Vascular calcification is an active and regulated process that is similar to bone formation. While calcium channel blockers (CCBs) have been shown to improve outcomes in atherosclerotic vascular disease, it remains unknown whether CCBs have an effect on the process of vascular calcification. Here we investigated whether CCBs inhibit osteogenic differentiation and matrix mineralization of vascular smooth muscle cells induced by 2, a key factor of vascular calcification. Human aortic smooth muscle cells (HASMCs) were transduced with adenovirus expressing 2 and were treated with 3 distinct CCBs. Azelnidipine, a dihydropyridine subclass of CCBs, significantly decreased alkaline phosphatase (ALP) activity of 2-overexpressed HASMCs, whereas verapamil and diltiazem had no effect. Furthermore, azelnidipine, but not verapamil and diltiazem, significantly decreased matrix mineralization of 2-overexpressing HASMCs. Azelnidipine significantly attenuated the induction of ALP gene expression by 2, a key transcription factor in osteogenesis, while it did not reduce enzymatic activity of ALP. Furthermore, azelnidipine inhibited the ability of 2 to activate the ALP gene, but had no effect on Notch-induced 2 expression. Given that L-type calcium channels are equally blocked by these CCBs, our results suggest that azelnidipine inhibits the 2-dependent process of vascular calcification by mechanisms other than inhibition of calcium channel activity.

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-1 is a member of the homeobox family, which plays important roles in industive tissue interactions during vertebrate organogenesis. As mice deficient in -1 demonstrated tooth morphogenesis failure, -1 could be a key factor controlling inductive signaling molecules between the dental epithelium and mesenchyme during tooth formation. Furthermore, it has been found that FGF8 expressed in dental mesenchyme induced -1 in dental mesenchyme in early tooth development. These results indicated that the relationship between FGF8 and -1 could be an important clue to elucidate dentinogenesis; however, few papers have studied the signaling pathways of -1 expression induced by FGF8 in dental mesenchyme. In this study, we found that MSW-1 was induced by FGF8 throughout mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) in cultured dental pulp cells isolated from rat lower incisors. When MAPK/ERK activation was inhibited with a specific inhibitor (PD98059), -1 expression was remarkably reduced. The specific inhibitor also significantly reduced -1 expression induced by FGF8. Interestingly, the inhibitor had no effect on alkaline phosphatase, but blocked the cell proliferation stimulated by FGF8. These results suggest that MAPK/ERK is necessary for -1 expression induced by FGF8, and plays important roles in dentinogenesis in rat dental mesenchymal cells.

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心血管の形成に重要とされる神経堤および心内膜床に発現が認められるホメオボックス遺伝子1および2のノックアウトマウスを作製し,心血管発生における1および2の機能を解析した.1および2の単独欠損マウスでは心血管の発生異常は認められなかったが,1・ 2両欠損マウスでは多様な心血管の発生異常が出現した.心血管の発生異常は,大別して,心室流出路の奇形(大血管転位,両大血管右室起始,総動脈幹症),心室流入路の奇形(三尖弁閉鎖,両房室弁左室挿入),大動脈弓の奇形(重複大動脈,左鎖骨下動脈起始異常など),心室中隔欠損などが認められた.これらの発生異常は,大血管転位の発生頻度が両大血管右室起始より高いという点を除けば,ニワトリ胚神経堤のアブレーションで出現する奇形とほぼ一致することより,1・2両欠損マウスの心血管の発生異常は神経堤細胞の異常に起因することが考えられた.一方,1・2両欠損マウスでは心内膜床の上皮-間葉形質転換の障害が考えられ,これに起因する心奇形も認められた.ノックアウトマウスにより1および2の個体レベルでの機能が明らかになったが,1および 2の標的遺伝子を含めた下流遺伝子を同定し, 1および2が関与する心血管形成の遺伝子機構を解明することが今後の課題になると思われる.

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Although much is known about the hormonal regulation of hard tissue development, much less is known about the nuclear regulatory molecules that affect the process. Homeobox-containing genes are thought to encode DNA-binding transcriptional factors which control the expression of other genes.
In this study, molecular cloning of homeobox-containing genes from a bovine odontoblast library and a human dental pulp-derived cells library was performed, and also the expression of mRNAs for Hox and homeobox-containing genes during ectopic bone formation induced by BMP was investigated.
Screening of a bovine odontoblast cDNA library and a human dental pulp-derived cells library with murine -1 and -2 cDNA probes led to the isolation of several positive clones. All of the clones from a bovine odontoblast library encoded the bovine counterpart for human -1. All of the clones from a human dental pulp-derived cells library encoded the human counterpart for murine -2. Northern blot analysis probed with a human -2 cDNA indicated the expression of 2.2kb and 1.2kb mRNA in human dental pulp-derived cells.
Polymerase chain reaction (PCR) -based analysis in the BMP-implanted tissue revealed that nine rat homologues of Hox homeobox-containing genes were expressed in the early cell migration stage and that two genes were expressed in the cartilage and bone differentiation stages. The PCR study provided evidence of dynamic changes in the BMP-induced homeobox gene expression.

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Aim: Vascular calcification is prevalent in patients with diabetes and chronic kidney disease. Receptor for advanced glycation end products (RAGE) and its multiple ligands have been implicated in the pathogenesis of accelerated atherosclerosis; however, little is known about the effects of RAGE activation on vascular calcification.
Methods and Results: Cultured rat and human aortic smooth muscle cells (HASMC) were transduced with adenovirus expressing RAGE. Expression of myocardin and the SMC-marker genes was significantly repressed in these cells. RAGE activation inhibited myocardin-induced expression of the SMC genes in mouse embryonic mesenchymal C3H10T1/2 cells. Interestingly, RAGE activation induced alkaline phosphatase (ALP) expression, calcium deposition, and 2 expression, a crucial transcription factor for osteogenic differentiation, in HASMC. RAGE-induced osteogenic differentiation was significantly inhibited by endogenous secretory RAGE. RAGE-induced ALP and 2 expression was completely abrogated by DAPT, an inhibitor of the Notch signaling pathway. PD98059 (MEK inhibitor) effectively blunted RAGE-induced Notch1 and 2 gene expression. Simultaneous stimulation with bone morphogenetic protein 2 (BMP2) and RAGE signaling synergistically induced expressions of 2 and ALP in HASMC. Immunohistochemistry revealed that the human calcifying atherosclerotic plaque expressed RAGE, Notch components and 2. The ALP activity induced in RAGE-overexpressing HASMCs by human serum was positively correlated with the serum creatinine level, but not with phosphate and hemoglobin A1c levels.
Conclusions: These results indicate that activation of RAGE not only inhibits myocardin-dependent SMC gene expression, but also induces osteogenic differentiation of vascular SMC through Notch/2 induction. These results provide a novel insight into the role of RAGE axis in vascular calcification.

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Quail-chick chimera experiments have shown a contribution of carnial neural crest cells to the craniofacial skeletal elements. Moreover, tissue interactions between epithelial-mesenchymal interaction during early facial process development are required for both skeletal differentiation and morphogenesis. In this study, it was observed that homeobox containing genes expressed in the facial process were important molecules of cartilage morphogenesis. Rat cDNAs were isolated and encoded by -1 and -2, and then the expression patterns using in situ hybridization were investigated during early rat face development. These genes were correlatively expressed in the cranial neural crest forming area (E 9.5 dpc) and the facial process (E 12.5 dpc) . Antisence inhibition of genes in the E12.5 mandibular process exhibited the alteration of their gene expression and cartilage patterns. Antisence inhibition of -1 induced lack of the medial portion of cartilage, and antisence inhibition of -2 enhanced chondrogenesis of mandibular process under the organ culture condition. Thus it was concluded that expression of genes during mandibular process development comprises important signals of chondrogenesis.

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我々は，EMT関連分子である2の膵腫瘍における役割を解析し，その悪性化への関与を検討した．2は膵癌細胞のEMTを誘導し膵癌の転移を促進していた．膵癌細胞における2の発現亢進は膵癌幹細胞数を増加させ，化学療法耐性能に関与していた．また，2はmiR126の発現を低下させEMTを誘導していた．2はIPMNの癌部と浸潤部に高頻度に発現しており，IPMNの進展に関与していることも明らかとなった．さらに，ERCP時に採取した膵管擦過細胞における2 mRNA量を測定すると，膵癌において慢性膵炎に比べ有意に高いことが確認された．以上のことから，膵腫瘍の進展に2の発現と，それによって誘導されたEMTが重要な役割を果たしていること，並びにEMT誘導分子の発現量測定による臨床応用の可能性が示唆された．

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Filamentous, non-heterocystous diazotrophic cyanobacterium Plectonema boryanum simultaneously photoproduced hydrogen (0.30μmol mg^-1 dry wth^-1) and ammonium (8.2nmol mg^-1 dry wth^-1) under microaerobic (A:CO₂, 96:4, v/v) conditions in the presence of 1mM sulfide. Nitrogen in the gas phase (A:CO₂:N₂, 72:4:24, v/v) enhanced the process more than two fold. A glutamine synthetase inhibitor, , stimulated ammonium excretion and H₂-production and pulses of rather than a single dose favoured both the processes. A higher dose (15μM) in comparison to lower dose (5μM) of , although it stimulated H₂-evolution and NH₄⁺-excretion, shortened the duration of both the processes. The stability of both the processes was prolonged in alginate-immobilized cyanobacterial cells besides enhancement in H₂-evolution and NH₄⁺-excretion. was less toxic under immobilized state than the free cells.

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Previously, we showed that HDAC1 prevents vascular calcification (VC), whereas its E3 ligase, MDM2 exaggerates it by inducing the polyubiquitination of HDAC1. We further investigated the roles of MDM2 in VC. We observed that VSMC-specific conditional knockout of Mdm2 blunted VC. TgMDM2 WT mice elicited VC, while TgMDM2 Y489A or TgMDM2 ΔR failed to do so. By promoter mapping analysis, we found that

-binding element at -1331～1327 upstream of transcription start site of MDM2 promoter is Pi-responsive element for the induction of MDM2 transactivation. Chromatin immunoprecipitation as well as mutant promoter analysis further confirmed that both 1 and 2 bound to and activated MDM2 promoter. Pi potentiated the binding of to its binding element of MDM2 promoter. Both 1 and 2 potentiated Pi-induced VC. -induced potentiation of Pi-mediated VC was attenuated by MDM2 siRNA. As an alternative key epigenetic regulator, non-coding RNAs are under extensive investigation in association with VC. By RNA sequencing, we found some candidate circular and long non-coding RNAs that are expected to affect the VC. Taken together, the epigenetic regulation of both /MDM2/HDAC1 axis and non-coding RNA participate in the development of VC, which will be novel therapeutic targets of the diseases.

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We cloned a porcine ortholog of homeodomain transcription factor from the porcine pituitary cDNA library. The amino acid sequence of 1 shows high conservation among mammalian species. RT-PCR for porcine fetal and postnatal pituitaries showed that is already expressed at early fetal day 40, decreases to a low level before birth and then remarkably decreases after birth. On the other hand, expression was observed in all pituitary-derived cell line tested, with most in a gonadotrope lineage LβT4. Transfection assay demonstrated that 1 markedly repressed the basal Cga and Fshb gene expression, while Lhb expression was affected slightly. Taken together, 1 may play a role in repressing gene expression in the fetal and postnatal periods.

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歯科矯正学的治療の免疫組織化学的基盤，すなわち歯科矯正学的メカニカルストレスを付与したマウスの歯根膜組織を用いて，その初期における病理組織学的変化を調べるとともに，骨芽細胞分化関連因子であるRunx2と2の免疫組織化学的発現の変化について展望した。当該の牽引側歯根膜線維芽細胞は，メカニカルストレスを受けた20分後には両者の強い発現があり，この傾向は時間の経過とともに増強していた。24時間後においては，歯根膜線維芽細胞，骨芽細胞，セメント芽細胞に強発現していた。また，ALPの発現も同様であった。以上の所見から，Runx2は初期の骨芽細胞への分化を誘導し，2はその際の促進因子として働く事が強く示唆された。

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Tooth agenesis is the congenital lack of permanent teeth, which is called oligodontia, when the number of missing teeth is 6 or more. Oligodontia affects more than 1 of 100 humans, but its pathogenesis is largely unknown. Tooth genesis depends on the complex interactions between environmental and genetic factors. The gene, a member of homeobox gene family, encodes a DNA-binding protein, which is involved in many epithelial-mesenchymal interactions, leading to vertebrate organogenesis, and appears to be most critical during early tooth development. The MSH1 gene has 2 exons, separated by an intron, and its mutations, such as missense or frame-shift mutations, have been reported to be associated with tooth agenesis. In the present study, we sequenced the gene of three unrelated patients with sporadic, non-syndromic oligodontia: 2 boys aged 8.5 and 15 years old and one girl aged 15.5 years old. We have thus identified a homozygotic deletion of 11 nucleotides in the intron, near the 5' splicing site, in two patients, who also carry a different exonic transition. The base changes we detected were not present in an open reading-frame of the gene, but the newly identified deletion of 11 nucleotides might interfere with the splicing of the gene. In contrast, the third patient, a 15-year boy, displayed no base change in the examined regions. Therefore, the identified 11-nucleotide deletion may decrease the expression level of the 1 protein, but the link with oligodontia needs further study.

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In recent years, many things have progressed and became clear in the study of genes involved in tooth development. However, the genes associated in the formation of periodontal tissues particular those studies involved in the formation of furcation are few. We have carried out a research to examine the gene expression of cementoblasts in the furcation area in porcine tooth germ. Porcine tooth germs were used as samples excised during the formation of mandibular molars and the cementoblasts around the root and at the furcation were prepared. Total RNA was isolated from the samples followed by PCR amplifying specific genes using PCR templates. Further, significant difference with respect to gene expression was determined by semi-quantitative PCR from the electrophoresis image. Also, after the removal of tooth germs, tissues were processed in routine tissue preparation, embedded in paraffin, sectioned into 5 um thickness, stained with H and E and examined under the light microscope.
Amelogenin, enamelin, ameloblastin, MMP-20, Klk-4 expressions were observed uniformly by the cementoblasts at the furcation of the tooth but only amelogenin and ameloblastin were observed by the cementoblasts around the root of the tooth. Also, a typical strong 2 homeobox gene by the cementoblasts at the furcation was observed with an expression level of approximately 64 times than the cementoblasts around the root.
The present study clearly showed that specific genes are involved in the formation of furcation. A strong 2 gene expression was observed in the tooth germ and even at the furcation suggesting that 2 plays an important role in the process of periodontal tissue formation and maturation of furcation.

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Epithelial to mesenchymal transition（EMT）は通常密に結合している上皮細胞がその極性を失って遊走能が亢進し間葉系細胞の形質を獲得する現象であり，上皮由来の癌が転移する際にも認められる．我々は，BMPシグナルの膵癌細胞に対するEMT誘導機構とその臨床応用の可能性について検討した．その結果，BMP4は膵癌細胞のEMTを誘導しその過程に標的遺伝子である2が必須であった．また，2それ自身が単独で膵癌のEMTを誘導し膵癌の転移を促進していた．さらに，ERCP時に採取した膵管擦過細胞における2 mRNA量を測定すると，膵癌において慢性膵炎に比べ有意に高いことが確認された．以上のことから，膵癌の進展にEMTが重要な役割を果たしていること，並びにEMT誘導シグナルに関与する分子の発現量測定による臨床応用の可能性が示唆された．

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Tooth agenesis is the failure of tooth bud development, causing definitive absence of the tooth. It is the most common dental anomaly, affecting up to one-quarter of the general population. The main cause is related to abnormal function of specific genes which play key roles during odontogenesis, particularly 1 and PAX9. 1 is a transcription factor highly expressed in the mesenchyme of developing tooth germs, whereas PAX9 is a transcription factor that shows a direct relationship with craniofacial development, particularly the formation of the palate and teeth. Despite the high frequency of tooth agenesis, there are as yet only a restricted number of mutations in 1 and PAX9 that have been associated with non-syndromic tooth agenesis. Thus, a deeper analysis of the gene networks underlying this anomaly is imperative. By means of a literature review based on Medline, PubMed, Lilacs, NCBI, and STRING, performed between 1991 and 2010 and focused on etiologically associated mutations, this work aimed to assess the latest advances in the genetic etiology of tooth agenesis and to offer an insight into how they can assist dental practice in the near future. A better knowledge of the genetic networks underlying tooth agenesis will lead to better treatment options and, perhaps, a tool for early diagnosis possibly related to DNA examination based on polymorphic variants. Such a test based on DNA analysis may be available to and accessible by clinicians, resulting in a more accurate diagnosis and allowing for a better approach to this anomaly.

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低価格・統一規格のパソコンに,ADコンバータやタイムカウンターを接続して,高校物理全般の実験で測定器として使う研究開発を行った.付加装置はカセットテープの大きさの基板にまとめ,ROMスロットに挿入して使う.プログラムはROM化して,オートスタートをかけることで,専用の測定器と同じ使い勝手とすることができた.各種センサーとプログラムにより,ほとんどの物理実験に有用な測定器として使える.

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In this paper, we examined the periodontal tissue reaction course of mice to mechanical stress according to the Waldo method, before examination of the transcription factor profile change. In the examination group, the arrangement of the periodontal ligament was irregular on specimen day 1. The extension and compression sites were observed at the opposite side of the roots. In day 1 and 3 specimens, the osteoclasts appeared in the compression sites. In day 7 specimens, the number of osteoclasts was reduced in number to less than that of day 3. Immunohistochemical examination revealed that the expression patterns of Runx2 and 2 were clearly dynamic changed compared to the control specimens. These results suggest that the appearance of transcription factors related to cell differentiation of periodontal ligament, which was due to the mechanical stress of insertion of elastic separator, happened within 24 hours.

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In this report, a basic principle of designing on effective man-machine interface is given to construct a speech training system based upon the information theory. Although the speech training system has been developed by our group, its evaluation in a field-testing indicated a need for correction in this system. In order to keep trainee's attention, a program on the display has to be attractive. Concerning these problems the newly designed inexpensive training system is constructed using the home computer 2 and the speech processing interface board. The signals are picked up by a microphone, and then pass through a low pass filter which cut-off frequency is controllable by the micro computer. The analogue signals are converted to digital ones by the A/D convertor. The signal processor (μPD 7720) is adopted to find the peaks of the power spectrum which is realized by the cascaded 4 signal processors arrangement. Using the 2 graphic functions, the result of this analysis is presented to trainee. This system could be utilized in the first three stage of speech training; basic vocalization;intensity, duration, pitch; pronounciation of specific vowels and dipthongs.

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ハイブリッド人工肝に適した人工肝細胞の構築を目的として, 遺伝子組換えにより動物細胞株にアンモニア除去能を付与することを試みた.組込み遺伝子として, 1段階反応でアンモニアを除去可能で, 遺伝子増幅現象を引き起こすグルタミン合成酵素遣伝子を選択した.SV40プロモーターを接続したグルタミン合成酵素遺伝子を組込んだpSV2-GSベクターを構築し, Chinese Hamster Ovary細胞に遺伝子導入した.得られた組換え細胞株をグルタミン合成酵素の阻害剤Methionine Sulfoximine () の存在下にて選択を行ない, グルタミン合成酵素活性の上昇した細胞株 (800μM 耐性株) を取得した.さらに, 得られた細胞株のアンモニア除去特性を検討した結果, 0, 1, 2mMいずれのアンモニア添加条件下においても培養中期 (増殖期) にアンモニア消費がおこり, そのアンモニア比消費速度の値は人工肝に汎用されている初代肝細胞の約1/5の値となった.

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