Biomedical subjects

Yusuke Takahashi

Publications and source records attributed to Yusuke Takahashi.

At least 19 recordsLinked to original sources

PubMed2026

Evaluation of methacrylic resin-modified calcium silicate cements for pulpal healing using an experimental pulpitis model.

Recently, vital pulp treatment (VPT), including direct pulp capping, which preserves pulp vitality and extends the functional lifespan of teeth, has garnered significant attention. The purpose of this study was to evaluate the performance of calcium silicate cement, the gold standard for VPT, alongside hydraulic calcium silicate cement (Pro-MTA), a material with extensive evidence of effectiveness, Bis-GMA resin-modified calcium silicate cement (TH), and a newly developed material, methacrylate resin-modified calcium silicate cement (RM-MTA), in a model of pulpitis induced by caries progression. Additionally, the calcium ion (Ca2+) release capacity of these materials and their comprehensive effects on pulpal wound healing were assessed using RNA sequencing (RNA-seq). In both sound pulp models and caries-induced pulpitis models, RM-MTA and Pro-MTA exhibited similar performances. Unlike TH, they induced significant tertiary dentin formation within the dental pulp beneath the material, without any residual inflammatory cells. Inflammation was specifically assessed with a focus on M1/M2 macrophages. While the timing of Ca2+ ion release differed among the materials, the total amount released was comparable, although the release of calcium ions (Ca2+) from TH was significantly lower compared to that observed for the above materials. Moreover, comprehensive genetic analysis revealed that the expression of cell proliferation-related genes was selectively reduced in TH, suggesting that differences in resin composition may account for these variations in behavior. These findings suggest that RM-MTA induces tertiary dentin and demonstrates biocompatibility comparable to that of Pro-MTA. This makes it suitable for the treatment of both sound and mildly inflamed pulp tissues. Additionally, its resin properties are expected to enhance both mechanical performance and clinical handling.

Journal Article↗

PubMed2006

Impacts of two point mutations of RPE65 from Leber's congenital amaurosis on the stability, subcellular localization and isomerohydrolase activity of RPE65.

RPE65, a membrane-associated protein in the retinal pigment epithelium, is the isomerohydrolase essential for regenerating 11-cis retinal, the chromophore for visual pigments. RPE65 mutations are associated with inherited retinal dystrophies. Here we report that single point mutations of RPE65, Y144D and P363T, identified in patients with Leber's congenital amaurosis (LCA), significantly decreased the stability of RPE65. Moreover, these mutations altered subcellular localization of RPE65 and abolished its isomerohydrolase activity. These observations suggest that the decreased protein stability and altered subcellular localization of RPE65 may represent a mechanism for these mutations to lead to vision loss in LCA patients.

Amino Acid Substitution↗

PubMed2006

Two point mutations of RPE65 from patients with retinal dystrophies decrease the stability of RPE65 protein and abolish its isomerohydrolase activity.

RPE65 is the isomerohydrolase in the retinoid visual cycle essential for recycling of 11-cis retinal, the chromophore for visual pigments in both rod and cone photoreceptors. Mutations in the RPE65 gene are associated with inherited retinal dystrophies with unknown mechanisms. Here we show that two point mutations of RPE65, R91W and Y368H, identified in patients with retinal dystrophies both abolished the isomerohydrolase activity of RPE65 after a subretinal injection into the Rpe65-/- mice and in the in vitro isomerohydrolase activity assay, independent of their protein levels. Further, the R91W and Y368H mutants showed significantly decreased protein levels but unchanged mRNA levels when compared with the wild-type RPE65 (wtRPE65). Protein stability analysis showed that wtRPE65 is a fairly stable protein, with an apparent half-life longer than 10 h, when expressed in 293A cells. Under the same conditions, mutants R91W and Y368H both showed substantially decreased protein stabilities, with half-lives less than 2 and 6 h, respectively. Subcellular fractionation and Western blot analysis demonstrated that wtRPE65 predominantly exists in the membrane fraction, while both of the mutants are primarily distributed in the cytosolic fraction, suggesting that these mutations disrupt the membrane association of RPE65. However, palmitoylation assay showed that wtRPE65 and both of the mutants were palmitoylated. These results suggest that these mutations may result in critical structural alterations of RPE65 protein, disrupt its membrane association, and consequently impair its isomerohydrolase activity, leading to retinal degeneration.

Animals↗

PubMed2006

An in vivo evaluation of bonding ability of comprehensive antibacterial adhesive system incorporating MDPB.

OBJECTIVES: This study examined the in vivo bonding ability to sound dentin of antibacterial adhesive systems incorporating an antibacterial monomer MDPB based on morphological evaluation of the resin-dentin interface. METHODS: Class V cavities were prepared on the buccal surfaces of the teeth of a beagle dog and a composite filling performed using (1) commercial self-etching system Liner Bond 2 (LB primer+LB bond), (2) experimental primer containing 5% MDPB and LB bond, (3) LB primer and experimental bonding-resin containing 2.5% MDPB, or (4) combination of experimental primer and bonding-resin. After 7 days, the tooth crown was cut and fixed in half-Karnovsky's solution, and the sectioned surface observed under scanning electron microscopy (SEM) after treatment with phosphoric acid and NaOCl. The ultrastructure of the bonding interface was also examined by transmission electron microscopy (TEM). Microtensile bond strengths (microTBS) of each group were measured using extracted teeth. RESULTS: SEM demonstrated that all groups produced a 1-2microm thick hybrid layer with funnel shaped resin tags, although the length of tags was shorter for the group in which MDPB-containing bonding-resin was used. TEM examination supported good adhesion of the comprehensive adhesive system employing MDPB-containing primer/bonding-resin, showing integrity between resin and dentin. There were no significant differences in microTBS among the four groups tested (p>0.05, ANOVA). SIGNIFICANCE: This study confirmed that the experimental antibacterial adhesive systems employing MDPB-containing primer or/and bonding-resin could produce an effective bond under in vivo conditions.

Animals↗

PubMed2006

Fimbria-dependent activation of pro-inflammatory molecules in Porphyromonas gingivalis infected human aortic endothelial cells.

Epidemiological studies support that chronic periodontal infections are associated with an increased risk of cardiovascular disease. Previously, we reported that the periodontal pathogen Porphyromonas gingivalis accelerated atherosclerotic plaque formation in hyperlipidemic apoE-/- mice, while an isogenic fimbria-deficient (FimA-) mutant did not. In this study, we utilized 41 kDa (major) and 67 kDa (minor) fimbria mutants to demonstrate that major fimbria are required for efficient P. gingivalis invasion of human aortic endothelial cells (HAEC). Enzyme-linked immunosorbent assay (ELISA) revealed that only invasive P. gingivalis strains induced HAEC production of pro-inflammatory molecules interleukin (IL)-1beta, IL-8, monocyte chemoattractant protein (MCP)-1, intracellular adhesion molecule (ICAM)-1, vascular cellular adhesion molecule (VCAM)-1 and E-selectin. The purified native forms of major and minor fimbria induced chemokine and adhesion molecule expression similar to invasive P. gingivalis, but failed to elicit IL-1beta production. In addition, the major and minor fimbria-mediated production of MCP-1 and IL-8 was inhibited in a dose-dependent manner by P. gingivalis lipopolysaccharide (LPS). Both P. gingivalis LPS and heat-killed organisms failed to stimulate HAEC. Treatment of endothelial cells with cytochalasin D abolished the observed pro-inflammatory MCP-1 and IL-8 response to invasive P. gingivalis and both purified fimbria, but did not affect P. gingivalis induction of IL-1beta. These results suggest that major and minor fimbria elicit chemokine production in HAEC through actin cytoskeletal rearrangements; however, induction of IL-1beta appears to occur via a separate mechanism. Collectively, these data support that invasive P. gingivalis and fimbria stimulate endothelial cell activation, a necessary initial event in the development of atherogenesis.

Aorta↗

PubMed2006

Pathogen-accelerated atherosclerosis occurs early after exposure and can be prevented via immunization.

Here we report on early inflammatory events associated with Porphyromonas gingivalis-accelerated atherosclerosis in apolipoprotein E knockout (ApoE-/-) mice. Animals challenged with P. gingivalis presented with increased macrophage infiltration, innate immune marker expression, and atheroma without elevated systemic inflammatory mediators. This early local inflammatory response was prevented in mice immunized with P. gingivalis. We conclude that localized up-regulation of innate immune markers early after infection, rather than systemic inflammation, contributes to pathogen-accelerated atherosclerosis.

Animals↗

PubMed2006

RPE65 gene delivery restores isomerohydrolase activity and prevents early cone loss in Rpe65-/- mice.

PURPOSE: Recent in vitro evidence has shown that RPE65 is the isomerohydrolase that converts all-trans retinyl ester to 11-cis retinal, the chromophore for visual pigments in vertebrates. Homozygous RPE65 knockout (Rpe65-/-) mice lack 11-cis retinoids and have early cone degeneration. The purpose of this study is to determine whether RPE65 gene delivery restores the isomerohydrolase activity and normal profile of endogenous retinoids in Rpe65-/- mice. METHODS: Adenovirus-expressing RPE65 (Ad-RPE65) was injected into the subretinal space of Rpe65-/- mice. The expression of RPE65 was determined by immunohistochemistry and Western blot analysis. The isomerohydrolase activity was measured in vitro in eyecup homogenates. Endogenous retinoid profile in the eyecups was analyzed by high-performance liquid chromatography (HPLC). Photoreceptor-specific gene expression was determined with real-time RT-PCR. Cone degeneration was determined by cone-specific staining and counting cones in flatmounted retina. RESULTS: High levels of RPE65 expression from the Ad-RPE65 injection generated robust isomerohydrolase activity in the eyecup of Rpe65-/- mice, at levels comparable to those in wild-type (wt) mice. Consequently, the RPE65 gene delivery resulted in substantial amounts of 11-cis retinal in Rpe65-/- mice. The RPE65 gene delivery prevented the downregulation of cone-specific genes, including both cone opsins and cone tranducin alpha subunit in Rpe65-/- mice. Moreover, the Ad-RPE65 injection also prevented massive cone degeneration at early ages of Rpe65-/- mice. CONCLUSIONS: RPE65 gene delivery generates isomerohydrolase activity and restores retinoid profile in Rpe65-/- mice. Regeneration of 11-cis retinal is essential for survival of cone photoreceptors.

Adenoviridae↗

PubMed2006

The roles of three palmitoylation sites of RPE65 in its membrane association and isomerohydrolase activity.

PURPOSE: RPE65, a membrane-associated protein predominantly expressed in the retinal pigment epithelium, is the isomerohydrolase in the retinoid visual cycle. Three cysteine (Cys) residues, Cys231, Cys329, and Cys330, in RPE65 have been shown to be palmitoylated and have been suggested to be responsible for its membrane association. The purpose of this study was to define the role of these Cys residues in palmitoylation, membrane association, and isomerohydrolase activity of RPE65. METHODS: The three Cys residues in RPE65 were replaced by Alanine (Ala) with site-directed mutagenesis. The mutant protein levels and subcellular localizations were determined by Western blot analysis and subcellular fractionation, respectively. Their enzymatic activities were evaluated with the in vitro isomerohydrolase activity assay. Palmitoylation of the mutants was examined by labeling of the protein with [3H]-labeled palmitic acid. RESULTS: Mutation of any single residue of these three Cys significantly reduced protein levels of RPE65. Similar to wild-type RPE65, however, all three single Cys mutants were predominantly present in the membrane fraction. Mutations of any one or two of these Cys substantially weakened the isomerohydrolase activity of RPE65, whereas mutations of all three Cys (triple mutant) completely abolished the enzymatic activity. However, this triple Cys mutant was still palmitoylated and associated with the membrane, although at a reduced level. CONCLUSIONS: There are additional yet to be identified palmitoylation sites in RPE65. The structural distortions induced by the Cys mutations may be responsible for the mislocalization and decreased isomerohydrolase activities of RPE65.

Alanine↗

PubMed2006

Roles of the host oxidative immune response and bacterial antioxidant rubrerythrin during Porphyromonas gingivalis infection.

The efficient clearance of microbes by neutrophils requires the concerted action of reactive oxygen species and microbicidal components within leukocyte secretory granules. Rubrerythrin (Rbr) is a nonheme iron protein that protects many air-sensitive bacteria against oxidative stress. Using oxidative burst-knockout (NADPH oxidase-null) mice and an rbr gene knockout bacterial strain, we investigated the interplay between the phagocytic oxidative burst of the host and the oxidative stress response of the anaerobic periodontal pathogen Porphyromonas gingivalis. Rbr ensured the proliferation of P. gingivalis in mice that possessed a fully functional oxidative burst response, but not in NADPH oxidase-null mice. Furthermore, the in vivo protection afforded by Rbr was not associated with the oxidative burst responses of isolated neutrophils in vitro. Although the phagocyte-derived oxidative burst response was largely ineffective against P. gingivalis infection, the corresponding oxidative response to the Rbr-positive microbe contributed to host-induced pathology via potent mobilization and systemic activation of neutrophils. It appeared that Rbr also provided protection against reactive nitrogen species, thereby ensuring the survival of P. gingivalis in the infected host. The presence of the rbr gene in P. gingivalis also led to greater oral bone loss upon infection. Collectively, these results indicate that the host oxidative burst paradoxically enhances the survival of P. gingivalis by exacerbating local and systemic inflammation, thereby contributing to the morbidity and mortality associated with infection.

Animals↗

PubMed2005

Tertiary structure and spectral tuning of UV and violet pigments in vertebrates.

Many vertebrate species use ultraviolet (UV) vision for such behaviors as mating, foraging, and communication. UV vision is mediated by UV-sensitive visual pigments, which have the wavelengths of maximal absorption (lambda max) at approximately 360 nm, whereas violet (or blue) vision is mediated by orthologous pigments with lambda max values of 390-440 nm. It is widely believed that amino acids in transmembrane (TM) I-III are solely responsible for the spectral tuning of these SWS1 pigments. Recent molecular analyses of SWS1 pigments, however, show that amino acids in TM IV-VII are also involved in the spectral tuning of these pigments through synergistic interactions with those in TM I-III. Comparisons of the tertiary structures of UV and violet pigments reveal that the distance between the counterion E113 in TM III and amino acid sites 87-93 in TM II is narrower for UV pigments than for violet pigments, which may restrict the access of water molecules to the Schiff base pocket and deprotonate the Schiff base nitrogen. Both mutagenesis analyses of E113Q and quantum chemical calculations strongly suggest that unprotonated Schiff base-linked chromophore is responsible for detecting UV light.

Amino Acid Sequence↗

PubMed2005

RPE65 is an iron(II)-dependent isomerohydrolase in the retinoid visual cycle.

The isomerization of all-trans-retinyl ester to 11-cis-retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle and is essential for normal vision. Recently, we have established that protein RPE65 is the isomerohydrolase catalyzing this reaction. The present study investigated if metal ions are required for the isomerohydrolase activity of RPE65. The conversion of all-trans-[3H]retinol to 11-cis-[3H]retinol was used as the measure for isomerohydrolase activity. Metal chelators 2,2'-bipyridine and 1,10-phenanthroline both showed dose-dependent inhibitions of the isomerohydrolase activity in bovine RPE microsomes, with IC50 values of 0.5 and 0.2 mm, respectively. In the same reaction systems, however, lecithin-retinol acyltransferase (LRAT) activity was not affected by these metal chelators. The isomerohydrolase activity inhibited by the metal chelators was restored by FeSO4 but not by CuSO4, ZnCl2, or MgCl2. Moreover, addition of Fe(III) citrate or FeCl3 did not restore the activity, indicating that Fe2+ is the metal ion essential for the isomerohydrolase activity. To confirm this result in recombinant RPE65, we expressed RPE65 in a 293A cell line stably expressing LRAT. In vitro activity assay showed that both metal chelators inhibited isomerohydrolase activity of recombinant RPE65. The addition of FeSO4 restored the enzymatic activity of the recombinant RPE65. Further, two specific iron-staining methods showed that purified RPE65 contains endogenous iron. Inductively coupled plasma mass spectrometry measurements showed that bovine RPE65 binds iron ion with a stoichiometry of 0.8 +/- 0.1. These results indicate that RPE65 is an iron-dependent isomerohydrolase in the visual cycle.

Animals↗

PubMed2005

Antibacterial effects and physical properties of glass-ionomer cements containing chlorhexidine for the ART approach.

OBJECTIVES: Since atraumatic restorative treatment (ART) involves removal of carious lesions with manual instruments, improvement of filling materials to guarantee greater success should be considered. This study aimed to evaluate antibacterial, physical, and bonding properties of glass-ionomer cements (GIC) containing chlorhexidine (CHX), and to determine optimal concentrations for incorporation of agents to obtain antibacterial GICs for use with the ART approach. METHODS: CHX diacetate combined with CHX dihydrochloride was added to control GIC powder to obtain concentration ratios of 1/0, 2/0, 3/0, 1/1, or 2/2% w/w. Antibacterial activity of each cement against Streptococcus mutans, Lactobacillus casei or Actinomyces naeslundii was examined using agar-diffusion methods, and release of CHX was analyzed by HPLC. Compressive strength, bond strength to dentin, and setting time were measured, and compared with those of control samples. RESULTS: All experimental GICs exhibited inhibition of three bacteria, but sizes of inhibition zones and concentrations of CHX released were not dependent upon CHX content. Incorporation of CHX diacetate at 2% or greater, significantly decreased compressive strength, and bond strength to dentin was adversely affected by addition of CHX diacetate at 2% or more (p<0.05, ANOVA, Fisher's PLSD test), although setting time was extended a little by addition of any concentrations of CHX. SIGNIFICANCE: The present results demonstrate that experimental GICs containing CHX are effective in inhibiting bacteria associated with caries, and incorporation of 1% CHX diacetate is optimal to give appropriate physical and bonding properties.

Actinomyces↗

PubMed2005

Identification of conserved histidines and glutamic acid as key residues for isomerohydrolase activity of RPE65, an enzyme of the visual cycle in the retinal pigment epithelium.

We have recently reported that RPE65 from the retinal pigment epithelium is the isomerohydrolase, a critical enzyme in the visual cycle for regeneration of 11-cis retinal, the chromophore for visual pigments. Here, we demonstrated that mutation of any one of the absolutely conserved four histidine and one glutamic acid residues to alanine in RPE65 abolished its isomerohydrolase activity. Substitution of the conserved glutamic acid with glutamine also resulted in loss of the activity. Moreover, these mutations significantly reduced protein stability of RPE65. These results indicate that these conserved residues are essential for the isomerohydrolase activity of RPE65 and its stability.

Carrier Proteins↗

PubMed2005

In vitro antibacterial effects of the dentin primer of Clearfil Protect Bond.

OBJECTIVES: This study aimed to investigate the antibacterial effects of the dentin primer of a commercially available self-etching adhesive system, Clearfil Protect Bond, which contains antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB). METHODS: Inhibitory effects against Streptococcus mutans, Lactobacillus casei, or Actinomyces naeslundii were examined by an agar-disc diffusion method using the Clearfil Protect Bond primer containing 5% MDPB and an acidic adhesion-promoting monomer MDP, the primer only with MDP, and the primer with 1% cetylpyridinium chloride. The minimum inhibitory/bactericidal concentrations (MIC/MBC) of each primer for the three bacterial species were determined by serial microdilution assays. For testing the bactericidal effects seen in dentin, the primer was applied to demineralized dentin blocks in which S. mutans had been impregnated, and numbers of viable bacteria were counted. RESULTS: For all three bacteria, the sizes of the inhibition zones produced by Clearfil Protect Bond primer were significantly greater than for the other primers (p<0.05, ANOVA and Scheffe's F-test). The MIC/MBC values of Clearfil Protect Bond primer were less than those of the primer without MDPB, and comparable to those of the primer containing cetylpyridinium chloride. No bacterial recovery was obtained after application of Clearfil Protect Bond primer to the bacteria-impregnated dentin, although the primer without MDPB showed some bactericidal effect. SIGNIFICANCE: Clearfil Protect Bond primer has strong antibacterial activity based upon MDPB against S. mutans, L. casei and A. naeslundii, and the capability to disinfect cavities containing residual bacteria.

Acid Etching, Dental↗

PubMed2005

RPE65 is the isomerohydrolase in the retinoid visual cycle.

RPE65 is an abundant protein in the retinal pigment epithelium. Mutations in RPE65 are associated with inherited retinal dystrophies. Although it is known that RPE65 is critical for regeneration of 11-cis retinol in the visual cycle, the function of RPE65 is elusive. Here we show that recombinant RPE65, when expressed in QBI-293A and COS-1 cells, has robust enzymatic activity of the previous unidentified isomerohydrolase, an enzyme converting all-trans retinyl ester to 11-cis retinol in the visual cycle. The initial rate for the reaction is 2.9 pmol/min per mg of RPE65 expressed in 293A cells. The isomerohydrolase activity of RPE65 requires coexpression of lecithin retinol acyltransferase in the same cell to provide its substrate. This enzymatic activity is linearly dependent on the expression levels of RPE65. This study demonstrates that RPE65 is the long-sought isomerohydrolase and fills a major gap in our understanding of the visual cycle. Identification of the function of RPE65 will contribute to the understanding of the pathogenesis for retinal dystrophies associated with RPE65 mutations.

Acyltransferases↗

PubMed2005

Genetic basis of spectral tuning in the violet-sensitive visual pigment of African clawed frog, Xenopus laevis.

Ultraviolet (UV) and violet vision in vertebrates is mediated by UV and violet visual pigments that absorb light maximally (lambdamax) at approximately 360 and 390-440 nm, respectively. So far, a total of 11 amino acid sites only in transmembrane (TM) helices I-III are known to be involved in the functional differentiation of these short wavelength-sensitive type 1 (SWS1) pigments. Here, we have constructed chimeric pigments between the violet pigment of African clawed frog (Xenopus laevis) and its ancestral UV pigment. The results show that not only are the absorption spectra of these pigments modulated strongly by amino acids in TM I-VII, but also, for unknown reasons, the overall effect of amino acid changes in TM IV-VII on the lambdamax-shift is abolished. The spectral tuning of the contemporary frog pigment is explained by amino acid replacements F86M, V91I, T93P, V109A, E113D, L116V, and S118T, in which V91I and V109A are previously unknown, increasing the total number of critical amino acid sites that are involved in the spectral tuning of SWS1 pigments in vertebrates to 13.

Amino Acid Substitution↗

PubMed2005

Expression profiling of the estrogen responsive genes in response to phytoestrogens using a customized DNA microarray.

Here, we examined phytoestrogens, isoflavones (genistein, daidzein, glycitein, biochanin A and ipriflavone), flavones (chrysin, luteolin and apigenin), flavonols (kaempferol and quercetin), and a coumestan, a flavanone and a chalcone (coumestrol, naringenin and phloretin, respectively) by means of a DNA microarray assay. A total of 172 estrogen responsive genes were monitored with a customized DNA microarray and their expression profiles for the above phytoestrogens were compared with that for 17beta-estradiol (E2) using correlation coefficients, or R values, after a correlation analysis by linear regression. While R values indicate the similarity of the response by the genes, we also examined the genes by cluster analysis and by their specificity to phytoestrogens (specific to genistein, daidzein or glycitein) or gene functions. Several genes were selected from p53-related genes (CDKN1A, TP53I11 and CDC14), Akt2-related genes (PRKCD, BRCA1, TRIB3 and APPL), mitogen-activated protein kinase-related genes (RSK and SH3BP5), Ras superfamily genes (RAP1GA1, RHOC and ARHGDIA) and AP-1 family and related genes (RIP140, FOS, ATF3, JUN and FRA2). We further examined the extracts from two local crops of soy beans (Kuro-daizu or Mochi-daizu) by comparing the gene expression profiles with those of E2 or phytoestrogens as a first step in utilizing the expression profiles for various applications.

Cell Line, Tumor↗

PubMed2005

A case of recurrent and synchronous fibrous hamartoma of infancy.

We retrospectively reviewed the chart of a patient with recurrent fibrous hamartoma of infancy (FHI). This lesion presented as a gradually enlarging mass on the child's buttock. The child, a 6-month-old male, was followed up, and at 19 months of age the mass was excised. Eighteen months after the initial operation, a local recurrence was noticed. Forty-one months after the initial operation, reexcision was performed. Both masses demonstrated the characteristic features of FHI, which should be considered in the differential diagnosis of subcutaneous mass in young children.

Buttocks↗