Novel genetic linkage of rat Sp6 mutation to Amelogenesis imperfecta

Background: Amelogenesis imperfecta (AI) is an inherited disorder characterized by abnormal formation of tooth enamel. Although several genes responsible for AI have been reported, not all causative genes for human AI have been identified to date. AMI rat has been reported as an autosomal recessive mutant with hypoplastic AI isolated from a colony of stroke-prone spontaneously hypertensive rat strain, but the causative gene has not yet been clarified. Through a genetic screen, we identified the causative gene of autosomal recessive AI in AMI and analyzed its role in amelogenesis. Methods: cDNA sequencing of possible AI-candidate genes so far identified using total RNA of day 6 AMI rat molars identified a novel responsible mutation in specificity protein 6 (Sp6). Genetic linkage analysis was performed between Sp6 and AI phenotype in AMI. To understand a role of SP6 in AI, we generated the transgenic rats harboring Sp6 transgene in AMI (Ami/Ami + Tg). Histological analyses were performed using the thin sections of control rats, AMI, and Ami/Ami + Tg incisors in maxillae, respectively. Results: We found the novel genetic linkage between a 2-bp insertional mutation of Sp6 gene and the AI phenotype in AMI rats. The position of mutation was located in the coding region of Sp6, which caused frameshift mutation and disruption of the third zinc finger domain of SP6 with 11 cryptic amino acid residues and a stop codon. Transfection studies showed that the mutant protein can be translated and localized in the nucleus in the same manner as the wild-type SP6 protein. When we introduced the CMV promoter-driven wild-type Sp6 transgene into AMI rats, the SP6 protein was ectopically expressed in the maturation stage of ameloblasts associated with the extended maturation stage and the shortened reduced stage without any other phenotypical changes. Conclusion: We propose the addition of Sp6 mutation as a new molecular diagnostic criterion for the autosomal recessive AI patients. Our findings expand the spectrum of genetic causes of autosomal recessive AI and sheds light on the molecular diagnosis for the classification of AI. Furthermore, tight regulation of the temporospatial expression of SP6 may have critical roles in completing amelogenesis

女子大学生の食事の満足感に与える要因の検討

本調査では､長崎県内の女子大学生384名を対象に食生活に関するｱﾝｹｰﾄ調査を実施し､L. Green ら1) の｢PRECEDE-PROCEED MODEL｣を参考に､準備要因､強化要因､実現要因､食行動､食情報､食事の満足感についての把握と食事の満足感を与える要因について検討を行い､以下の結論を得た｡(1) 準備要因､強化要因､実現要因､食行動､食情報､食事の満足感の各分野で望ましいまたは積極的な考え方を持つ者は､他分野でも望ましいまたは積極的な考え方を持っていた｡特に準備要因および食行動においてそれらの傾向が強くみられた｡(2) 強化要因､実現要因､食行動､食情報､食事の満足感において望ましいまたは積極的な考え方を持つことにより準備要因の食品選択行動のｾﾙﾌｴﾌｨｶｼｰおよび食行動のｾﾙﾌｴﾌｨｶｼｰに関しても望ましいまたは積極的な考え方を持つ傾向がみられた｡以上の結果より､食事の満足感を高めるためには､食情報に対して活用できる能力やｾﾙﾌｴﾌｨｶｼｰを高める食のｽｷﾙの向上を目指す教育の必要性が示されたものと考える｡A questionnaire was conducted on eating habits of 384 students at women\u27s colleges in Nagasaki Prefecture. The “PRECEDE-PROCEED MODEL” of L. Green et al was used as a reference to grasp predisposing factors, reinforcing factors, enabling factors, eating behaviors, food information, the feeling of satisfaction with meals and study factors that give the feeling. The following conclusions were obtained:(1) Those who had desirable or positive ideas about predisposing factors, reinforcing factors, enabling factors, eating behaviors, food information, and the feeling of satisfaction with meals also had desirable or positive ideas about other eating habits.(2) Desirable or positive ideas about reinforcing factors, enabling factors, eating behaviors, food information, and the feeling of satisfaction with meals tended to lead the students toward desirable or positive ideas about self-efficacy in such predisposing factors as food selection and eating behaviors.It is thought that the necessity of the education that aims at the improvement of the skill of food that improves the ability and self-efficacy that can use food information was shown to improve the satisfaction of the meal

Dissection of morphological and metabolic differentiation of amelobrasts via ectopic SP6 expression

Tooth enamel is the hardest organ in the body. In rodent incisor, the enamel is exclusively produced by ameloblasts with yellowish-brown pigmentation, indicating normal enamel formation. However, the molecular mechanisms of ameloblast differentiation and amelogenesis are not fully understood. Specificity protein (Sp) 6 has been reported as one of the critical factors for tooth development. To explore SP6 function, we generated Sp6 transgenic (Tg) rats. Unexpectedly, the enamel surfaces of the incisors in Tg rats were discolored, even though enamel formation and serum iron concentrations were normal. Histological analysis of incisors from 6-week-old Tg rats demonstrated that the ameloblast layer at the pigmentation stage was elongated up to the gingival margin with ectopic SP6 expression in longitudinal incisor sections. In contrast, the incisors from 10-week-old Tg rats revealed that the pigmented ameloblasts were morphologically changed to those of the reduced stage, concomitant with the sporadic disappearance of ectopic SP6 expression. Here we report that morphological differentiation and metabolism of the iron-containing pigment in ameloblasts are independently regulated during amelogenesis by means of ectopic SP6 expression

Isolation and Characterization of Mouse Specificity Protein 6 Promoter

Specificity protein 6 (SP6)is a member of the SP/Kruppel-like transcription factor family and plays key roles in tooth development. Tostudy its biological roles, it is important to understand the spatiotemporal regulation of Sp6gene expression. Forthis purpose, we first identified two separate 5\u27 ends of the Sp6cDNA by 5\u27 RACE analysis using mouse mandibular RNA. Next, we isolated mouse genomic DNA fragments covering the Sp6 gene including two putative mouse Sp6 promoter regions and generated a series of luciferase reporter constructs. We confirmed the activity of both promoters by a luciferase assay and found strong second promoter activity in dental epithelial cells. Unexpectedly,we also detected potential third promoter activity in the intron 2 of the Sp6 gene. Last,we also found that bone morphogenetic protein and wingless signals could enhance Sp6promoter activity in dental epithelial cells, suggestingthe regulatory roles of two cytokines in Sp6 gene expression during tooth development. Our findings may shed new light on the regulatory mechanisms of Sp6 gene expression and provide a possible linkage between cytokine regulation of Sp6 expression and inductive epithelial and mesenchymal interactions. Keywords:Gene expressionluciferase assaypromoter activitySp

Isolation and Characterization of Mouse Specificity Protein 6 Promoter

Specificity protein 6 (SP6) is a member of the SP/Krüppel-like transcription factor family and plays key roles in tooth development. To study its biological roles, it is important to understand the spatiotemporal regulation of Sp6 gene expression. For this purpose, we first identified two separate 5' ends of the Sp6 cDNA by 5' RACE analysis using mouse mandibular RNA. Next, we isolated mouse genomic DNA fragments covering the Sp6 gene including two putative mouse Sp6 promoter regions and generated a series of luciferase reporter constructs. We confirmed the activity of both promoters by a luciferase assay and found strong second promoter activity in dental epithelial cells. Unexpectedly, we also detected potential third promoter activity in the intron 2 of the Sp6 gene. Last, we also found that bone morphogenetic protein and wingless signals could enhance Sp6 promoter activity in dental epithelial cells, suggesting the regulatory roles of two cytokines in Sp6 gene expression during tooth development. Our findings may shed new light on the regulatory mechanisms of Sp6 gene expression and provide a possible linkage between cytokine regulation of Sp6 expression and inductive epithelial and mesenchymal interactions

Isolation and Characterization of Mouse Specificity Protein 6 Promoter

Specificity protein 6 (SP6) is a member of the SP/Krüppel-like transcription factor family and plays key roles in tooth development. To study its biological roles, it is important to understand the spatiotemporal regulation of Sp6 gene expression. For this purpose, we first identified two separate 5' ends of the Sp6 cDNA by 5' RACE analysis using mouse mandibular RNA. Next, we isolated mouse genomic DNA fragments covering the Sp6 gene including two putative mouse Sp6 promoter regions and generated a series of luciferase reporter constructs. We confirmed the activity of both promoters by a luciferase assay and found strong second promoter activity in dental epithelial cells. Unexpectedly, we also detected potential third promoter activity in the intron 2 of the Sp6 gene. Last, we also found that bone morphogenetic protein and wingless signals could enhance Sp6 promoter activity in dental epithelial cells, suggesting the regulatory roles of two cytokines in Sp6 gene expression during tooth development. Our findings may shed new light on the regulatory mechanisms of Sp6 gene expression and provide a possible linkage between cytokine regulation of Sp6 expression and inductive epithelial and mesenchymal interactions

女子大学生の食生活に与える要因の検討

長崎県内の女子大学生384名を対象に､食生活に関するｱﾝｹｰﾄ調査を実施し､女子大学生の食生活に与えている影響について探り､食生活に与える要因の検討を行った｡(1) 食生活について因子分析を行った結果､｢栄養･食情報の活用｣､｢食事の満足感｣､｢食意識｣の3つの因子が得られた｡(2) 因子の関連については､｢食事の満足感｣と｢食意識｣､｢栄養･食情報の活用｣と｢食事の満足感｣､｢栄養･食情報の活用｣と｢食意識｣の順に強い関連となる傾向が認められた｡特に｢食事の満足感｣は､他の2つの因子との関連が相対的に強くなっていた｡(3) 潜在変数間及び潜在変数と観測変数の関連の因果ﾓﾃﾞﾙを作成した結果､第1因子の｢栄養･食情報の活用｣は､｢栄養成分表示の利用｣に大きく影響を与えていたほか､｢食情報の獲得能力の有無｣｢献立作成｣､｢食品の安全性の確認｣に影響を与えていた｡第2因子の｢食事の満足感｣は､｢食事時間の楽しさ｣や｢食事の待ち遠しさ｣および｢食事内容の満足感｣｢食べたいものを食べている｣に影響を与えていた｡第3因子の｢食意識｣は､｢現在の食生活｣に正の大きな影響を､｢食生活改善意欲｣は負の影響を与え､｢健康への配慮｣には､正の弱い影響を与えていた｡以上の結果より､食生活において､食事の満足感は､単に食欲を満たすだけでなく､精神的な豊かさをもたらす食事時間の楽しさにより得ると考えられた｡また､食事の満足感は､栄養･食情報の活用によるｾﾙﾌｴﾌｨｶｼｰや食生活を振り返るなどの食意識との関連が明確となった｡A questionnaire on dietary life was conducted with 384 college women in Nagasaki Prefecture to search for and study factors that influenced their dietary life.(1) A factor analysis on dietary life yielded the following three factors:“utilization of information on nutrition and food”,“feeling of satisfaction with meals”, and“awareness of diet”. (2) With respect to relationships among the factors, there was a strong tendency of correlations between“feeling of satisfaction with meals”and“awareness of diet”, between“utilization of information on nutrition and food”and“feeling of satisfaction with meals”, and between“utilization of information on nutrition and food”and“awareness of diet”in the descending order.“Feeling of satisfaction with meals”in particular was strongly correlated with the other two factors.(3) A cause and effect model was prepared to relate the three factors. As a result, factor 1,“utilization of information on nutrition and food”, was found to exert a great influence on“utilization of nutrient labels”. It also influenced“acquisition of food-related information”,“menu preparation”, and“confirmation of food safety”. Factor 2,“feeling of satisfaction with meals”, was found to influence“enjoying meal time”,“eagerly waiting for meals”,“feeling of satisfaction with meal contents”, and“eating what they want to eat”. Factor 3,“awareness of diet”, exerted a great positive influence on“judgment of present dietary life”, a negative influence on“intention to improve dietary life”, and a weak positive influence on“attention to health”.From the above results, the feeling of satisfaction with meals in dietary life was inferred to originate not only from the satisfaction of appetite but also from the perception of pleasure at meal time, which enriches spirit. In addition, the feeling of satisfaction with meals was clearly related to the sense of self-efficacy resulting from the utilization of information on nutrition and food and to the awareness of diet in looking back on one,s dietary life

大学生の児童期の家庭での食教育が現在の食生活に与える影響

長崎県内の大学生 389名を対象に､大学生の児童期(小学校高学年時)に家庭で受けた食に対してのしつけや教育及び食環境､食生活状況について調査した｡あわせて､現在の食生活実態調査を行い､児童期の家庭での食生活が大学生の現在の食生活への影響について分析し､以下のような結果を得た｡1)児童期における家庭での食に関わるしつけが大学生になっての食習慣や食行動が良い傾向になる関連性を示唆した｡2)因子分析の結果､第1因子｢食事での健康への配慮｣､第2因子｢現在の食生活の改善意欲｣､第3因子｢食事づくりへの興味｣､第4因子｢現在の健康観｣､第5因子｢児童期の家庭の食環境｣の5つの因子が抽出され､現在の食生活に児童期の食生活の影響は小さいながら影響を及ぼす要素であることを示した｡本調査から､児童期の食環境が現在の大学生の食生活や生活習慣と関連がある可能性が示された｡特に児童期の食行動や食習慣は家庭における保護者の意識が影響していることが示唆した｡過去の食環境は未来の健康な食習慣の確立や QOL の向上に影響すると考えられ､そのために児童期の家庭の食環境を充実させることが必要であると考えられた｡We surveyed the disciplines and education on eating learned at families during childhood(at a higher grade of a primary school)of university students as well as the food environment and the circumstances related to eating habits, for 389 students residing in Nagasaki Prefecture. At the same time, we investigated the actual situations of eating habits of the present to analyze the effect of eating habits at families during childhood upon the present eating habits of university students, and obtained the following results: 1)It was suggested that the discipline given at families on eating habits during childhood would have a relationship with the tendency to make the eating habit and behavior better at the time when grown to university students. 2)As a result of the factor analysis, the 5 factors, i.e. 1st factor of“a concern of eating for health”, 2nd factor of“desire for improved eating habits of the present”, 3rd factor of“interest in cooking”, 4th factor of“present view on health”and 5th factor of“eating environment at family during childhood”, were extracted, which showed that, although the effect of eating habits at childhood upon the present dietary life was small, it still constituted an influencing element. From this survey, it was suggested that the eating environment at childhood has a potential relationship with the dietary life and eating habits of the university students of the present. The eating behavior and habits dwelt particularly at childhood was suggested to be influenced by the conscious attitude of a guardian at a family. As the eating environment in the past is thought to influence the establishment of healthy eating habits and the improvement of QOL in the future, it may be necessary to enrich the eating environment at a family during childhood

Sp6 downregulation of follistatin gene expression in ameloblasts

Sp6 is a member of the Sp family of transcription factors that regulate a wide range of cellular functions, such as cell growth and differentiation. Sp6, also called epiprofin, is specifically expressed in tooth germ, limb bud, and hair follicle, but there is little information on its function. To investigate the possible role of Sp6 in tooth development, first we established an Sp6- overproducing clone, CHA9, and analyzed the features of the cell, including cell proliferation and gene expression. The parental cells of CHA9 are the ameloblast-lineage G5 cells that we previously established from rat dental epithelia of lower incisor. Sp6 overproduction accelerated cell proliferation and induced the expression of ameloblastin mRNA, a marker of ameloblast differentiation. Second, we performed genome-wide screening of Sp6 target genes by microarray analysis. Out of a total 20,450 genes, 448 genes were up-regulated and 500 genes were down-regulated by Sp6. We found the expression of follistatin, a BMP antagonist, to be 22.4-fold lower in CHA9 than in control cells. Transfection of the Sp6-antisense construct into CHA9 cells restored follistatin expression back to equivalent levels seen in control cells, indicating that Sp6 regulates follistatin gene expression in ameloblasts. Our findings demonstrate that the follistatin gene is one of the Sp6 target genes in ameloblasts and suggest that Sp6 promotes amelogenesis through inhibition of follistatin gene expression

Isolation and Characterization of Mouse Specificity Protein 6 Promoter

Specificity protein 6 (SP6) is a member of the SP/Krüppel-like transcription factor family and plays key roles in tooth development. To study its biological roles, it is important to understand the spatiotemporal regulation of Sp6 gene expression. For this purpose, we first identified two separate 5\u27 ends of the Sp6 cDNA by 5\u27 RACE analysis using mouse mandibular RNA. Next, we isolated mouse genomic DNA fragments covering the Sp6 gene including two putative mouse Sp6 promoter regions and generated a series of luciferase reporter constructs. We confirmed the activity of both promoters by a luciferase assay and found strong second promoter activity in dental epithelial cells. Unexpectedly, we also detected potential third promoter activity in the intron 2 of the Sp6 gene. Last, we also found that bone morphogenetic protein and wingless signals could enhance Sp6 promoter activity in dental epithelial cells, suggesting the regulatory roles of two cytokines in Sp6 gene expression during tooth development. Our findings may shed new light on the regulatory mechanisms of Sp6 gene expression and provide a possible linkage between cytokine regulation of Sp6 expression and inductive epithelial and mesenchymal interactions