Determination of the Hydroxyapatite-Nucleating Region of Bone Sialoprotein

Bone sialoprotein (BSP) was shown to be a potent nucleator of hydroxy apatite (HA) in a steady-state agarose gel system (Hunter and Goldberg, 1993, PNAS 90: 8562). Nucleation of HA was also demonstrated with the homopolymer poly-glutamic acid but not with poly-aspartic acid or osteopontin. Since BSP contains contiguous sequences of glutamic acid, it is reasonable to suggest that the HA-nucleating activity of BSP resides within these regions. Purified porcine BSP was treated with trypsin and digests fractionated by gel filtration. In addition to small peptides (P3–5), two peptides of 38 kDa (PI) and 25 kDA (P2) were recovered, and after characterization assigned to the regions within BSP encompassing residues 133–272 (PI) and 42–125 (P2). Each of these peptides contained one of the two glutamic acid-rich regions of porcine BSP. In the steady-state agarose gel system, BSP, PI and P2 induced HA formation, whereas the pooled small BSP-derived peptides (P3–5) did not. Analysis by circular dichroism spectroscopy revealed that the homopolymer poly-L-glutamic acid assumes a helical structure, while poly-L-aspartic acid does not. These findings suggest that the nucleating activity does not require intact molecules, that the nucleation of HA by BSP appears to require glutamic acid-rich sequences in a helical conformation and that there are two domains in porcine BSP that are each capable of nucleating HA.     

Binding of Bone Sialoprotein,Osteopontin and Synthetic Polypeptides to Hydroxyapatite

The phosphorylated acidic glycoproteins bone sialoprotein (BSP) and osteopontin (OPN) bind to hydroxyapatite (HA) crystals and may be involved in the regulation of bone mineralization. The HA-binding properties of these proteins have been attributed to glutamic acid-rich sequences in BSP and aspartic acid-rich sequences in OPN. The present study examines the roles of these polycarboxylate sequences in the binding of BSP and OPN to HA.

Porcine BSP, OPN and the synthetic polypeptides poly-L-glutamic acid [Poly(Glu)] and poly-L-aspartic acid [Poly(Asp)] were labeled with fluorescein isothiocyanate and their binding to HA determined by fluorimetry. From the binding isotherms, dissociation constants (K_Ds) for all the reagents tested were determined to be in the micromolar range. The saturation binding capacities of HA for Poly(Glu), Poly(Asp), BSP and OPN were similar (500–600 μg/m²). To investigate the role of glutamic acid-rich and aspartic acid-rich sequences in the binding to HA of BSP and OPN, respectively, competitive binding studies with Poly(Glu) and Poly(Asp) were performed. Poly(Glu) was able to displace a maximum of 100% of Poly(GIu), 81% of OPN, 68% of BSP and 65% of Poly(Asp). Poly(Asp) was able to displace a maximum of 100% of Poly(Glu), 99% of Poly(Asp), 95% of OPN and 89% of BSP. These results are consistent with the view that BSP and OPN bind to HA via their polycarboxylate sequences, but suggest a complex mode of interaction between polyelectrolytes and ionic crystals.     

Role of Two Mineral-Associated Adhesion Molecules,Osteopontin and Bone Sialoprotein,during Cementogenesis

Adhesion molecules and their cell membrane receptors are known to play important regulatory roles in cell differentiation. Consequently, the following experiments were conducted to determine the role of two adhesion molecules, bone sialoprotein (BSP) and osteopontin (OPN) in tooth root formation. Developing murine molar tooth germs at sequential stages of development (developmental days 21–42) were analyzed using immunohistochemical and in situ hybridization techniques. While BSP was localized to alveolar bone and odontoblasts early in development, BSP was distinctly localized to the cemental root surface at latter periods coincident with the initiation of root formation and cementogenesis. Conversely, OPN was distributed in a nonspecific fashion throughout the PDL and the eruption pathway of the forming tooth. In situ hybridization confirmed that cells lining the root surface express BSP. The fact that BSP is specifically localized to the cemental surface suggests that this protein is involved in cementoblast differentiation and/or early mineralization of the cementum matrix. Localization of OPN to non-mineralized tissues further suggests that OPN functions as an inhibitor of mineralization during periodontal ligament formation. These findings collectively suggest that BSP and OPN are intimately involved in the sequence of cellular and molecular events accompanying cementogenesis.     

Generation and Use of Recombinant Human Bone Sialoprotein and Osteopontin for Hydroxyapatite Studies

Bone sialoprotein (BSP) and osteopontin (OPN) are two extracellular bone matrix proteins that have the ability to modulate the growth of hydroxyapatite in vitro. Studies of BSP/OPN hydroxyapatite interactions in the past have been directed toward the identification of essential structural elements that allow these two proteins to modulate hydroxyapatite growth. However, these studies are limited by the finite quantities of purified extracellular matrix proteins. I have utilized a recombinant E. coli expression system to obtain milligram quantities of human bone sialoprotein and human osteopontin that may be used to study extracellular matrix protein interactions with hydroxyapatite.     

Evidence of Steroid Receptors in Human Osteoblast-Like Cells

A nuclear binding assay was used to demonstrate steroid receptors in normal human osteoblast-like cells. Nuclear binding of [³H] estradiol was found in 27 of 30 (90%) cell strains and nuclear binding of [³H] androgen was identified in 21 of 25 (84%) separate osteoblast cell strains. The nuclear binding was saturable and steroid-specific. Estrogen and androgen receptor gene expression was confirmed by RNA blot analysis. These data suggest that circulating sex steroids may act directly on normal human osteoblasts through receptor-mediated mechanisms.     

骨形成蛋白介导的基因治疗研究进展

基因治疗是指通过导入基因的功能片段改善机体生理状况或者治疗疾病.利用基因治疗可在骨缺损或骨折局部释放骨形成蛋白（bone morphogenetic proteins,BMP）,并且无需异体载体,方法包括体内法和离体法.BMP基因治疗可以促进骨和软骨形成、脊柱融合、颌面骨和牙齿修复、肌腱韧带形成,此外对椎间盘退变性疾病也可采用BMP基因治疗.总之BMP基因治疗方法经济有效,是有前景的治疗手段.     

uPA基因表达调控研究进展

uPA的高表达与肿瘤侵袭转移密切相关。研究表明在乳腺癌、肺癌、结肠癌等肿瘤组织中都有uPA的高表达。已经证实细胞内uPA蛋白合成增加主要由基因的转录水平引起 ,uPA基因启动子上含有AP1、NFκB、PEA3等增强子元件 ,可与相应转录因子结合增强uPA的基因转录 ,许多调节分子如细胞内cAMP、生长因子和佛波酯等都是通过核内转录因子起调节作用的。另外肿瘤细胞内外信号传导通路的某种改变也导致了uPA表达的增高 ,促进细胞浸润转移。     

应激状态下环加氧酶-2基因表达调控的研究进展

环加氧酶-2是体内催化花生四烯酸合成前列腺素的关键酶。它在细胞因子、有丝分裂原、生长因子等诱导刺激下可以大量表达,并在炎症和肿瘤的发生发展过程中发挥着重要作用。近年来的研究表明,应激状态下,环加氧酶-2转录增强和(或)mRNA稳定性增强,导致其蛋白表达量增加及产物前列腺素增多,从而引起机体一系列的生理和病理反应。     

基因突变和转基因技术评价TLR-4信号受体在流体切应力诱导血管内皮细胞IL-8基因转录激活中的作用

用基因突变和转基因技术，评价TLR-4信号受体在层流低切应力刺激诱导血管内皮细胞IL-8基因转录激活中的作用，RT-PCR，Northern杂交和免疫荧光细胞化学染色均显示脐静脉血管内皮细胞表达TLR-4，同时RT-PCR和Northern杂交显示，层流切应力刺激1h后血管内皮细胞TLR-4表达增强，用RT-PCR技术从血管内皮细胞扩增出胞内区段缺失突变TLR-4cDNA，用PCR技术从其基因组DNA中扩增出IL-8上游调控序列，分别克隆于真核表达质粒pcDNA3和绿色荧光增强蛋白报告基因pEGFP1质粒，构建出重组TLR-4缺失突变基因真核表达质粒pcDNA3-mTLR4和IL-8报告基因表达质粒pEGFP1-IL8USCS。用pEGFP1-IL8USCS转染或pcD-NA3-mTLR4和pEGFP1-IL8USCS共转染ECV304细胞，4.2dyne/cm^2层流切应力刺激3h后，流式细胞仪观察荧光蛋白表达强度变化，用pEGFP1-IL8USCS转染细胞，经层流切应力刺激3h后荧光蛋白表达增强（1.06:2.71)，同样用pcDNA3-mTLR4和pEGFP1-IL8USCS共转染细胞，层流切应力刺激3h后荧光蛋白表达未明显增强，提示TLR4/NF-кB信号传导通路可能介导层流切应力诱导血管内皮细胞IL-8基因的表达。     

人牙髓细胞骨基质蛋白表达的研究进展

骨基质蛋白在骨骼和牙齿形成，以及损伤修复过程中起着重要作用。本对骨涎蛋白(BSP)、骨桥蛋白(OPN)、牙本质涎蛋白(DSP)、牙本质磷蛋白(DPP)、牙本质涎磷蛋白(DSPP)的表达，以及在牙齿形成、损伤修复，对组织工程中成骨组织作用等研究进展进行了概述。     

Regulation of gene expression dynamics during developmental transitions by the Ikaros transcription factor

The DNA-binding protein Ikaros is a potent tumor suppressor and hematopoietic regulator. However, the mechanisms by which Ikaros functions remain poorly understood, due in part to its atypical DNA-binding properties and partnership with the poorly understood Mi-2/NuRD complex. In this study, we analyzed five sequential stages of thymocyte development in a mouse strain containing a targeted deletion of Ikaros zinc finger 4, which exhibits a select subset of abnormalities observed in Ikaros-null mice. By examining thymopoiesis in vivo and in vitro, diverse abnormalities were observed at each developmental stage. RNA sequencing revealed that each stage is characterized by the misregulation of a limited number of genes, with a strong preference for stage-specific rather than lineage-specific genes. Strikingly, individual genes rarely exhibited Ikaros dependence at all stages. Instead, a consistent feature of the aberrantly expressed genes was a reduced magnitude of expression level change during developmental transitions. These results, combined with analyses of the interplay between Ikaros loss of function and Notch signaling, suggest that Ikaros may not be a conventional activator or repressor of defined sets of genes. Instead, a primary function may be to sharpen the dynamic range of gene expression changes during developmental transitions via atypical molecular mechanisms that remain undefined.     

产单核细胞李斯特菌诱导胸腺细胞凋亡及其基因调控

为研究产单核细胞李斯特菌 (LM )感染对小鼠胸腺细胞凋亡的诱导作用及胸腺细胞凋亡过程中的基因调控 ,小鼠经尾静脉注射LM后 ,以DNA琼脂糖凝胶电泳和流式细胞仪 (FCM )检测细胞凋亡及凋亡细胞的基因产物表达水平。结果表明LM能诱导小鼠胸腺细胞凋亡 ,琼脂糖凝胶电泳分析显示胸腺细胞出现典型的DNA“梯状带” ,FCM分析显示特征性的凋亡峰。胸腺细胞凋亡于LM (5× 10 5CFU )感染后 8h出现 ,48h达高峰。胸腺细胞凋亡百分率随LM感染剂量增加而增高。LM诱导小鼠胸腺细胞凋亡中p5 3、Bax及c myc基因表达产物明显增加 ,而Bcl 2基因表达产物水平无明显改变。提示LM以时间和剂量依赖方式诱导小鼠胸腺细胞凋亡 ,p5 3、Bax及c myc基因在LM诱导小鼠胸腺细胞凋亡的基因调控中起重要作用。     

采用粒子群优化的基因转录差异分析模型

高通测序技术可以更加全面地检测实验样本中基因转录水平,这使得对样本间基因转录差异的分析精度越来越准确。根据通过DNA高通测序技术获得的基因转录区内PolⅡ蛋白个数,提出了两个样本间基因转录差异分析模型。该模型在考虑同一基因间转录差异的同时,引入了全基因的转录分布特性以提高模型分析精度。模型采用预测概率最大化统计算法进行参数求取。在概率最大化步骤中,由于难以得到模型参数解析解和提高算法效率,采用粒子群优化算法直接进行求取模型参数数值解。乳腺癌实验数据测试表明,该模型可有效分析不同样本间基因转录差异。     

Regulation of the Caenorhabditis elegans posterior hox gene egl‐5 by microRNA and the polycomb‐like gene sop‐2

In Caenorhabditis elegans, the domains of Hox gene expression are controlled by the novel global regulatory gene sop‐2. We identified a region located 3′ of the Hox gene egl‐5 that promotes ectopic expression of an egl‐5 reporter gene in a sop‐2 mutant. SOP‐2 could directly block positive regulatory factors acting in this region, or it could block their expression. We identified three possible miRNA binding sites within the egl‐5 3′ untranslated region (UTR). Cognate microRNAs are expressed in relevant tissues and can block egl‐5 expression when expressed from a transgene. Mutation of the putative binding sites in the egl‐5 3′UTR resulted in a modest degree of misexpression of a minimal egl‐5 reporter gene, suggesting that microRNAs may contribute to the tight restriction of egl‐5 expression to particular cell lineages. Developmental Dynamics 238:595–603, 2009. © 2009 Wiley‐Liss, Inc.