转录因子Osterix对成骨细胞Col1a1基因的反式激活作用

Osterix（Osx）是一种具有锌指结构的转录因子,对骨形成十分重要. 但到目前为止,直接接受Osterix调控的靶基因尚不清楚.用骨形态发生蛋白2（bone morphogenetic protein 2,BMP2）诱导原代培养小鼠成骨细胞的骨分化,定量RT PCR检测Ⅰ型胶原蛋白(collagen Ⅰ a 1, Col1a1)与Osx的转录水平.结果发现,二者的转录时相具有相同的变化模式.为了确定二者之间的关系,采用腺病毒表达系统在原代成骨细胞中过表达Osx. 数据表明,Osx能够明显上调Col1a1的转录水平.用EMSA（electromobility shift assay）检测这些过表达Osx基因的成骨细胞核抽提物,迁移条带的出现表明,Osx能够直接与Col1a1的启动子相结合.结果提示,在原代培养的成骨细胞中,Osterix通过直接与启动子相结合,调控Col1a1基因的转录.     

A new family with autosomal dominant porencephaly with a novel Col4A1 mutation. Are arachnoid cysts related to Col4A1 mutations?

Porencephaly is an extensively encountered condition in pediatric neurology practice and leads to serious morbidity with its complications. Important etiological factors are trauma, hemorrhage, infection and thrombophilic factors that may cause destruction in the developing brain. Col4A1 mutations were also shown in familial porencephaly cases. We describe two siblings with porencephaly, hemiparesis, epilepsy, atrophic kidney in one of the siblings and asymptomatic mothers with an arachnoid cyst. We performed Col4A1 gene mutation screening and detected a novel mutation in mother and both of the children. This family has some features previously undescribed in patients with mutations of Col4A1 gene like atrophic kidney in one sibling and arachnoid cyst in the mother. We discuss here the possible relationship between these abnormalities and the mutation.     

猪肌肉组织Col3a1基因表达的发育性变化及其对肌内胶原特性的影响

以30—90妇体重莱芜猪和40—100kg体重鲁莱黑猪共84头去势公猪为试验对象（每组6头）,采用相对定量RT-PCR方法,以β-actin作为内标,研究肌肉中编码Ⅲ型胶原的Col3al基因表达的发育性变化及其对肌肉中胶原蛋白含量和性质（溶解度）的影响。结果表明：研究的两个品种猪肌肉中Col3al基因表达的发育性变化基本一致,即随体重的增加,肌肉中Col3al mRNA表达呈逐渐增加趋势,但莱芜猪和鲁莱黑猪分别在70妇和80妇体重组表达量略有下降。总体上莱芜猪肌肉组织Col3al mRNA表达丰度显著高于鲁莱黑猪（P〈0．05）。相关分析表明,莱芜猪肌肉组织Col3al mRNA表达的发育性变化与总胶原和不溶性胶原含量呈极显著正相关（P〈0．01）,与胶原溶解度呈极显著负相关（P〈0．01）。鲁莱黑猪肌肉组织Col3al mRNA表达的发育性变化与不溶性胶原和胶原溶解度分别呈显著正相关和负相关妒〈0．05）。研究结果提示：猪肌肉组织中Col3al基因表达具有明显的体重发育和品种特征,其mRNA表达对于肌内胶原的含量和性质有重要影响。     

小鼠Bcl2a1a蛋白的生物信息学预测分析

目的:分析小鼠Bcl2a1a全长基因的核苷酸序列及其编码蛋白的氨基酸序列,利用软件预测其蛋白的二、三级结构与特征。方法:运用生物信息学相关软件分析和预测人类BCL2A1和小鼠Bcl2a1a基因的同源区段,预测小鼠Bcl2a1a基因的启动子区域及蛋白跨膜区域与信号肽;利用软件模拟生成蛋白三级结构图像,并了解小鼠Bcl2a1a蛋白与其他蛋白的相互作用关系。结果:小鼠Bcl2a1a基因全长5497 bp,编码的蛋白含有172个氨基酸残基,相对分子质量为460 087.23,属于不稳定的疏水性蛋白。小鼠Bcl2a1a基因与人类BCL2A1基因的同源区域在≥200 bp的位置。2个软件预测的小鼠Bcl2a1a基因启动子最可能在3439～3543 bp和4600 bp,但由于没有预测到CpG岛存在,所以结果准确率较低。小鼠Bcl2a1a蛋白无跨膜结构域,无信号肽;在二级结构预测中,α螺旋为Bcl2a1a蛋白的主要折叠形式;该蛋白仅含有1个BCL结构域,且与Bbc3、Apaf1、Bcl2l2、Trp53、Bak1、Bid、Nfkb1、Jun、Rel、Rela等蛋白形成相互作用网络。结论:Bcl2a1a基因及蛋白的生物信息学分析为相关研究奠定了重要的信息基础。     

猪肌肉组织Col3a1基因表达的发育性变化及其对肌内胶原特性的影响(英文)

以30~90kg体重莱芜猪和40~100kg体重鲁莱黒猪共84头去势公猪为试验对象(每组6头),采用相对定量RT-PCR方法,以β-actin作为内标,研究肌肉中编码Ⅲ型胶原的Col3a1基因表达的发育性变化及其对肌肉中胶原蛋白含量和性质(溶解度)的影响。结果表明:研究的两个品种猪肌肉中Col3a1基因表达的发育性变化基本一致,即随体重的增加,肌肉中Col3a1 mRNA表达呈逐渐增加趋势,但莱芜猪和鲁莱黑猪分别在70kg和80kg体重组表达量略有下降。总体上莱芜猪肌肉组织Col3a1 mRNA表达丰度显著高于鲁莱黑猪(P<0.05)。相关分析表明,莱芜猪肌肉组织Col3a1 mRNA表达的发育性变化与总胶原和不溶性胶原含量呈极显著正相关(P<0.01),与胶原溶解度呈极显著负相关(P<0.01)。鲁莱黑猪肌肉组织Col3a1 mRNA表达的发育性变化与不溶性胶原和胶原溶解度分别呈显著正相关和负相关(P<0.05)。研究结果提示:猪肌肉组织中Col3a1基因表达具有明显的体重发育和品种特征,其mRNA表达对于肌内胶原的含量和性质有重要影响。     

К ФАУНЕ КОРОЕДОВ(Col.Ipidae)КИТАЙСКОЙ НАРОДНОЙ РЕСПУБЛИКИ

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Col1A1 Production and Apoptotic Resistance in TGF-β1-Induced Epithelial-to-Mesenchymal Transition-Like Phenotype of 603B Cells

Recent studies have suggested that proliferating cholangiocytes have an important role in the induction of fibrosis, either directly via epithelial-to-mesenchymal transition (EMT), or indirectly via activation of other liver cell types. Transforming growth factor beta 1 (TGF-β1), a critical fibrotic cytokine for hepatic fibrosis, is a potent EMT inducer. This study aimed to clarify the potential contributions of TGF-β1-induced EMT-like cholangiocyte phenotype to collagen production and cell survival of cholangiocytes in vitro. Mouse cholangiocytes (603B cells) were treated with TGF-β1 and EMT-like phenotype alterations were monitored by morphological changes and expression of EMT-associated genes. Alterations in Col1A1 gene, Col1A1-associated miR-29s, and pro-apoptotic genes were measured in TGF-β1-treated 603B cells. Snail1 knockdown was achieved using shRNA to evaluate the contribution of EMT-associated changes to Col1A1 production and cell survival. We found TGF-β1 treatment induced partial EMT-like phenotype transition in 603B cells in a Snail1-dependent manner. TGF-β1 also stimulated collagen α1(I) expression in 603B cells. However, this induction was not parallel to the EMT-like alterations and independent of Snail1 or miR-29 expression. Cells undergoing EMT-like changes showed a modest down-regulation of multiple pro-apoptotic genes and displayed resistance to TNF-α-induced apoptosis. TGF-β1-induced apoptosis resistance was attenuated in Snail1 knockdown 603B cells. TGF-β1-induced Col1A1 production seems to be independent of EMT-like transition and miR-29 expression. Nevertheless, TGF-β1-induced EMT may contribute to the increased survival capacity of cholangiocytes via modulating the expression of pro-apoptotic genes.     

2′-Deoxy-1-methylpseudocytidine,a stable analog of 2′-deoxy-5-methylisocytidine

2′-Deoxy-5-methylisocytidine is widely used in assays to personalize the care of patients infected with HIV, hepatitis C, and other infectious agents. However, oligonucleotides that incorporate 2′-deoxy-5-methylisocytidine are expensive, because of its intrinsic chemical instability. We report here a C-glycoside analog that is more stable and, in oligonucleotides, pairs with 2′-deoxyisoguanosine, contributing to duplex stability about as much as a standard 2′-deoxycytidine and 2′-deoxyguanosine pair.     

1∶1 and 2∶1 phase entrainment in a system of two coupled limit cycle oscillators

A model of a pair of coupled limit cycle oscillators is presented in order to investigate the extent of, and the transition between, 11 and 21 phase entrainment, a phenomenon exhibited by numerous diverse biological systems. The mathematical form of the model involves a flow on a phase torus given by two coupled first order nonlinear ordinary differential equations which govern the oscillators' phase angles (i.e. their respective positions around their limit cycles). The regions corresponding to 11 and 21 phase entrainment in an appropriate parameter space are determined analytically and numerically. The bifurcations occurring during the transition from 11 to 21 phase entrainment are discussed.     

Cdk1 and Plk1 mediate a CLASP2 phospho-switch that stabilizes kinetochore–microtubule attachments

Accurate chromosome segregation during mitosis relies on a dynamic kinetochore (KT)–microtubule (MT) interface that switches from a labile to a stable condition in response to correct MT attachments. This transition is essential to satisfy the spindle-assembly checkpoint (SAC) and couple MT-generated force with chromosome movements, but the underlying regulatory mechanism remains unclear. In this study, we show that during mitosis the MT- and KT-associated protein CLASP2 is progressively and distinctively phosphorylated by Cdk1 and Plk1 kinases, concomitant with the establishment of KT–MT attachments. CLASP2 S1234 was phosphorylated by Cdk1, which primed CLASP2 for association with Plk1. Plk1 recruitment to KTs was enhanced by CLASP2 phosphorylation on S1234. This was specifically required to stabilize KT–MT attachments important for chromosome alignment and to coordinate KT and non-KT MT dynamics necessary to maintain spindle bipolarity. CLASP2 C-terminal phosphorylation by Plk1 was also required for chromosome alignment and timely satisfaction of the SAC. We propose that Cdk1 and Plk1 mediate a fine CLASP2 “phospho-switch” that temporally regulates KT–MT attachment stability.     

HCV 1a／1b型嵌合体能在HepG2细胞内复制与表达

采用HCV 1a／1b嵌合体cDNA构建表达质粒转染HepG2细胞,以免疫组化和Westem blotting检测HCV蛋白表达,RT-PCR检测HCV正、负链RNA,研究丙型肝炎病毒(HCV) 1a和1b型嵌合体全长cDNA在HepG2细胞中的复制和表达。结果证明,转染细胞中检测到分子量约70kDa的HCV NS3蛋白,转染细胞连续传20代,仍能检测到HCV正、负链RNA。表明该HCV嵌合体可以在细胞中复制和表达,HCV1b型的RNA依赖的RNA聚合酶(RdRp)可以起始含1a型非编码区的病毒复制。HCV5′端非翻译区第11、12、13、34和35位核苷酸改变可不影响其与核糖体结合。3′非翻译区9400,9403和9407位核苷酸改变,9435位缺失“A”,9409,9410位及9495,9496,9497位分别插入“TT”和“AAT”可不影响RdRp的生物活性。本研究对阐明HCV复制和翻译机制有重要意义。     

HCV 1a/1b型嵌合体能在HepG2细胞内复制与表达

采用HCV 1a/1b嵌合体cDNA构建表达质粒转染HepG2细胞,以免疫组化和Western blotting检测HCV蛋白表达,RT-PCR检测HCV正、负链RNA,研究丙型肝炎病毒(HCV)1a和1b型嵌合体全长cDNA在HepG2细胞中的复制和表达.结果证明,转染细胞中检测到分子量约70 kDa的HCV NS3蛋白, 转染细胞连续传20代, 仍能检测到HCV正、负链RNA.表明该HCV嵌合体可以在细胞中复制和表达,HCV 1b型的RNA依赖的RNA聚合酶(RdRp)可以起始含1a型非编码区的病毒复制. HCV 5′端非翻译区第11、12、13、34和35位核苷酸改变可不影响其与核糖体结合.3′非翻译区9400,9403和9407位核苷酸改变,9435位缺失"A",9409,9410位及9495,9496,9497位分别插入"TT"和"AAT"可不影响RdRp的生物活性.本研究对阐明HCV复制和翻译机制有重要意义.     

Upregulated Expression of Integrin α1 in Mesangial Cells and Integrin α3 and Vimentin in Podocytes of Col4a3-Null (Alport) Mice

Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV) networks found in mature kidney glomerular basement membrane (GBM). The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV). This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV) in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type), and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that overexpression of mesangial integrin α1 and podocyte vimentin and integrin α3 may be important features of glomerular Alport disease, possibly affecting cell-signaling, cell shape and cellular adhesion to the GBM.