Biomedical subjects

Yusuke Takahashi

Publications and source records attributed to Yusuke Takahashi.

32 records · Page 2Linked to original sources

PubMed2005

Sensitization of human aortic endothelial cells to lipopolysaccharide via regulation of Toll-like receptor 4 by bacterial fimbria-dependent invasion.

Toll-like receptors (TLRs) are differentially up-regulated in response to microbial infection and chronic inflammatory diseases such as atherosclerosis. Epidemiological data support the idea that periodontal disease may be a risk factor for acceleration of atherosclerosis. Porphyromonas gingivalis, the etiological agent of periodontal disease, invades endothelium, has been detected in human atheromatous tissue, and accelerates atheroma formation in apolipoprotein E-/- mice with concurrent induction of TLRs in the aorta. As endothelial cells can present antigen via TLRs and play an important role in the development of atherosclerosis, we examined TLR expression in human aortic endothelial cells (HAEC) cultured with wild-type P. gingivalis, a fimbria-deficient mutant, and purified antigens. We observed increased TLR expression in HAEC infected with wild-type P. gingivalis by fluorescence-activated cell sorter, but not with noninvasive, fimbria-deficient mutant or purified P. gingivalis antigens. Following a wild-type P. gingivalis challenge, functional TLR2 and TLR4 activation was assessed by subsequent stimulation with TLR agonists Staphylococcus aureus lipoteichoic acid (SLTA; TLR2 ligand) and Escherichia coli lipopolysaccharide (LPS; TLR4 ligand). Unchallenged HAEC failed to elicit monocyte chemoattractant protein 1 (MCP-1) in response to LPS or SLTA but did so when cultured with wild-type P. gingivalis. P. gingivalis-induced TLR2 and -4 expression on HAEC functionally reacted to SLTA and E. coli LPS as measured by a further increase in MCP-1 production. Furthermore, MCP-1 expression elicited by E. coli LPS was inhibitable with TLR4-specific antibody and polymyxin B. These results indicate that invasive P. gingivalis stimulates TLR expression on the surface of endothelium and these primed cells respond to defined TLR-specific ligands.

Antigens, Bacterial↗

PubMed2005

Porphyromonas gingivalis fimbria-dependent activation of inflammatory genes in human aortic endothelial cells.

Epidemiological and pathological studies have suggested that infection with the oral pathogen Porphyromonas gingivalis can potentiate atherosclerosis and human coronary heart disease. Furthermore, infection with invasive, but not noninvasive P. gingivalis has been demonstrated to accelerate atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice and to accelerate local inflammatory responses in aortic tissue. In the present study, using high-density oligonucleotide microarrays, we have defined the gene expression profile of human aortic endothelial cells (HAEC) after infection with invasive and noninvasive P. gingivalis. After infection of HAEC with invasive P. gingivalis strain 381, we observed the upregulation of 68 genes. Genes coding for the cytokines Gro2 and Gro3; the adhesion molecules intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule (VCAM)-1, and ELAM-1 (E-selectin); the chemokine interleukin-8 (IL-8); and the proinflammatory molecules IL-6 and cyclooxygenase-2 were among the most highly upregulated genes in P. gingivalis 381-infected HAEC compared to uninfected HAEC control. Increased mRNA levels for signaling molecules, transcriptional regulators, and cell surface receptors were also observed. Of note, only 4 of these 68 genes were also upregulated in HAEC infected with the noninvasive P. gingivalis fimA mutant. Reverse transcription-PCR, enzyme-linked immunosorbent assay, and fluorescence-activated cell sorting analysis confirmed the expression of ICAM-1, VCAM-1, E-/P-selectins, IL-6, and IL-8 in HAEC infected with invasive P. gingivalis. We also demonstrated that increased expression of ICAM-1 and VCAM-1 in aortic tissue of ApoE(-/-) mice orally challenged with invasive P. gingivalis but not with the noninvasive P. gingivalis fimA mutant by immunohistochemical analysis. Taken together, these results demonstrate that P. gingivalis fimbria-mediated invasion upregulates inflammatory gene expression in HAEC and in aortic tissue and indicates that invasive P. gingivalis infection accelerates inflammatory responses directly in the aorta.

Aorta↗

PubMed2005

Genetic and environmental etiology of effortful control.

We examined whether effortful control (EC), a temperament proposed by Rothbart and Bates (1998), has genetically coherent structure. A self-report measure of EC was administered to 450 Japanese twins (151 males and 299 females, ages 17 to 32 years) including 152 monozygotic and 73 dizygotic pairs. Univariate genetic analysis revealed that AE model fit best for the total EC as well as its subscales. The heritability estimate for total EC was 49%, and the estimates for subscales ranged between 32% and 45%. Multivariate genetic analysis revealed that the subscales of EC were genetically correlated to a high degree and environmentally correlated to a moderate degree. These results suggest that EC has substantial genetic basis and genetically coherent structure, supporting the validity of the construct. The implications to molecular genetic study and study of psychopathology were discussed.

Adolescent↗

PubMed2004

The C-terminal domains of the gingipain K polyprotein are necessary for assembly of the active enzyme and expression of associated activities.

The Porphyromonas gingivalis lysine-specific cysteine protease (gingipain K, Kgp) is expressed as a large precursor protein consisting of a leader sequence, a pro-fragment, a catalytic domain with a C-terminal IgG-like subdomain (IgSF) and a large haemagglutinin/adhesion (HA) domain. In order to directly study the role of these non-catalytic domains in pro-Kgp processing and maturation in P. gingivalis, the wild-type form of the gene was replaced with deletion variants encoding C-terminally truncated proteins, including KgpDeltaHA3/4 (Delta1292-1732 aa), KgpDeltaHA2-4 (Delta1157-1732 aa), KgpDeltaHA1-4 (Delta738-1732 aa), KgpDeltaC-term/HA (Delta681-1732 aa) and KgpDeltaIg/C-term/HA (602-1732 aa). Northern blot and reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that all truncated variants of the kgp gene were transcribed in P. gingivalis. Despite high levels of kgpDeltaC-term/HA and kgpDeltaIg/C-term/HA transcripts, no Kgp-specific antigen was detected in cultures of these mutants as determined by Western blot analysis with monoclonal antibodies specific for the Kgp catalytic domain. Furthermore, only barely measurable amounts of Kgp-specific activity were detected in these two mutants. The remaining mutants expressed significant Kgp activity, however, at lower levels when compared with the parental strain. The decreased activity most probably resulted from altered folding and/or hindered secretion of the protein. The kgp gene truncation was also demonstrated to alter the distribution of the gingipain protein between membrane-associated and -secreted forms. While both gingipain K activity and the protein were cell membrane-associated in the parental strain, the mutants released significant amounts of both protein and activity into the media. Taken together, these results suggest that the C-terminal HA domains of Kgp are not only essential for full expression of gingipain activity, but also for proper processing of the multiprotein complex assembly on the P. gingivalis outer membrane. Moreover, our results indicate that the immunoglobulin-like subdomain is indispensable for proper folding and expression of the gingipains.

Adhesins, Bacterial↗

PubMed2004

[A case of extrapontine myelinolysis precipitated by correction of a hyper-osmolar state].

We report a case of extrapontine myelinolysis (EPM) precipitated by correction of a hyper-osmolar state. A 7-year-old boy presented with disturbed consciousness. Serum BUN and Na level were 48 mg/dl and 196 mmol/L, respectively. T2-weighted images and FLAIR images of MRI visualized symmetrical bilateral high signal intensity areas in the external capsule and in the thalamus. In addition, the splenium of the corpus callosum showed high signal intensity. All these lesions showed high signal intensity on diffusion-weighted images. Extrapontine myelinolysis is precipitated not only by the rapid correction of a hypo-osmolar state but also by that of a hyper-osmolar state.

Child↗

PubMed2003

Molecular basis of spectral tuning in the newt short wavelength sensitive visual pigment.

Previously we reported the sequence of the member of the short wavelength sensitive 2 (SWS2) family of vertebrate visual pigments from the retina of the Japanese common newt, Cynops pyrrhogaster[Takahashi, Y. et al. (2001) FEBS Lett. 501, 151-155]. Now we have expressed the apopigment and regenerated it with A1 retinal. Its absorption maximum, 474 nm, is greatly red shifted compared to other known SWS2 pigments (418-455 nm). To determine the amino acid residues that control its spectral tuning, we replaced the residues that were near the chromophore and which differed between the newt and the bullfrog (lambda(max) = 430 nm) wild-type SWS2 pigments: Pro91Ser, Ser94Ala, Ile122Met, Cys127Ser, Ser211Cys, Tyr261Phe, and Ala292Ser. Each of these site-directed mutants led to blue shifts of the newt pigment with five of them causing substantial shifts; their sum was about equal to the difference between the absorption maximum of the bullfrog and newt pigments, 44 nm. The 32 nm shift of the absorption maximum of the multiple seven-residue mutant to 442 nm is fairly close to that of the wild-type bullfrog pigment. Thus, the seven amino acid residues that we replaced are the major cause of the red shift of the newt SWS2 pigment's spectrum. Two of the residues, 91 and 94, have not previously been identified as wavelength regulating sites in visual pigments. One of these, 91, probably regulates color via a new mechanism: altering of a hydrogen bonding network that is connected via a water to the chromophore, in this case its counterion, Glu113.

Amino Acid Sequence↗

PubMed2003

Site-directed mutagenesis of benzalacetone synthase. The role of the Phe215 in plant type III polyketide synthases.

Benzalacetone synthase (BAS) and chalcone synthase (CHS) are plant-specific type III polyketide synthases (PKSs) that share approximately 70% amino acid sequence identity. BAS catalyzes a one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce a diketide benzalacetone, whereas CHS performs sequential condensations with three malonyl-CoA to generate a tetraketide chalcone. A homology model suggested that BAS has the same overall fold as CHS with cavity volume almost as large as that of CHS. One of the most characteristic features is that Rheum palmatum BAS lacks active site Phe-215; the residues 214LF conserved in type III PKSs are uniquely replaced by IL. Our observation that the BAS I214L/L215F mutant exhibited chalcone-forming activity in a pH-dependent manner supported a hypothesis that the absence of Phe-215 in BAS accounts for the interruption of the polyketide chain elongation at the diketide stage. On the other hand, Phe-215 mutants of Scutellaria baicalensis CHS (L214I/F215L, F215W, F215Y, F215S, F215A, F215H, and F215C) afforded increased levels of truncated products; however, none of them generated benzalacetone. These results confirmed the critical role of Phe-215 in the polyketide formation reactions and provided structural basis for understanding the structure-function relationship of the plant type III PKSs.

Acyltransferases↗

PubMed2003

Enzymatic formation of unnatural novel polyketides from alternate starter and nonphysiological extension substrate by chalcone synthase.

[reaction: see text] In the chalcone synthase (CHS) enzyme reaction, both the starter molecule and the extension unit of the polyketide chain elongation reaction were simultaneously replaced with nonphysiological substrates. When incubated with benzoyl-CoA and methylmalonyl-CoA as substrates, recombinant CHS from Scutellaria baicalensis afforded an unnatural novel triketide, 4-hydroxy-3,5-dimethyl-6-phenyl-pyran-2-one, along with a tetraketide, 4-hydroxy-3,5-dimethyl-6-(1-methyl-2-oxo-2-phenyl-ethyl)-pyran-2-one. On the other hand, the enzyme also accepted hexanoyl-CoA and methylmalonyl-CoA as substrates to produce an unnatural novel triketide, 4-hydroxy-3,5-dimethyl-6-pentyl-pyran-2-one.

Acyl Coenzyme A↗

PubMed2003

Antibacterial activity of bactericide-immobilized filler for resin-based restoratives.

This study examined the antibacterial activity of prepolymerized resin filler, in which the bactericide quaternary ammonium was immobilized. The experimental filler was prepared by grinding prepolymerized resin blocks of methacrylate monomers, silica particles, and the antibacterial monomer methacryloyloxydodecylpyridinium bromide (MDPB). The number of Streptococcus mutans after incubation for 18h in contact with the experimental filler with or without protein adsorption by saliva treatment was determined, and adherence of bacteria to the filler surface was evaluated by scanning electron microscopy. Elution of unpolymerized MDPB from the filler and its influence on bacterial growth were also investigated. The growth of S. mutans was completely inhibited by contact with the experimental filler without saliva treatment. Although the effects were attenuated, the saliva-treated filler still exhibited growth inhibition at >99.9%. Less bacteria attached to the experimental filler than the control filler without MDPB, indicating that the reduction in bacterial number after contact with the experimental filler was not due to bacterial adherence to the particles. Unpolymerized MDPB at 1 microg/ml was eluted from the filler particles but was confirmed to have little effect on bacterial growth. The results indicate that the bactericide-immobilized filler containing MDPB shows significant bacteriostatic effects without releasing antibacterial components, and is useful for incorporation into various resin-based restoratives.

Adsorption↗

PubMed2003

Effects of chlorhexidine, minocycline, and metronidazole on Porphyromonas gingivalis strain 381 in biofilms.

BACKGROUND: Porphyromonas gingivalis is found in subgingival biofilm and is associated with periodontal disease. Bacteria in biofilms are able to resist higher antimicrobial concentrations than in suspension. Little is known about the susceptibility of P. gingivalis in biofilms to antimicrobial agents. The effects of chlorhexidine gluconate, minocycline hydrochloride, and metronidazole on P. gingivalis biofilms were examined in vitro. METHODS: P. gingivalis strain 381 biofilms were prepared on 32 hydroxyapatite disks. At 0, 24, 72, and 144 hours after perfusion of the three antimicrobial agents, two disks from each device were used to assess the antimicrobial effects by adenosine triphosphate (ATP) bioluminescence, and for morphological investigation by scanning electron microscopy (SEM). RESULTS: Close relationships were found between the results of the ATP analyses and the SEM observations in all groups examined. A significant decrease (P < 0.001) in ATP content was found between the chlorhexidine-treated and control groups. The extracellular matrix structure and P. gingivalis cells were altered in the presence of chlorhexidine. Minocycline hydrochloride also caused a decrease (P < 0.05) in the ATP content and morphological change on P. gingivalis biofilms. Metronidazole showed no significant efficacy against P. gingivalis biofilms. CONCLUSION: Chlorhexidine gluconate was effective at reducing the viability of P. gingivalis 381 cells in biofilms, while minocycline hydrochloride and metronidazole exhibited weaker effects.

Adenosine Triphosphate↗

PubMed2003

Inhibitory effects of incadronate on the progression of rat experimental periodontitis by porphyromonas gingivalis infection.

BACKGROUND: Incadronate (YM175, disodium cycloheptylaminomethylenediphosphonate monohydrate), a bisphosphonate, has been suggested to prevent the bone resorption associated with periodontitis by inhibiting osteoclast activity. The purpose of this study was to investigate the effect of incadronate in preventing periodontal destruction in rats with Porphyromonas gingivalis-induced periodontitis. METHODS: Periodontitis was induced in 35 Wister rats by inoculating P. gingivalis into the oral cavity and feeding the rats a soft diet for 4 weeks. Incadronate or placebo was administered to the oral cavity of the rats 2 days per week for 2, 4, or 8 weeks. RESULTS: P. gingivalis infection resulted in destruction of the periodontal ligament, reduced bone density, and caused inflammatory cell migration. Radiographic, morphometric, and histological results showed that incadronate had the ability to increase the bone mineral density (quantum level score; cortex 518.9 [placebo 612.8]; sponge 579.8 [placebo 672.0]) and to prevent periodontal ligament destruction (width 0.16 mm [placebo 0.20 mm]; area 0.36 mm2 [placebo 0.54 mm2]) after 8 weeks' administration. Furthermore, the polymorphonuclear leukocyte (PMN) infiltration in gingival tissue was significantly decreased. CONCLUSION: These results showed that incadronate inhibits bone resorption and PMN migration in P. gingivalis-induced periodontitis.

Alveolar Bone Loss↗

PubMed2002

Enzymatic formation of an unnatural C(6)-C(5) aromatic polyketide by plant type III polyketide synthases.

[reaction: see text] Substrate specificities of plant polyketide synthases (PKSs) were investigated using analogues of malonyl-CoA, the extension unit of the polyketide chain elongation reactions. When incubated with methylmalonyl-CoA and 4-coumaroyl-CoA, plant PKSs (chalcone synthase from Scutellaria baicalensis, stilbene synthase from Arachis hypogaea, and benzalacetone synthase from Rheum palmatum) afforded an unnatural C(6)-C(5) aromatic polyketide, 1-(4-hydroxyphenyl)pent-1-en-3-one, formed by one-step decarboxylative condensation of the two substrates. In contrast, succinyl-CoA was not accepted as a substrate by the enzymes.

Butanones↗

PubMed2002

Opsin expression in adult, developing, and regenerating newt retinas.

Japanese common newts (Cynops pyrrhogaster) have an ability to regenerate their neural retina even as adults. Although extensive research has been carried out attempting to understand this retinal regeneration, the molecules characterized in newt retina are limited. We isolated cDNAs encoding three putative opsins (Cp-Rh, -LWS and -SWS1), in addition to Cp-SWS2 [Takahashi et al., FEBS Lett. 501 (2001) 151-155] from a cDNA library of adult newt retina. Our immunohistochemical and in situ hybridization studies demonstrated that Cp-Rh is selectively expressed in rods, whereas the other opsins are expressed in cones. The distribution of opsin mRNAs in normal and regenerated retinas is very similar. In both developing and regenerating retinas, Cp-Rh and its mRNA first appeared in immature rods at the beginning or just after the formation of plexiform layers. Cp-Rh was initially found isotropically in the plasma membrane, and then translocalized to the apical region along with the maturation of regenerating rods. This suggests that reorganization of the intracellular structure takes place during maturation of the regenerating newt photoreceptors.

Amino Acid Sequence↗

PubMedDate not supplied

In vivo antibacterial effects of dentin primer incorporating MDPB.

This study examined the hypothesis that experimental primer containing the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB), which was previously reported to show bactericidal effects in vitro, inhibits bacteria in cavities under in vivo conditions. The number of bacteria resulting from applying primer solution to cavities in dog teeth infected with Streptococcus mutans was determined. The infected cavities were also restored using primer and the pulp response was histopathologically examined after 7, 30 and 75 days. No bacteria were recovered after applying the experimental primer, although the bactericidal effects of the proprietary primer were insignificant. Restoration with the experimental primer resulted in little or no pulpal inflammation for all periods; whereas, mild to moderate inflammatory response was observed when using proprietary primer. These results indicate that the experimental primer containing MDPB could exhibit in vivo antibacterial effects, suggesting its possible clinical benefit.

Analysis of Variance↗