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Biomedical subjects

Akira Murakami

Publications and source records attributed to Akira Murakami.

At least 55 records · Page 3Linked to original sources
PubMed2005

Preparation and properties of ProNectin F-coated biodegradable hollow fibers.

ProNectin F-coated biodegradable hollow fibers were newly prepared and their cytocompatibility was evaluated in vitro. Although the coating efficiency onto poly(L-lactic acid) (PLLA) and poly(lactide-co-caprolactone) [p(LA/CL)] matrices was similar, the cell adhesion properties were greatly affected by the nature of the polymer substrate. ProNectin F-coated PLLA showed about seven times higher cytocompatibility than ProNectin F-coated p(LA/CL). The single-extruded melt spinning method and the core-sheath bicomponent melt spinning method were employed to prepare PLLA hollow fibers. The effect of the spinning conditions, such as the melt draw ratio, spinneret temperature, and take-up speed, on the diameter and wall thickness of the spun fibers was studied in detail. For single-extruded melt spinning, a segmented type of spinneret was used, and the effect of the flow rate of nitrogen, which was confined in the hollow part of fibers, was studied. X-ray photographs of the drawn hollow fibers, clarified the significant molecular orientation, which was much higher than that in drawn solid PLLA fiber under identical drawing conditions. The morphology and mechanical properties of hollow fibers demonstrated an increase in the tensile strength and a decrease in the thickness of the PLLA wall with increased nitrogen flow rates and melt draw ratios for single-extruded melt spinning. These results indicate the unique characteristics of ProNectin F-coated PLLA hollow fibers, which can be successfully utilized as a biodegradable substrate.

Absorbable Implants↗
PubMed2005

Macular hole secondary to epiretinal membrane in a juvenile patient.

PURPOSE: To report a macular hole secondary to an idiopathic epiretinal membrane in a 14-year-old girl. DESIGN: Interventional case report. METHODS: A 3-year-old girl who failed a public visual-screening test was examined. Since then, she had been followed-up for her epiretinal membrane, which began to peel spontaneously in the left eye for 11 years until a macular hole was observed. She was treated by vitrectomy and membranectomy, and the membrane was histologically examined. RESULT: The macular hole was successfully closed, and her visual acuity improved. The membrane appeared to have a homogenous structure and contained no cells. CONCLUSIONS: The juvenile idiopathic epiretinal membrane sometimes peels spontaneously. However, long-term follow-up is needed because vitreous traction of the membrane by intraocular aging change may result in a macular hole.

Child, Preschool↗
PubMed2005

Mast cell chymase decreases the barrier function and inhibits the migration of corneal epithelial cells.

PURPOSE: We investigated the in vitro effects of human mast cell chymase on corneal epithelial cells. METHODS: Human corneal epithelial cells were incubated with human chymase at activity levels that were likely to exist in the tears of patients with vernal keratoconjunctivitis. RESULTS: Incubation of chymase resulted in a decrease of barrier function of corneal epithelium. Occludin protein was cleaved by chymase. In the wound assay, incubation with chymase resulted in an inhibition of cell migration. CONCLUSION: Human chymase causes the proteolysis of occludin and fibronectin, resulting in a decrease of barrier function and inhibition of the migration of corneal epithelial cells.

Blotting, Western↗
PubMed2005

Mast cell chymase induces conjunctival epithelial cell apoptosis by a mechanism involving degradation of fibronectin.

PURPOSE: To determine the mechanism of conjunctival epithelial injury in vernal keratoconjunctivitis, we investigated the effects of human chymase on conjunctival epithelial cells in vitro. METHODS: Human conjunctival epithelial cells were incubated with human chymase for 24 or 48 hr at levels of activity that were likely to exist in the tear fluid of patients with vernal keratoconjunctivitis. Morphologic changes of the cells were observed by phase contrast microscopy. To determine the number of detached cells, we used an automated cell counter, while apoptotic cells were quantitated by flow cytometry. The level of soluble fibronectin in conditioned medium was measured by ELISA. RESULTS: Most of the cells in the incubation with chymase were detached by 24 hr. However, chymase-mediated apoptosis was a slower process and was only detected after incubation of cells with chymase for 36 to 48 hr. Both cell detachment and apoptosis were blocked when cells were incubated in fibronectin-coated plates. The increase of soluble fibronectin was dependent on the amount of chymase added and the exposure time. A caspase inhibitor (antiapoptotic agent) rescued cells from apoptosis but did not prevent cell detachment. These results indicate that chymase-induced apoptosis of conjunctival epithelial cells represents anoikis, which is a slowly occurring apoptotic process induced by lack of adhesion to an extracellular matrix. CONCLUSIONS: Human mast cell chymase caused conjunctival epithelial cell detachment by degrading fibronectin, and this led to secondary apoptosis.

Apoptosis↗
PubMed2005

Novel bioassay system for evaluating anti-oxidative activities of food items: use of basolateral media from differentiated Caco-2 cells.

Reactive oxygen and nitrogen species, including superoxide and nitric oxide (NO), are known to be mediators of oxidative stress and play pivotal roles in the onset of numerous life style-related diseases. While a number of studies have shown that naturally occurring anti-oxidants may be applicable for prevention and therapy for those diseases, most in vitro anti-oxidation tests reported have not provided significant insight into the absorption efficiency or metabolism of dietary anti-oxidants in the gastrointestinal tract. In the present study, we established a novel assay system by focusing on the bioconversion of food constituents using differentiated Caco-2 cells as a model of human intestinal epithelial cells. Various fresh food preparations [ginger, garlic, shimeji (Hypsizigus marmoreus), onion, carrot] were added to the apical side of differentiated Caco-2 monolayers. After incubation, the medium was recovered and tested for its inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced generation in differentiated HL-60 cells, and on combined lipopolysaccharide (LPS)- and interferon (IFN)-gamma -induced NO generation in RAW 264.7 macrophages. The garlic preparation (25% v/v) basolateral medium abolished generation without any cytotoxicity toward HL-60 cells, though it was cytotoxic to Caco-2 cells. In the NO generation tests, all of the food preparations showed notable inhibitory activity, while the garlic preparation (5% v/v) basolateral medium inhibited NO generation with substantial cytotoxicity toward RAW 264.7 cells. Interestingly, the carrot preparation (1% v/v) basolateral medium inhibited NO generation in both a concentration- and time-dependent manner without any cytotoxicity toward RAW 264.7 or Caco-2 cells, and its activities were higher than those of the carrot preparation alone (1% v/v). Our results indicate that the present assay system is appropriate and reliable for determination of the anti-oxidative efficacy of dietary phytochemicals in vivo.

Animals↗
PubMed2005

Zingiberaceous and citrus constituents, 1'-acetoxychavicol acetate, zerumbone, auraptene, and nobiletin, suppress lipopolysaccharide-induced cyclooxygenase-2 expression in RAW264.7 murine macrophages through different modes of action.

In the present study, we explored the suppressive activities of 1'-acetoxychavicol acetate (ACA), auraptene, nobiletin, and zerumbone toward LPS-induced cyclooxygenase (COX)-2 mRNA expression in mouse macrophages and the underlying molecular mechanisms. Pretreatment of RAW264.7 cells with LPS led to the activation of mitogen-activated protein kinase (MAPK)s [p38, extracellular signal-regulated kinase (ERK)1/2, c-Jun NH2-terminal kinase (JNK)1/2] and Akt, together with degradation of the inhibitor of nuclear factor-kappaB (IkappaB)-alpha protein and nuclear translocation of nuclear factor (NF)-kappaB p65, and the resultant activation of activator protein (AP)-1, NF-kappaB, and cAMP-responsive element-binding protein (CREB) transcription factors. ACA abrogated ERK1/2 and JNK1/2, but not p38 MAPK, as well as the activation of those transcription factors. Although it allowed LPS-triggered phosphorylation of those MAPKs and NF-kappaB nuclear translocation, nobiletin suppressed the activation of AP-1, NF-kappaB, and CREB. Zerumbone had no effect on those transcription factors, though it attenuated COX-2 mRNA expression, suggesting that it disrupts the stabilization of COX-2 mRNA. Conversely, zerumbone significantly accelerated spontaneous COX-2 mRNA decay, the potency of which was comparable with that of SB203580, an inhibitor of p38 MAPK, whose activation has key roles in the proinflammatory mRNA stabilization processes. Because SB203580 but not zerumbone suppressed LPS-induced p38 MAPK activation, the molecular targets of zerumbone may be MAPK-activated protein kinase-2 or located downstream. However, auraptene suppressed the expression of COX-2 protein but not mRNA, implying that it targets translation. We propose that these phytochemicals are promising chemopreventive agents for inflammation-associated carcinogenesis. Their use in combination may enhance their efficacy because of their different modes of action.

Animals↗
PubMed2005

Synthesis of antisense oligonucleotides containing a photocleavable protecting group on a guanine base and their photoinduced duplex formation.

An oligonucleotide containing a photocleavable protecting group at a guanine base was synthesized to induce the duplex formation by photo-irradiation. Alpha-methyl-2-nitropiperonyl (MeNP) group was used for the photocleavable protecting group at O6 position of deoxyguanosine. The oligonucleotide containing MeNP group (MeNP-ODN:5'-dTTCTG(MeNP)TCTGT-3') was synthesized by phosphoramidite method. The MeNP group was found to be removable by UV irradiation at wavelength of 365 nm for 5 min in 98% yield. UV-melting temperature (Tm value) analysis indicated that the duplex of MeNP-ODN with the complementary RNA was significantly unstable compared with the unmodified DNA/RNA duplex (deltaTm = -25 degrees C). After UV irradiation at 365 nm, the Tm value of the mixture increased to the same as that of the unmodified duplex. These results suggest that the RNA binding ability of the MeNP-ODN can be induced by photocleavage of the MeNP group.

Benzodioxoles↗
PubMed2005

Luminescence anisotropy-based detection of nucleic acids and proteins using long-lifetime Ru(II) complex as a luminescent label.

Luminescence anisotropy-based methods are powerful tool for the detection of biomolecules in homogeneous physiological media without Bound/Free separation. However, analyses based on the method are always disturbed by the autofluorescence from biological specimen. This disturbance limits the sensitivity of the method. To improve the detection limit of the method, a long-lifetime luminophore was adopted as the label. Because of the lifetime of the autofluorescence is below 10 ns, it is expected that the use of the long-lifetime luminophore enables us to avoid the disturbance by adopting time-resolved luminescence anisotropy measurement. To verify this concept, oligodeoxyribonucleotides and Staphylococcus aureus protein A were labeled with Ru(II) complex (tau approximately 500 nsec) and used for the detection of Escherichia coli 16S ribosomal RNA (16S rRNA) and immunoglobulin G (IgG), respectively. Results indicated that the methods were useful for the detection of rRNA and IgG without disturbance of the autofluorescence.

Anisotropy↗
PubMed2005

Synthesis and properties of photo-reactive antisense oligonucleotides containing 2'-O-psoralen-conjugated adenosine.

In order to selectively regulate mRNA having a point mutation, the photo-reactive antisense oligonucleotides were developed. Two types of photo-reactive oligonucleotides containing adenosine whose 2'-OH was modified with 4,5',8-trimethylpsoralen (psoralen) were synthesized (2'-Ps-oligo). One contains psoralen via a methylene linkage (2'-Ps-met), and the other via an amidomethylene linkage (2'-Ps-amd). 2'-Ps-oligos were then subjected to the photo-cross-linking reaction. 2'-Ps-met cross-linked to the complementary RNA and scarcely did to the RNA having a single mismatch base. Contrarily, 2'-Ps-amd did not cross-link to both RNA strands. These results suggest the structure of the linkage might affect the efficiency of the photo-cross-linking.

Adenosine↗
PubMed2005

The destabilization of polyplexes facilitates intranuclear transcription efficiency.

We have been focusing on the last step of transfection, that is, the intranuclear recognition of polyplexes by transcription factors. In this work, we studied about the relationship between the intracellular destabilization of polyplexes and the transcription efficiency. Polypeptides containing specific sequences which are digested by intracellular proprotein convertase (PC), furin (Fur-polypeptide) were synthesized as novel carriers. When Fur-polypeptide/plasmid DNA polyplexes were incubated with furin in the cell-free transcription/translation system, the sequence specific expression was observed as time. Additionally, Fur-polypeptides/plasmid DNA polyplexes led to site-specific effective transgene expression even in COS-1 cells. These results indicate that shortened carriers lose their abilities to form polyplexes in response to furin activity in the cells, and it seems to enhance the destabilization of polyplexes, the recognition of transgene in polyplexes by transcription factors, and the expression efficiency.

Animals↗
PubMed2005

Viral FLIP enhances Wnt signaling downstream of stabilized beta-catenin, leading to control of cell growth.

Death receptor-mediated apoptosis is potently inhibited by viral FLIP (FLICE/caspase 8 inhibitory protein), which is composed of two tandemly repeated death effector domains (DEDs), through reduced activation of procaspase 8. Here, we show that equine herpesvirus 2-encoded viral FLIP E8 enhances Wnt/beta-catenin signaling in a variety of cell lines. E8 was shown to strikingly augment Wnt3a signaling, as shown both in a luciferase assay for T-cell factor/beta-catenin and through induction of endogenous cyclin D1. The effect of E8 was independent of its direct binding activity with DED-containing signaling molecules, including caspase 8 and FADD, in death receptor-mediated apoptosis. E8 enhanced Wnt signaling downstream of stabilized beta-catenin, while a long form of cellular FLIP (c-FLIP(L)) enhanced stabilization of beta-catenin in 293T cells. Consequently, coexpression of E8 and c-FLIP(L) synergistically increased Wnt signaling in 293T cells. Moreover, E8-mediated stimulation of Wnt signaling induced dramatic growth retardation in untransformed cell lines but not in transformed cell lines. Thus, viral FLIP E8 not only inhibits death receptor-mediated apoptosis but also enhances Wnt signaling pathways that are closely related to those of both ontogenesis and oncogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Nobiletin, a citrus flavonoid, down-regulates matrix metalloproteinase-7 (matrilysin) expression in HT-29 human colorectal cancer cells.

Overexpression of matrix metalloproteinases (MMPs) is associated with cancer metastasis. We assessed mRNA expression of MMPs in six human colorectal cancer cell lines and found a considerable level of MMP-7 expression in HT-29 cells. Next, we searched for natural and synthetic compounds that cause a reduction in the production of proMMP-7 protein, and found that nobiletin (NOB), quercetin, valeryl salicylate, and sulindac sulfone demonstrated marked inhibition. Importantly, NOB attenuated proMMP-7 protein and its mRNA expression both concentration- and time-dependently via a reduction of activator protein-1 (AP-1) DNA binding activity, suggesting it as a promising agent for suppression of cancer cell invasion and metastasis through MMP-7 gene repression.

Colorectal Neoplasms↗
PubMed2005

Evaluation of dynamic features of Escherichia coli 16S ribosomal RNA in homogeneous physiological solution.

There is no methodology for the estimation of the dynamic features of large-molecular-weight RNAs in homogeneous physiological media. In this report, a luminescence anisotropy-based method using a long-lifetime luminescent oligonucleotide probe for the estimation of the dynamic features of large-molecular-weight RNA is described. As a luminescent probe, Ru(II) complex-labeled oligonucleotides, which have a complementary sequence to the single-stranded regions of Escherichia coli 16S rRNA, were synthesized. After the hybridization of the probe to single-stranded regions of 16S rRNA, the segmental motions of the regions were evaluated by time-resolved luminescence anisotropy analysis. In 16S rRNA, the L2 site (323-332 nt) was found to be the most flexible among the seven sites chosen. From a comparison between the hybridization kinetics of oligonucleotides to these single-stranded regions and the rotational correlation times, it was suggested that the flexibility of the single-stranded region was closely correlated with the hybridization kinetics. Furthermore, results of the luminescence lifetime measurement and luminescence quenching experiments suggested that the highly flexible region was located on the surface of the 16S rRNA and that the less flexible region was located in the depths of 16S rRNA.

Body Fluids↗
PubMed2005

[Analysis of gene mutation in Chinese patients with Reis-Bücklers corneal dystrophy].

OBJECTIVE: To identify the mutation of the TGFBI gene in Chinese patients with Reis-Bücklers corneal dystrophy, and to study the relationship between the gene mutation and the clinical appearance. METHODS: Ten patients and 2 unaffected family members from 2 unrelated families with corneal dystrophy were studied. Molecular genetic analysis was performed on DNA extracted from peripheral leucocytes, and exons 4 and 12 of the TGFBI gene were amplified by polymerase chain reaction for direct sequencing. RESULTS: Both pedigrees showed an autosomal dominant inheritance. The clinical appearance of the cornea consisted of fine granular, subepithelial opacities which spread and become confluent with time, and resembled geographic type of Reis-Bücklers corneal dystrophy. Direct sequencing of all affected members revealed a G-to-T transition at codon 124 (CGC to CTC), producing R124L mutation of TGFBI gene. CONCLUSIONS: R124L mutation of the TGFBI gene is found in two Chinese families with Reis-Bücklers corneal dystrophy. The phenotype of Reis-Bücklers corneal dystrophy in both families belongs to the geographic type. Molecular genetic approach may be useful for the proper diagnosis of this type of corneal dystrophy.

Adolescent↗
PubMed2005

Suppression of beta-catenin mutation by dietary exposure of auraptene, a citrus antioxidant, in N,N-diethylnitrosamine-induced hepatocellular carcinomas in rats.

Alteration of beta-catenin expression in hepatocellular carcinomas (HCCs) may play an important role in tumor progression by stimulating cell proliferation. We have previously reported that auraptene (AUR), an antioxidant agent isolated from citrus fruit, effectively inhibits chemically induced hepatocarcinogenesis in rats. In this study, we investigated the molecular mechanism of the inhibitory effects of dietary supplementation with AUR on N,N-diethylnitrosamine (DEN)-initiated hepatocarcinogenesis. Male F344 rats initiated with DEN were fed the AUR-containing diet during either the initiation stage (initiation feeding for 7 weeks) or post-initiation phase (post-initiation feeding for 25 weeks) of hepatocarcinogenesis. Liver tumors >5 mm in diameter were used for the analysis of beta-catenin gene mutation and beta-catenin protein expression. After PCR amplification of exon 2 of the beta-catenin gene, the products were sequenced directly. Mutations in the beta-catenin gene were detected in 8 of 24 HCCs (33.3%) in the DEN alone group, 7 of 15 HCCs (46.7%) in the DEN + AUR (initiation feeding) group, and 0 of 8 HCCs (0%) in the DENright curved arrow AUR (post-initiation feeding) group. No mutations of beta-catenin gene were detected in liver cell adenomas of any group. These results demonstrate that AUR exposure in post-initiation period suppresses the occurrence of HCCs with beta-catenin mutation, presumably by negative selection for neoplastic cells harboring the mutation.

Animals↗
PubMed2004

Dietary rutin, but not its aglycone quercetin, ameliorates dextran sulfate sodium-induced experimental colitis in mice: attenuation of pro-inflammatory gene expression.

Oxidative stress has been shown to play a pivotal role in the onset of inflammatory bowel disease (IBD) and carcinogenesis. We evaluated the effects of two dietary anti-oxidants, rutin and its aglycone quercetin, on dextran sulfate sodium (DSS)-induced experimental colitis in mice. Female ICR mice were fed a diet containing 0.1% rutin or 0.1% quercetin for 2 weeks, and given 5% DSS in drinking water during the second week to induce colitis. We also examined the dose-dependency of rutin and quercetin (0.01% and 0.001% each) as well as their therapeutic efficacy, which was evaluated following DSS administration, on DSS-induced colitis. The protein level of interleukin (IL)-1 beta in both colonic mucosa and peritoneal macrophages was quantified by enzyme-linked immunosorbent assay. Further, mRNA expression levels of IL-1 beta, tumor necrosis factor-alpha, IL-6, granulocyte macrophage-colony stimulating factor, inducible nitric oxide synthase, and cyclooxygenase (COX)-1 and COX-2 in colonic mucosa were determined by reverse transcription-polymerase chain reaction. A diet containing 0.1% rutin, but not quercetin, attenuated DSS-induced body weight loss and shortening of the colorectum (P<0.01 and <0.05, respectively), and dramatically improved colitis histological scores. Further, DSS-induced increases in colonic mucosal IL-1 beta levels were blunted significantly in rutin-, but not quercetin-, fed mice (P<0.01), while dietary rutin attenuated the expressions of IL-1 beta and IL-6 mRNA in colonic mucosa (each, P<0.01). As for dose dependency, 0.01%, but not 0.001%, dietary rutin significantly reduced mucosal IL-1 beta levels (P<0.01). Notably, a 0.1% rutin diet given 3 days after DSS treatment significantly suppressed both colorectal shortening and IL-1 beta production (P<0.05 and <0.01, respectively). Dietary rutin ameliorates DSS-induced colitis, presumably by suppressing the induction of pro-inflammatory cytokines. Our results suggest that rutin may be useful for the prevention and treatment of IBD and colorectal carcinogenesis via attenuation of pro-inflammatory cytokine production.

Animals↗
PubMed2004

Truncation and mutagenesis analysis of the human X-arrestin gene promoter.

X-arrestin (arrestin-3) is an arrestin present specifically in the outer segments of red-, green-, and blue-cone photoreceptors. The X-arrestin gene is on Xcen-q22, and consists of 17 exons with a promoter containing a TATA box and elements important for photoreceptor expression, including three CRX and one PCE-1-like element. In order to delineate the promoter structure necessary for the pan-cone-specific expression of X-arrestin, the expression of the gene in retinoblastoma cell lines was investigated, and a structure-function analysis of the promoter was conducted in the appropriate cellular substrate. Expression of X-arrestin was detected at a low level in the Y79 retinoblastoma cell line but not in the WERI retinoblastoma cell line. Truncation and expression analysis of the X-arrestin promoter in Y79 showed maximal activity in the proximal 378-bp region containing the CRX and PCE-1-like elements upstream of the TATA and CAAT boxes and a negative regulator in the distal 1-2-kbp region. Mutagenesis of the three CRX and PCE-1-like elements and expression analysis demonstrated complete elimination of the promoter activity. Mutagenesis of the TATA box and PCE-1-like element individually resulted in similar decrease in promoter activity, but the decrease in the promoter activity was greater when the CRX elements were mutagenized with a 5' to 3' spatial gradient in the negative effect, suggesting a cooperative effect of the three CRX elements. The regulation of expression from this promoter may involve the binding of a multi-protein enhanceosome complex at the CRX triplet and the PCE-1-like element, resulting in the recruitment and activation of the RNA polymerase II complex at the downstream TATA box.

Arrestins↗