptake. Together, our results suggest that nobiletin is a promising phytochemical for regulating atherosclerosis with reasonable action mechanisms.

Carbocyanines↗

PubMed2006

Three-dimensional cell seeding and growth in radial-flow perfusion bioreactor for in vitro tissue reconstruction.

Radial-flow perfusion bioreactor systems have been designed and evaluated to enable direct cell seeding into a three-dimensional (3-D) porous scaffold and subsequent cell culture for in vitro tissue reconstruction. However, one of the limitations of in vitro regeneration is the tissue necrosis that occurs at the central part of the 3-D scaffold. In the present study, tubular poly-L-lactic acid (PLLA) porous scaffolds with an optimized pore size and porosity were prepared by the lyophilization method, and the effect of different perfusion conditions on cell seeding and growth were compared with those of the conventional static culture. The medium flowed radially from the lumen toward the periphery of the tubular scaffolds. It was found that cell seeding under a radial-flow perfusion condition of 1.1 mL/cm2 x min was effective, and that the optimal flow rate for cell growth was 4.0 mL/cm2 x min. At this optimal rate, the increase in seeded cells in the perfusion culture over a period of 5 days was 7.3-fold greater than that by static culture over the same period. The perfusion cell seeding resulted in a uniform distribution of cells throughout the scaffold. Subsequently, the perfusion of medium and hence the provision of nutrients and oxygen permitted growth and maintenance of the tissue throughout the scaffold. The perfusion seeding/culture system was a much more effective strategy than the conventional system in which cells are seeded under a static condition and cultured in a bioreactor such as a spinner flask.

Animals↗

PubMed2006

Real-time monitoring of antibody secretion from hybridomas on a microchip by time-resolved luminescence anisotropy analysis.

This article presents a real-time monitoring system for cellular analysis using micro total analysis systems technology. Time-resolved luminescence anisotropy analysis was adopted for real-time detection of small amounts of a target protein produced by a small number of cells. The system was tested by real-time monitoring of the antibody secretion by hybridomas. The cells were successfully cultivated in a micro-incubation chamber (240 nl) fabricated on a microchip. The quantification of the antibody was achieved using the Ru(II) complex-labeled Staphylococcus aureus protein A probe, which can bind specifically to the Fc region of the antibody. Using this system, we detected as little as 24 fmol of immunoglobulin G under physiological conditions without the bound/free separation protocol. We successfully achieved real-time and quantitative monitoring of small amounts of antibody production by approximately 200 hybridoma cells. This method could be applied to various cellular analyses using small numbers of cells.

Animals↗

PubMed2006

Paraoxonase 1 gene polymorphisms influence clinical features of open-angle glaucoma.

BACKGROUND: The purpose of this study was to determine whether genetic polymorphisms affecting high-density lipoprotein (HDL)-associated antioxidant enzymes were associated with open-angle glaucoma (OAG). The rationale for this study was that the modification of low-density lipoprotein (LDL) by HDL prevents oxidative modification which can then cause dysfunction of endothelial cells. METHODS: We studied 284 normal Japanese controls and 555 Japanese patients with OAG, including primary open-angle glaucoma (POAG) and normal-tension glaucoma (NTG). The possible associations of polymorphisms of PON1/L55M, PON1/Q192R, PON2/S311C, and PAF-AH/V279F with OAG were investigated. We compared the genotype distributions and allele frequency in controls and patient groups. The age at diagnosis, intraocular pressure (IOP) at diagnosis, and visual field score at diagnosis were examined for association with polymorphisms. RESULTS: The distributions of genotypes and allele frequency for the four polymorphisms were not significantly different between any patient group and controls. In NTG patients, 55M carriers of the PON1 gene were significantly older at diagnosis than 55M non-carriers (P=0.001). The IOP at diagnosis was significantly higher in glaucoma patients carrying 192R in the PON1 gene than in patients not carrying 192R (P=0.006). No significant differences were seen in clinical characteristics of OAG patients in relation to other polymorphisms. CONCLUSION: PON1 gene polymorphisms may influence the features of Japanese patients with OAG.

Adult↗

PubMed2006

Synthesis and cross-linking activities of oligodeoxynucleotides containing a 4-oxoalkenal group.

The 4-oxoalkenal group, which has been characterized as part of a novel lipid-peroxidation-derived genotoxin, reacts with dG, dC, and dA, yielding etheno adducts. To develop a new interstrand cross-linking system based on the covalent bond formed between the 4-oxoalkenal group and DNA bases, we prepared 4-oxoalkenal-derivatized oligodeoxynucleotides. The protected 4-oxoalkenal derivative with a primary amino group was synthesized and incorporated to the 5' end of the oligodeoxynucleotide. In the presence of the complementary strand, cross-linking products were observed by HPLC and MALDI-MS analysis.

Chromatography, High Pressure Liquid↗

PubMed2006

Solid-phase detection of RNA using bispyrene-modified RNA probe.

Bispyrene modified 2'-O-methyl oligoRNA (OMUpy2), which is a useful fluorescent probe for specific RNA detection, was immobilized on a glass substrate via various linker molecules to develop a more convenient RNA detection chip for gene expression study. As the fluorescence intensity of OMUpy2 enhanced greatly when it hybridized with its complementary RNA, it was expected that RNA detection by OMUpy2-immobilized chip (RNA-chip) required neither fluorescent labels to target nucleic acid nor washing protocol after the hybridization. As the linker molecules, oligo(dT), agarose, and poly(ethylene glycol) (PEG) were chosen, and the fluorescence from the chip was measured by a fluorescence microscope. Results indicated that the RNA-chip was able to detect the complementary oligoribonucleotide (cORN) without target labeling and washing protocols. Furthermore, the background emission was reduced as the length of the linker increased. In the case of PEG linker, 25-fold enhancement of the fluorescence intensity of the OMUpy2 was observed upon the addition of cORN.

Fluorescent Dyes↗

PubMed2006

Properties of novel antisense oligonucleotides containing 2'-O-modified adenosine with a photo-reactive group.

In order to enhance the efficiency of antisense molecules for target RNA regulation, a novel photo-reactive antisense oligonucleotide was developed. We designed and synthesized a photo-reactive antisense oligonucleotide containing an adenosine in which 2'-OH was modified with 4,5',8-trimethylpsoralen (Ps) via an ethoxymethylene linkage (2'-Ps-eom). We evaluated the photo-cross-linking efficiency and sequence specificity toward complementary RNA (match-RNA). 2'-Ps-eom was selectively photo-cross-linked to the match-RNA. The photo-cross-linking efficiency was about 75% upon UVA-irradiation (365 nm) for 10 min. Previously, we reported oligonucleotides that had an adenosine anchoring Ps at 2'-O-position via a methylene linkage (2'-Ps-met). The photo-cross-linking efficiency of 2'-Ps-met and match-RNA was about 35% upon UVA-irradiation for 120 min. The photo-cross-linking efficiency of 2'-Ps-eom was dramatically enhanced in comparison with the one of 2'-Ps-met.

Adenosine↗

PubMed2006

Various properties of polymeric carriers improved the transfection efficiency.

In order to develop novel efficient gene carriers, we have been focusing on the transcription of transgene in the nucleus among various steps in the gene transfer system. Optimal carrier properties for improving the transgene recognition by transcription factors have not been clarified so far. In the present study, we established a novel evaluation system for the intranuclear transcription efficiency of the transgene using microinjection technique. Polyplexes composed of polypeptides with different molecular weights were microinjected into the cytoplasm or nucleus of COS-1 cells, and the relationship between the carrier properties, such as molecular weight, and the intranuclear transcription efficiency was evaluated. The molecular weight (Mw) dependency of the transgene transcription in the living cells was successfully quantified, and the low Mw polymers were found not to suppress the transcription but high Mw polymers allowed almost no transcription. Interestingly, the transcription efficiencies of poly(arginine) (PR) and poly(lysine) (PK) were almost same although the PR is widely reported to be more efficient gene carrier than PK. The difference in the various properties and intracellular trafficking of PR/DNA and PK/DNA polyplexes will be discussed.

Animals↗

PubMed2006

The interaction and compatibility between a soft contact lens and an ophthalmic drug.

PURPOSE: To investigate the interaction and compatibility between a soft contact lens (SCL) and an ophthalmic drug. METHODS: Samples were prepared with the nonionic SCL (groups I and II), the anionic SCL (group IV), and the zwitter ionic SCL (group IV). Chlorpheniramine maleate and sodium cromoglycate were used to measure the drug uptake into the SCL. RESULTS: The results showed the largest drug uptake into the anionic SCL accompanied with dimensional changes and lower drug uptakes into the nonionic and the zwitter ionic SCL with dimensional stability. The cell permeability of the substances when instilled in the eye over the SCL was also measured. The zwitter ionic SCL allowed cell permeability comparable to that by oral administration. CONCLUSIONS: These results indicated the possibility for the zwitter ionic SCL as a lens to be worn concomitantly with ophthalmic drug instillation.

Animals↗

PubMed2006

Reduced expression of laminin-5 in corneal epithelial cells under high glucose condition.

PURPOSE: Laminin-5 (alpha3, beta3, gamma2 chains) is a major component of corneal basement membrane and has a crucial role in corneal epithelial cell adhesion. On the other hand, diabetic keratopathy has a varied degree of adhesive disturbance in corneal epithelial cells. Therefore, in this study, we investigated whether a high glucose condition altered the expression of laminin-5 in corneal epithelial cells in vitro. METHODS: Human corneal epithelial (HCE) cells were cultured in either normal (5 mmol/L) or high glucose (30 mmol/L) medium for 5 passages before being used in experiments. We first examined the effect of a high glucose condition on the expression of mRNA and proteins for 3 chains of laminin-5 in HCE cells by semiquantitative reverse transcriptase-polymerase chain reaction, Western blotting analysis, and immunofluorescence staining. Second, we tried a cell detachment assay. After 5 days of incubation in high or normal glucose medium, HCE cells were treated with a solution of 0.05% trypsin and EDTA (0.2 mmol/L), pH = 8. The number of detached cells at different times after treatment was determined using a cell count machine. RESULTS: We found that alpha3 chain expression was reduced at the mRNA level in a high glucose condition, whereas beta3 and gamma2 chains showed no change. The high glucose condition induced the inhibition in the synthesis of 190-kd, 160-kd alpha3 chain and 105-kd gamma2 chain proteins of laminin-5. The adhesion capacity of HCE cells in the high glucose medium was weaker than that of HCE cells in normal glucose medium. Soluble laminin-5 rescued the detachment of HCE cells in high glucose medium. CONCLUSION: The loss of homeostatic levels of laminin-5 under a high glucose condition may correlate to weaken epithelial cell adhesion, resulting in the clinical manifestation of diabetic keratopathy.

Blotting, Western↗

PubMed2006

1'-Acetoxychavicol acetate inhibits RANKL-induced osteoclastic differentiation of RAW 264.7 monocytic cells by suppressing nuclear factor-kappaB activation.

Osteoclastogenesis is commonly associated with various age-related diseases, including cancer. A member of the tumor necrosis factor superfamily, receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL), has been shown to play a critical role in osteoclast formation and bone resorption. Thus, agents that suppress RANKL signaling have a potential to suppress bone loss. In this report, we investigated the effect of 1'-acetoxychavicol acetate (ACA), a component of Alpina galanga, on RANKL signaling and consequent osteoclastogenesis in RAW 264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated NF-kappaB, and coexposure of the cells to ACA completely suppressed RANKL-induced NF-kappaB activation in a time- and concentration-dependent manner. The suppression of NF-kappaB by ACA was mediated through suppression of RANKL-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, and IkappaBalpha degradation. Furthermore, incubation of monocytic cells with RANKL induced osteoclastogenesis, and ACA suppressed it. Inhibition of osteoclastogenesis was maximal when cells were simultaneously exposed to ACA and RANKL and minimum when ACA was added 2 days after RANKL. ACA also inhibited the osteoclastogenesis induced by human breast cancer MCF-7 cells, multiple myeloma MM1 cells, and head and neck squamous cell carcinoma LICR-LON-HN5 cells. These results indicate that ACA is an effective blocker of RANKL-induced NF-kappaB activation and of osteoclastogenesis induced by RANKL and tumor cells, suggesting its potential as a therapeutic agent for osteoporosis and cancer-associated bone loss.

Animals↗

PubMed2006

SNPs and interaction analyses of noelin 2, myocilin, and optineurin genes in Japanese patients with open-angle glaucoma.

PURPOSE: To evaluate the noelin 2 gene as a disease-causing factor for open-angle glaucoma (OAG) and the interactions between the noelin 2 (OLFM2), optineurin (OPTN), and myocilin (MYOC) genes. METHODS: OLFM2 was analyzed in 770 Japanese subjects including 215 patients with elevated intraocular pressure (IOP), 277 with normal IOP, 38 with juvenile open-angle glaucoma, and 240 control subjects. Two single-nucleotide polymorphisms (SNPs) in OPTN (c.412G-->A and c.603T-->A) and one SNP in MYOC (c.227G-->A) were examined. Single genes were investigated by univariate analysis and the gene-gene interactions by logistic regression analysis. Associations between genotypes and clinical characteristics at the time of diagnosis were examined. RESULTS: In OLFM2, 12 sequence variants were identified in 770 Japanese subjects. Arg144Gln (exon 4) was identified in two (0.3%) of the patients and in none of the control subjects. Combinations of OLFM2/317A and OPTN/412A or OLFM2/1281T and OPTN/412A were associated with patients with elevated IOP (P = 0.018 or P = 0.012, respectively). The combination of OLFM2/317G and OPTN/603A was significantly associated with elevated IOP (P = 0.018). No significant association was detected between SNPs in OLFM2 and in MYOC. Patients with normal IOP and with OLFM2/678A+OPTN/412G or OLFM2/1281C+OPTN/412G had significantly worse visual field scores (P = 0.022 or 0.030, respectively). CONCLUSIONS: The Arg144Gln mutation in OLFM2 is a possible disease-causing mutation in Japanese patients with OAG. Common polymorphisms in OLFM2 and OPTN may interactively contribute to the development of OAG, indicating a polygenic etiology.

Adult↗