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Biomedical subjects

Yoichi Yamada

Publications and source records attributed to Yoichi Yamada.

At least 19 recordsLinked to original sources
PubMed2006

Diverse DNA methylation statuses at alternative promoters of human genes in various tissues.

We characterized the DNA methylation status at 144 tissue-biased and 37 non-tissue-biased alternative promoters of 61 human genes in five normal tissues. Analysis of the collected data revealed that (i) DNA methylation status differed greatly among alternative promoters belonging to the same gene; (ii) DNA methylation status differed between tissues for the majority of the individual promoters, and (iii) 80-90% of CpG-island-containing promoters were not methylated on either allele throughout the five tissues examined. Furthermore, although the statistical significance was not as clear as for the above features, we also found that (iv) the DNA methylation patterns of tissue-biased promoters changed more drastically than those of non-tissue-biased promoters; (v) tissue-biased promoters tended to be less methylated than their respective alternative promoters in the tissues where they were preferentially expressed, and (vi) the 'null' methylation pattern of a given promoter was enriched in the tissues where the transcription was most active. These findings together indicate that there are dynamic physiological changes of DNA methylation. DNA methylation appears to play a significant role in differential usage of alternative promoters and may be related to functional diversification between CpG-island-containing promoters and CpG-island-less promoters.

Brain↗
PubMed2006

A single amino acid mutation attenuates rundown of voltage-gated calcium channels.

The activity of voltage-gated calcium channels (VGCCs) decreases with time in whole-cell and inside-out patch-clamp recordings. In this study we found that substituting a single amino acid (I1520) at the intracellular end of IIIS6 in the alpha(1) subunit of P/Q-type Ca(2+) channels with histidine or aspartate greatly attenuated channel rundown in inside-out patch-clamp recordings. The homologous mutations also slowed rundown of N- and L-type Ca(2+) channels, albeit to a lesser degree. In P/Q-type channels, the attenuation of rundown is accompanied by an increased apparent affinity for phosphatidylinositol-4,5-bisphosphate, which has been shown to be critical for maintaining Ca(2+) channel activity [L. Wu, C.S. Bauer, X.-G. Zhen, C. Xie, J. Yang, Dual regulation of voltage-gated calcium channels by PtdIns(4,5)P2. Nature 419 (2002) 947-952]. Furthermore, the histidine mutation significantly stabilized the open state, making the channels easier to open, slower to close, harder to inactivate and faster to recover from inactivation. Our finding that mutation of a single amino acid can greatly attenuate rundown provides an easy and efficient way to slow the rundown of VGCCs, facilitating functional studies that require direct access to the cytoplasmic side of the channel.

Amino Acid Sequence↗
PubMed2006

Cluster analysis and gene expression profiles: a cDNA microarray system-based comparison between human dental pulp stem cells (hDPSCs) and human mesenchymal stem cells (hMSCs) for tissue engineering cell therapy.

We investigated gene expression patterns and functional classifications regarding the clusters of human dental pulp stem cells (hDPSCs) and human mesenchymal stem cells (hMSCs)--which possess a multipotent ability--because little is known about the precise moleculobiological clues by which these cells activate their differentiating ability or functionality to eventually form dentin and bone, respectively. We first verified the expressions of the alkaline phosphatase (ALP) gene, dentin matrix protein 1 (DMP-1), and dentinsialophosphoprotein (DSPP) by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and consequently discovered the high expressions of these genes. Total RNA was also followed by hybridization with a human microarray system consisting of 12,814 genes. Analyses of gene expression patterns indicated several genes which encode extracellular matrix components, cell adhesion molecules, growth factors, and transcription regulators. Functional and clustering analyses of differences in gene expression levels revealed cell signaling, cell communication, or cell metabolism. In the future, information on the gene expression patterns of hDPSCs and hMSCs might be useful in determining the detailed functional roles of the relevant genes and applicable to stem cell therapies, and these cells could also be used as multipotent cell sources for gene technology and tissue engineering technology.

Adult↗
PubMed2006

CSN5/Jab1 inhibits cardiac L-type Ca2+ channel activity through protein-protein interactions.

L-type Ca(2+) channels have a wide tissue distribution and play essential roles in physiological responses. Recent studies have indicated that regulation of L-type Ca(2+) channels involves the assembly of macromolecular signaling complexes such as the beta(2)-adrenergic receptor signaling complex, the small G-protein kir/Gem and the BK channel. Here, we report the previously unidentified role of another protein in binding to the II-III linker of the alpha(1C) subunit of the L-type Ca(2+) channel. This protein is COP9 signalosome subunit 5 (CSN5)/Jun activation domain-binding protein 1 (Jab1). We have demonstrated that CSN5 interacts specifically with the II-III linker of the alpha(1C) subunit in a yeast two-hybrid system. The alpha(1C) subunit and CSN5 were coimmunoprecipitated in rat heart and both proteins were colocalized in sarcolemmal membranes and transverse tubules of cardiac myocytes. Silencing of CSN5 mRNA using siRNA decreased the endogenous protein level of CSN5 and activated L-type Ca(2+) channels expressed in COS7 cells. These data indicate that CSN5 is a protein that plays a newly defined functional role in association with the cardiac L-type Ca(2+) channel.

Animals↗
PubMed2006

Musidunin and musiduol, insect antifeedants from Croton jatrophoides.

Two novel limonoids, musidunin (1) and musiduol (2), were isolated from a methanol extract of Croton jatrophoides by bioassay-guided fractionation. Their structures were established by extensive NMR experiments. Interestingly, A,B-seco limonoid 1 contains a unique acetal annulation of A, A', and B' rings. Both limonoids exhibited antifeedant activities against two pests, Pectinophora gossypiella and Spodoptera frugiperda.

Animals↗
PubMed2006

Simultaneous implant placement and bone regeneration around dental implants using tissue-engineered bone with fibrin glue, mesenchymal stem cells and platelet-rich plasma.

This study was undertaken to evaluate the use of tissue-engineered bone as grafting material for alveolar augmentation with simultaneous implant placement. Twelve adult hybrid dogs were used in this study. One month after the extraction of teeth in the mandible region, bone defects on both sides of the mandible were induced using a trephine bar with a diameter of 10 mm. Dog mesenchymal stem cells (dMSCs) were obtained via iliac bone biopsy and cultured for 4 weeks before implantation. After installing the dental implants, the defects were simultaneously implanted with the following graft materials: (i) fibrin, (ii) dMSCs and fibrin (dMSCs/fibrin), (iii) dMSCs, platelet-rich plasma (PRP) and fibrin (dMSCs/PRP/fibrin) and (iv) control (defect only). The implants were assessed by histological and histomorphometric analysis, 2, 4 and 8 weeks after implantation. The implants exhibited varying degrees of bone-implant contact (BIC). The BIC was 17%, 19% and 29% (control), 20%, 22% and 25% (fibrin), 22%, 32% and 42% (dMSCs/fibrin) and 25%, 49% and 53% (dMSCs/PRP/fibrin) after 2, 4 and 8 weeks, respectively. This study suggests that tissue-engineered bone may be of sufficient quality for predictable enhancement of bone regeneration around dental implants when used simultaneous by with implant placement.

Alkaline Phosphatase↗
PubMed2006

A novel approach to periodontal tissue regeneration with mesenchymal stem cells and platelet-rich plasma using tissue engineering technology: A clinical case report.

Tissue engineering represents one of the most exciting advances in regenerative medicine. However, little has been reported on the application of tissue engineering for regeneration of periodontal tissues. Therefore, the aim of this study was to show how a technique based on tissue engineering principles can be applied to periodontology. Mesenchymal stem cells (MSCs) were isolated from a patient's iliac crest marrow aspirates. Platelet-rich plasma (PRP) was isolated from peripheral blood. Full-thickness periodontal flaps were elevated and the root surfaces were scaled and planed. A MSCs-PRP gel was prepared and applied to the root surface and adjacent defect space. The primary outcome measures were changes in pocket depth, clinical attachment level, bleeding on probing, and defect bone fill. Re-examination demonstrated that the treatment, including the application of MSCs-PRP gel at periodontal sites with angular defects, resulted in a 4-mm reduction in probing depths and a 4-mm clinical attachment gain, while bleeding and tooth mobility disappeared. Radiographic assessments showed that the bone defect had been reduced in depth. Interdental papillae supported by this tissue engineering technology regenerated. The use of MSCs in PRP gel might be helpful for periodontal tissue regeneration, treatment of esthetically sensitive sites, and reduction of patient morbidity.

Alveolar Bone Loss↗
PubMed2005

Osteogenic potential of injectable tissue-engineered bone: a comparison among autogenous bone, bone substitute (Bio-oss), platelet-rich plasma, and tissue-engineered bone with respect to their mechanical properties and histological findings.

Recently, tissue engineering has become available as a regenerative treatment for bone defects. However, the evaluation of its success is limited to histological analysis, and its effects on mechanical hardness remain to be investigated. This study investigated mechanical strength in support of histological findings, specifically for tissue-engineered bone with mesenchymal stem cells (MSCs) and platelet-rich-plasma (PRP). Initially, teeth were extracted, and bone defects on both sides of the mandible were prepared with a trephine bar. The defects were implanted by using the following graft materials: 1) PRP, 2) PRP and dog MSCs (dMSCs), 3) autogenous bone (PCBM), 4) bone substitute (Bio-Oss), and 5) control (defects only). After 2, 4, 8, and 12 weeks of implantation, the defects were histologically assessed to examine their mechanical properties. According to histological observations, the dMSCs/PRP groups had well-formed mature bone compared with the control (defects only), Bio-Oss, and PRP groups. The Vickers hardness test values were 8 (control), 9 (PRP), not detected (Bio-Oss), 11 (PCBM), and 17 (dMSCs/PRP) after 2 weeks. Therefore, tissue-engineered bone can be used for early stage bone regeneration from the viewpoint of histology and mechanical properties.

Animals↗
PubMed2005

Magnetic force-based mesenchymal stem cell expansion using antibody-conjugated magnetoliposomes.

Recently, there has been an accumulation of evidence indicating that human mesenchymal stem cells (MSCs, multipotent cells resident in the bone marrow) are useful for autologous cell transplantation. However, only small numbers of MSCs have been obtained in bone marrow aspirates. We have developed a novel methodology for enriching and proliferating MSCs from bone marrow aspirates using antibody-conjugated magnetoliposomes (AMLs). The AMLs are liposomes conjugated to anti-CD105 antibody (immunoliposomes) and contain magnetite nanoparticles (diameter 10 nm). In the present study, the AMLs were added to a small volume (1 mL) of human bone marrow aspirate. After a 1-h incubation period, the bone marrow aspirates containing AMLs were seeded into 10-cm tissue culture dishes, and a disk-shaped magnet (diameter 2.2 cm; height 1 cm; 4000 Gauss) was positioned under the dish to enrich MSCs by magnetic force. The MSCs proliferated, forming colonies at the site where the magnet was positioned. In contrast, no colonies and very few viable cells were observed in ordinary culture based on plastic-adherent tendencies of cells without use of AMLs. These results suggest that this AML culture method can rapidly and efficiently expand a small number of MSCs into numbers suitable for clinical application.

Animals↗
PubMed2005

Sinus floor elevation applied tissue-engineered bone. Comparative study between mesenchymal stem cells/platelet-rich plasma (PRP) and autogenous bone with PRP complexes in rabbits.

In the present study, we compared bone regeneration ability in sinus floor elevation between a tissue engineering method using mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP), and a promising new method using particulate cancellous bone and marrow (PCBM) and PRP. Bilateral sinus floor elevation procedures were performed in 18 adult Japanese white rabbits. MSCs/PRP or PCBM/PRP complexes were grafted to each maxillary sinus in the same rabbits. The MSCs were isolated from rabbit iliac crest marrow, and PRP was obtained from peripheral blood. PCBM were collected from the rabbit iliac crest and mixed with PRP. The animals were sacrificed at 2, 4, and 8 weeks after transplantation, and the bone formation ability of each implant was evaluated histologically and histometrically. According to the histological observations, both sites (MSCs/PRP and PCBM/PRP) showed well newly formed bone and neovascularization at 2 and 4 weeks. However, at 8 weeks, the lamellar bone was observed to be occupied by fatty marrow in large areas in both sites. There was no significant difference in bone volume or augmented height between MSCs/PRP and PCBM/PRP groups each week, but there were significant differences in bone volume and augmented height between 2 and 8 weeks in PCBM/PRP or MSCs/PRP groups and in bone volume between 4 and 8 weeks in the PCBM/PRP group (P<0.05). These results suggest that the MSCs/PRP complex may well be used for bone regeneration in sinus floor elevation, compared with the PCBM/PRP complex.

Animals↗
PubMed2005

Clinical case reports of injectable tissue-engineered bone for alveolar augmentation with simultaneous implant placement.

This clinical study was undertaken to evaluate the use of tissue-engineered bone, mesenchymal stem cells, platelet-rich plasma, and beta-tricalcium phosphate as grafting materials for maxillary sinus floor augmentation or onlay plasty with simultaneous implant placement in six patients with 3- to 5-mm alveolar crestal bone height. All 20 implants were clinically stable at second-stage surgery and 12 months postloading. A mean increase in mineralized tissue height of 7.3+/-4.6 mm was evident when comparing the pre- and postsurgical radiographs. Injectable tissue-engineered bone provided stable and predictable results in terms of implant success.

Absorbable Implants↗
PubMed2004

An evolutionary scenario for genomic imprinting of Impact lying between nonimprinted neighbors.

Mouse Impact is the sole imprinted gene mapped to chromosome 18 to date. Despite its remarkable evolutionary conservation, human IMPACT was shown to escape genomic imprinting. Here we identified Hrh4 and Osbpl1 as the distal and proximal nearest neighbors of Impact, respectively, and found that both genes are expressed biallelically. Thus, in contrast with most imprinted genes, Impact fails to show apparent physical clustering with other imprinted genes. Since Impact not only lies in an intergenic region but also consists of 11 exons, it does not seem to be an imprinted gene generated by a retrotransposition. Hazardous effects of overexpressed Impact, a genomic segment containing paralogues of Hrh4 and Osbpl1 but not of Impact, and enhanced promoter activity in the mouse led us to propose an alternative model. This model assumes that segmental duplication followed by enhancement of the promoter activity in the lineage to mouse is responsible for the species-specific imprinting of Impact.

Alleles↗
PubMed2004

Phosphatidylinositol [correction] 4,5-bisphosphate signals underlie receptor-specific Gq/11-mediated modulation of N-type Ca2+ channels.

Modulation of voltage-gated Ca2+ channels via G-protein-coupled receptors is a prime mechanism regulating neurotransmitter release and synaptic plasticity. Despite extensive studies, the molecular mechanism underlying Gq/11-mediated modulation remains unclear. We found cloned and native N-type Ca2+ channels to be regulated by phosphatidylinositol [correction] 4,5-bisphosphate (PIP2). In inside-out oocyte patches, PIP2 greatly attenuated or reversed the observed rundown of expressed channels. In sympathetic neurons, muscarinic M1 ACh receptor suppression of the Ca2+ current (ICa) was temporally correlated with PIP2 hydrolysis, blunted by PIP2 in whole-cell pipettes, attenuated by expression of PIP2-sequestering proteins, and became irreversible when PIP2 synthesis was blocked. We also probed mechanisms of receptor specificity. Although bradykinin also induced PIP2 hydrolysis, it did not inhibit ICa. However, bradykinin receptors became nearly as effective as M1 receptors when PIP2 synthesis, IP3 receptors, or the activity of neuronal Ca2+ sensor-1 were blocked, suggesting that bradykinin receptor-induced intracellular Ca2+ increases stimulate PIP2 synthesis, compensating for PIP2 hydrolysis. We suggest that differential use of PIP2 signals underlies specificity of Gq/11-coupled receptor actions on the channels

1-Phosphatidylinositol 4-Kinase↗
PubMed2004

Structural basis of the alpha1-beta subunit interaction of voltage-gated Ca2+ channels.

High-voltage-activated Ca2+ channels are essential for diverse biological processes. They are composed of four or five subunits, including alpha1, alpha2-delta, beta and gamma (ref. 1). Their expression and function are critically dependent on the beta-subunit, which transports alpha1 to the surface membrane and regulates diverse channel properties. It is believed that the beta-subunit interacts with alpha1 primarily through the beta-interaction domain (BID), which binds directly to the alpha-interaction domain (AID) of alpha1; however, the molecular mechanism of the alpha1-beta interaction is largely unclear. Here we report the crystal structures of the conserved core region of beta3, alone and in complex with AID, and of beta4 alone. The structures show that the beta-subunit core contains two interacting domains: a Src homology 3 (SH3) domain and a guanylate kinase (GK) domain. The AID binds to a hydrophobic groove in the GK domain through extensive interactions, conferring extremely high affinity between alpha1 and beta-subunits. The BID is essential both for the structural integrity of and for bridging the SH3 and GK domains, but it does not participate directly in binding alpha1. The presence of multiple protein-interacting modules in the beta-subunit opens a new dimension to its function as a multi-functional protein.

Amino Acid Sequence↗
PubMed2004

Single-channel activity of L-type Ca2+ channels reconstituted with the beta2c subunit cloned from the rat heart.

We recently cloned the beta(2c) subunit of the L-type Ca(2+) channel as a functional type of beta subunit from the rat heart. In order to clarify the contribution of the beta(2c) subunit to native Ca(2+) channel function, we investigated the single-channel properties of Ca(2+) channels reconstituted with beta(2a) or beta(2c) subunits and compared them with the properties of native channels. In contrast to the Ca(2+) channel with beta(2a) subunit, long-lasting closings were dominant in the Ca(2+) channel with beta(2c) subunit and the native channel. The ensemble-averaged current of the cells with beta(2c) subunits was comparable to that of the native cardiomyocytes. Many high P(o) sweeps (mode 2) were observed in the cells with beta(2a) subunits, while only a few high P(o) sweeps were observed in the cells with beta(2c) subunits and the native cells. These findings suggest that the beta(2c) subunit is one of the functional beta subunits in the rat heart.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2004

A yeast one-hybrid system to detect methylation-dependent DNA-protein interactions.

We developed a method for site-selective CpG methylation of the budding yeast genome. The method recruits LexA-fused M.SssI DNA methyltransferase to LexA operator sequences integrated adjacent to the target site. Microarray analysis of methylated DNAs indicated that the tethered enzyme selectively methylates the region around the target site. Exploiting this method to methylate bait DNA in the one-hybrid system, we demonstrated methylation-dependent DNA binding of methyl-CpG binding proteins, MBD1 and Kaiso, in vivo. This methylation-dependent one-hybrid system would provide a versatile tool for the search and analysis of proteins that recognize methylated DNA to participate in epigenetic regulation.

Base Sequence↗
PubMed2004

Elevated transcript level of hyaluronan synthase1 gene correlates with poor prognosis of human colon cancer.

Hyaluronan plays important roles in the complex processes of tumor invasion and metastasis. It is now known that three hyaluronan synthase (HAS) isoforms catalyze hyaluronan synthesis, which raises the question of how they are involved in malignant tumor progression. In this study, we examined the correlation between tumor progression and transcriptional levels of three HAS isoforms in specimens of human colon cancers. Tumor tissues from 31 patients with different diagnostic grades were assessed to determine the level of each HAS isoform by real time RT-PCR. The mean expression coefficients for HAS1, HAS2 and HAS3 in the cancerous parts were 0.82-, 0.91- and 1.22-fold, respectively; of those in the noncancerous parts at Dukes' stage A; 1.00-, 0.95- and 1.06-fold, respectively, at stage B; and 1.95-, 1.16- and 1.19-fold, respectively, at stage C. In survival analysis, a significant correlation was observed between poor survival and the HAS1 transcript level. When the ratio of tumor to normal tissue in the HAS1 level was compared with that of the HA receptor transcript level, there was a positive correlation with that of the CD44 variant 6 level at Dukes' stage C. Our current results therefore suggest that HAS1 plays a role in the malignant progression of human colon cancer cells.

Base Sequence↗