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Biomedical subjects

Yuji Inoue

Publications and source records attributed to Yuji Inoue.

At least 19 recordsLinked to original sources
PubMed2006

AFM and TEM observations of alpha-helix to beta-sheet conformational change occurring on carbon nanotubes.

Bacteriorhodopsin (BR), which is rich in alpha-helical structure, was spread onto water with single-wall carbon nanotubes (SCNTs). After a Langmuir trough was used to apply compressive surface pressure to maintain the alpha-helices monolayer of denatured BR, the composite films comprising alpha-helices and SCNTs were transferred horizontally onto substrates. Atomic force microscopy (AFM) and fluorescence microscopy observation suggested that alpha-helices in contact with SCNTs changed into beta-sheets. High-resolution transmission electron microscopy (HR-TEM) showed 0.54 nm periodicity characteristic of the turn of alpha-helical structure in the SCNTs-free alpha-helix monolayer region and showed the 0.70 nm periodicity of beta-sheet pleated structure in the region where SCNTs were covered with unfolded BR. Unique features of carbon nanotubes that trigger conformational changes of a protein were revealed.

Bacteriorhodopsins↗
PubMed2006

Effects of yellow intraocular lenses on light-induced upregulation of vascular endothelial growth factor.

PURPOSE: To investigate the protective effect of a blue-light filtering intraocular lens (yellow IOL) (YA60BB, Hoya) and an ultraviolet (UV)-absorbing IOL (VA60BB, Hoya) on light-induced phototoxicity to retinal pigment epithelial (RPE) cells laden with the lipofuscin fluorophore A2E and on the production of vascular endothelial growth factor (VEGF) after light exposure. SETTING: University of Tokyo, Tokyo, Japan. METHODS: The A2E-laden ARPE-19 cells were exposed to white light and a UV-absorbing IOL or a blue-light filtering IOL was placed over the light beam. After 48 hours of irradiation, the viability of the cells was determined with WST-1 (a sodium salt of 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay, and the secreted protein level of VEGF was determined by enzyme-linked immunosorbent assay. RESULTS: Without an IOL, the white-light exposure decreased cell viability to 28% of the nonirradiated control. Although the UV-absorbing IOL tended to reduce light-induced cell death, the decrease was not significant. However, the presence of the blue-light filtering IOL significantly attenuated light-induced cell damage, increasing cell viability to 42%. The secreted VEGF protein level increased 3.2-fold after the A2E-laden RPE cells were exposed to white light. In the presence of the UV-absorbing IOL, the VEGF protein level decreased, but not significantly. The presence of the blue-light filtering IOL significantly attenuated the upregulated VEGF expression compared to upregulation without an IOL. CONCLUSION: This study supports the theory that a blue-light filtering IOL may be more protective against A2E-induced photochemical damage and inhibit more light-induced VEGF production than a conventional UV-absorbing IOL.

Cell Line↗
PubMed2006

In vitro assay for fragmentation of amyloid fibers of yeast prion protein.

In prion propagation, fragmentation of amyloid fibers, as well as conformational conversion of prion protein, is critical: the latter increases the net amount of abnormal prion proteins and the former multiplies number of seeds. We present here a method for in vitro measurement of fragmentation of amyloid fibers of yeast Sup35 prion protein. In this method, amyloid fibers are tethered to the surface of magnetic beads. Fragmentation of the fibers results in release of fiber fragments into the medium, which are then quantified by immunoblotting. This method is versatile for other amyloid fibers.

Algorithms↗
PubMed2006

[Contributions of university hospitals to local health integration networks].

The use of ICT (Information and Communication Technology) has supported the widespread sharing of healthcare data. Collaboration among medical or health facilities in the community using a telemedicine infrastructure has been promoted by the Strategy of IT-related Structural Reform (the Acceleration and Advancement of e-Japan Priority Policy Program) of the Japanese government. Many university hospitals have been playing leading roles of facilitating collaboration with community healthcare. Through advances in broadband networking and security technology, we have made great progress from the trial stage to the practical stage in the electronic sharing of clinical data. Although many efforts are being made spread the cooperation areas or widen the scope of information sharing for utilization in disease prevention or clinical trials of newly developed drugs as well as medical information, university hospitals have to continue working on these areas to support patients and staff as core hospitals in the community.

Computer Communication Networks↗
PubMed2005

Light-induced gene transfer from packaged DNA enveloped in a dendrimeric photosensitizer.

The control of gene transfection in the body is a core issue in gene therapy. Photochemical internalization is a technology that allows light-induced delivery of DNA, drugs or other biological factors directly inside cells. Usually it requires that a photosensitizer be added to the drug-delivery system to photochemically destabilize the endosomal membrane. Here we present a system for in vivo DNA delivery in which these two components are assembled into one structure. This is a ternary complex composed of a core containing DNA packaged with cationic peptides and enveloped in the anionic dendrimer phthalocyanine, which provides the photosensitizing action. The ternary complex showed more than 100-fold photochemical enhancement of transgene expression in vitro with reduced photocytotoxicity. In an animal experiment, subconjuctival injection of the ternary complex followed by laser irradiation resulted in transgene expression only in the laser-irradiated site. This work demonstrates a new biomedical application for dendrimers, and the first success in the photochemical-internalization-mediated gene delivery in vivo.

Animals↗
PubMed2005

Properties of growth and molecular profiles of rat progenitor cells from ciliary epithelium.

Recent studies have demonstrated that multipotent retinal stem or progenitor cells can be isolated from the ciliary epithelium (CE) of the eye using a neurosphere culture. In this study, we investigated the properties of growth and differentiation, and molecular profiles of rat adult ciliary epithelium (CE)-derived retinal progenitors and forebrain (FB) derived neurospheres. Under clonogenic culture conditions, we found that the CE-derived neurospheres contained fewer undifferentiated cells compared with the FB-derived neurospheres, and that CE-derived neurospheres initially expressed the set of Notch pathway molecules genes including Notch 1 and Delta 1, HES-1 and HES-5, but partially lose their expression after passaging. Furthermore, we found that the CE-derived neurospheres did not express several markers for in vivo embryonic retinal progenitors. Additionally, when the eye was divided into four subregions along its dorsoventral and nasotemporal axes and progenitor cells were obtained from the subregions, the progenitor cells did not express the subregion specific transcription factors, suggesting that subregional specificity is not maintained in vitro. Together, our results demonstrate that CE-derived progenitor cells may have intrinsic limitations in the production of cell types.

Animals↗
PubMed2005

Cortical intrinsic circuits can support activity propagation through an isofrequency strip of the guinea pig primary auditory cortex.

A pure tone evokes propagating activities in a strip of the primary auditory cortex (AI), an isofrequency strip (IS). A fundamental issue concerns the roles that thalamocortical input and intracortical connectivity play in generating the activities. Here we addressed this issue in guinea pigs using in vivo and in vitro real-time optical imaging techniques. As reported previously, tone-evoked activity propagated dorsoventrally along a strip (an IS) in AI. We found that an electrical pulse applied focally within the strip, triggered activity propagation with a spatiotemporal pattern highly similar to tone-evoked activation. The propagation velocity of electrically evoked activity was significantly slower than that of tone-evoked activity, but was comparable to the velocity of lateral activity propagation in cortical slices, suggesting that the electrically evoked activity propagation in vivo is mediated by intracortical circuits. To test this notion, we lesioned the auditory thalamus chemically; in such animals, electrically evoked activity in AI was not affected, although tone-evoked activity was abolished. Further, in slices of the AI, the extent of electrically evoked activity propagation in layer II/III was significantly larger in coronal slices than in horizontal slices. Together, our results suggest that intracortical connectivity in AI enables a focally evoked activity to propagate throughout an IS.

Acoustic Stimulation↗
PubMed2005

Identification of an AraC-like regulator gene required for induction of the 78-kDa ferrioxamine B receptor in Vibrio vulnificus.

We previously reported that the 78-kDa outer membrane receptor for ferrioxamine B is induced in iron-starved Vibrio vulnificus cells when desferrioxamine B was supplied exogenously. Based on its N-terminal amino acid sequence, a candidate gene for the ferrichrome B receptor was detected in the V. vulnificus CMCP6 genomic database. Here, two contiguous genes, named desR and desA, encoding a member of the AraC family of transcriptional activators and the ferrioxamine B receptor, respectively, were cloned from V. vulnificus M2799 and characterized. Primer extension analysis mapped the iron-regulated transcription initiation sites for desR and desA, and demonstrated involvement of desferrioxamine B in the induction of desA transcription. Insertion mutation of desR resulted in no production of DesA under iron-limiting conditions even in the presence of desferrioxamine B. The DesA production under the same conditions was restored to wild-type levels when the desR mutant was complemented with desR in trans. These results suggest that the desR gene is required for desferrioxamine B-inducible production of DesA in iron-starved cells.

Amino Acid Sequence↗
PubMed2005

Clonogenic analysis of ciliary epithelial derived retinal progenitor cells in rabbits.

In lower vertebrates, multipotential retinal stem cells reside in a far peripheral retinal zone known as the ciliary marginal zone while more fate-restricted progenitor cells are located immediately adjacent to this retinal margin. To determine whether mammalian ciliary epithelium contains heterogenous stem cell and progenitor cell populations similar to lower vertebrates, we investigated the heterogeneity of the retinal progenitor (or stem) cells isolated from adult rabbit eyes, which are large and suited for precise identification of the region. Under clonogenic culture conditions for a neurosphere assay, spheres were generated from approximately 0.1% of the ciliary epithelial cells and expressed retinal selective markers when plated under conditions producing differentiation. Subsequently, we found that 13.7% of the primary spheres gave rise to a secondary sphere and that 77% of the primary sphere colonies were unipotential progenitors that generated either neurons or glial cells exclusively, while 23% were at least bipotential that could give rise to both cell types using double-labelled immunocytochemistry. Taken together, our results demonstrate that the ciliary epithelium of adult rabbits contains both (at least) bipotential retinal progenitor (or stem) cells and unipotential fate-restricted progenitor cells, similar to lower vertebrates.

Animals↗
PubMed2005

A novel aerobactin utilization cluster in Vibrio vulnificus with a gene involved in the transcription regulation of the iutA homologue.

We demonstrated that Vibrio vulnificus M2799 utilizes aerobactin for growth as an exogenous siderophore under iron-limiting conditions, concomitant with enhanced production of the 76-kDa iron-repressible outer membrane protein. Subsequently, by applying the Fur titration assay method to the M2799 genomic libraries followed by further cloning of the regions surrounding the candidate genes, we identified the 8.4-kb aerobactin utilization gene cluster which consists of five genes arranged in three distinct transcriptional units. It was confirmed by disruption of the corresponding genes that the first unit forming a three-gene operon (vatCDB) and the third unit of a single gene (iutA) encode an ATP-binding cassette transport component and the 76-kDa ferric aerobactin receptor, respectively. The second unit of another single gene (iutR), encodes a homologue of the GntR family of transcriptional repressors. Although transcription of the first and third units was iron-regulated, the iutR gene was transcribed regardless of iron status in the growth medium. Construction of an iutR disruptant coupled with genetic complementation experiments suggested that the gene encodes a transcriptional repressor for iutA. This is the first example of a regulator gene involved in aerobactin-enhanced production of IutA.

ATP-Binding Cassette Transporters↗
PubMed2005

Infection route-independent accumulation of splenic abnormal prion protein.

The accumulation kinetics of the abnormal form of prion protein (PrP(Sc)) in spleens and brains of scrapie (Obihiro-1)-infected mice at various times after intracerebral (i.c.), intraperitoneal (i.p.), or oral inoculation were studied. PrP(Sc) was first detected by Western blotting with anti-prion protein antibodies on days 70 and 116 after i.c. (3 microg) in spleens and brains, respectively. Although the amount of cerebral PrP(Sc) gradually increased to the maximum level on day 152 after i.c. inoculation, splenic PrP(Sc) established the maximum level on day 116 after i.c. inoculation then registered slight decreases up to day 152 with further incubation. The detectable levels of cerebral PrP(Sc) by Western blotting were established on day 231 or 259, whereas those of splenic PrP(Sc) were detected on day 94 or 93, after i.p. and oral infection, respectively. The splenic PrP(Sc) decreased slightly thereafter. These results indicate that splenic PrP(Sc) increased before cerebral PrP(Sc) established a detectable level in a manner independent of the inoculation route.

Animals↗
PubMed2004

Hsp104 binds to yeast Sup35 prion fiber but needs other factor(s) to sever it.

The interaction of Hsp104 with yeast prion fibers made of Sup35NM, a prion-inducing domain of Sup35, was tested. When fluorescently labeled Hsp104 was added to the preformed fibers, individual fibers were fluorescently decorated uniformly along the fiber length. However, the density of fluorescence differed from one fiber to another, indicating the presence of subspecies of Sup35NM fibers. The time course of fiber formation from monomer Sup35NM was delayed by Hsp104. Hsp104-mediated fragmentation of fibers was tested using bead-tethered fibers. In contrast with the recent report (Shorter, J., and Lindquist, S. (2004) Science 304, 1793-1797), Hsp104 alone was unable to sever the fibers. Yeast cell lysate or the Hsp104-deficient cell lysate plus Hsp104 caused ATP-dependent, guanidine hydrochloride-sensitive fragmentation of the fibers. Thus, in our experimental setup, Hsp104 plus other factor(s) in the yeast cytosol are required for severing yeast prion fiber. The reason of discrepancy from the above report is unknown but is possibly caused by different conformational subspecies of prion fibers.

Adenosine Triphosphate↗
PubMed2004

Cost-effectiveness of ribavirin plus interferon alpha-2b for either interferon relapsers or non-responders in chronic hepatitis C: a Japanese trial.

The aim of this study was to examine the cost-effectiveness of combination therapy with ribavirin plus interferon alpha-2b compared with interferon monotherapy for hepatitis C-infected Japanese patients who had either temporarily responded or not responded to initial interferon treatment. Data from a recent randomized clinical trial among relapsed or non-responding patients comparing combination therapy to interferon alone were applied to a computer cohort simulation Markov process model to project lifelong clinical and economic outcomes. Combination therapy for 24 weeks should increase life expectancy by 1.6 quality-adjusted life years and should reduce discounted (3% per year) lifetime costs by yen 121,000 when compared to retreatment with interferon alone. For the subgroup of patients with genotype 1b and high viral load, combination therapy should be cost-effective ( yen 187,000 per QALY gained with a 3% annual discount rate) by well-accepted international standards. These results were robust with combination therapy remaining cost-effective or cost saving in sensitivity analysis involving reasonable variation in all parameters. For patients similar to those enrolled in the interferon alpha-2b and ribavirin trials in Japan, combination therapy should be considered cost-effective with the higher drug treatment costs nearly completely offset by future savings through reductions in future liver complications from hepatitis C.

Journal Article↗
PubMed2004

Effects of indocyanine green on retinal ganglion cells.

PURPOSE: Recently, indocyanine green (ICG) has been used to visualize the internal limiting membrane during vitrectomy. After intraocular administration, ICG accumulates at the optic disc. In this study, the effects of ICG on retinal ganglion cells (RGCs) were examined. METHODS: In vitro, rat RGCs were purified by a two-step immunopanning procedure, briefly exposed to ICG (2.5 x 10(3) mg/L), and irradiated with an endoilluminator for 15 minutes or incubated in the presence of ICG (concentration: 2-250 mg/L) without irradiation. The number of viable RGCs was counted after 3 days in culture. In vivo, after rats received an intravitreal injection of 3 micro L ICG (0.25 and 2.5 mg/L), the distribution of ICG was observed with a fundus camera, and the number of viable RGCs was examined by a DiI (1,1'-dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-retrolabeling technique. RESULTS: In vitro, a brief exposure to ICG and light did not affect RGC survival. However, ICG reduced the number of viable RGCs in a dose-dependent manner when the cells were exposed for 3 days. In vivo, the dye was initially distributed on the retinal surface and around the optic disc. At day 7, the fluorescence became invisible in the 0.25-mg/L group, whereas the staining of the optic disc contour was evident in the 2.5-mg/L group. The number of viable RGCs decreased significantly in the 2.5-mg/L group 14 days after the injection. CONCLUSIONS: ICG showed an inherent toxicity to RGCs in a dose-dependent manner. Lower concentration and shorter staining time of ICG should be used for dye-assisted vitrectomy.

Animals↗
PubMed2004

Evaluation of factors to decrease bioavailability of cyclosporin A in rats with gentamicin-induced acute renal failure.

Focusing on the disposition of cyclosporin A (CsA) in the liver and intestine, effects of gentamicin-induced acute renal failure (ARF) on the decreased oral bioavailability of CsA were evaluated in rats. The area under the CsA concentration-time curve (AUC) in ARF rats after oral administration (5 mg/kg) significantly decreased by 43% as compared to the control, while the apparent oral clearance significantly increased by 76% of the control. The portal AUC of CsA in ARF rats with bile flow decreased by 67% as compared to the control rats. Without bile flow, the portal AUC of CsA in control rats decreased by 50% as compared to those with bile flow, whereas ARF rats without bile flow showed no notable change as compared to those with bile flow. The AUC of CsA mono-oxidative metabolite via CYP3A (M-OH) in ARF rats after oral or intravenous administration increased significantly by 84% or 241%, respectively, while there was no difference in the portal M-OH between control and ARF rats, suggesting that the elimination of M-OH was prolonged because of nephrotoxicity. Although the exsorption clearance of CsA from the blood circulation to the intestine after intravenous administration to ARF rats decreased significantly as compared to the control; and basolateral-to-apical transport of CsA through Caco-2 monolayers was significantly retarded in the presence of uremic toxins, there was no significant change in the total body clearance of CsA between ARF and control rats. Moreover, there were no effects of uremic toxins on the protein binding of CsA in plasma. These observations suggest that hepatic or intestinal CYP3A and P-glycoproteine (P-gp) are not likely to be concerned with lowering the oral bioavailability of CsA, and that bile function under the ARF condition induced by gentamicin is responsible for a marked decrease in the fraction absorbed of CsA in the small intestine.

Acute Kidney Injury↗