用DNA扩增法检测镰状细胞基因

本文报道应用DNA扩增技术对国内首例镰状细胞特征患者(Hb s杂合子)进行基因诊断。方法是从患者干血标本中微量抽提基因组DNA,通过聚合酶链反应(PCR)扩增其β珠蛋白基因,经限制性内切酶MstⅡ消化后作电泳分析直接检测Hb S基因。本文介绍的DNA诊断技术快速、灵敏、简便,它不需要放射性同位素标记的探针,可以采用干血抽提的DNA,因此,对遗传病基因诊断和携带者的筛查具有重要价值。     

Carcinogen-Mediated Amplification of Specific DNA Sequences

A model experimental system based on SV40-transformed Chinese hamster embryo cells and a highly sensitive in situ hybridization procedure was designed. Exposure of the cells to different categories of chemical and physical carcinogens resulted in the induction of SV40 DNA synthesis in the treated cells. Although the carcinogen-mediated amplification of SV40 DNA sequences is regulated by the viral “A” gene, neither infectious virus nor complete viral DNA molecules were rescued from the treated cells. A heterogenous collection of DNA molecules containing SV40 sequences was generated following treatment with DMBA. Restriction enzyme analysis of the amplified DNA molecules in the Hirt supernatant revealed that not all sequences in the integrated SV40 inserts are present. The possibility that the amplification of SV40 sequences is a reflection of a general gene amplification phenomenon mediated by carcinogens is discussed.     

Arabidopsis ARGONAUTE 1 Binds Chromatin to Promote Gene Transcription in Response to Hormones and Stresses

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Src Induces Podoplanin Expression to Promote Cell Migration

Nontransformed cells can force tumor cells to assume a normal morphology and phenotype by the process of contact normalization. Transformed cells must escape this process to become invasive and malignant. However, mechanisms underlying contact normalization have not been elucidated. Here, we have identified genes that are affected by contact normalization of Src-transformed cells. Tumor cells must migrate to become invasive and malignant. Src must phosphorylate the adaptor protein Cas (Crk-associated substrate) to promote tumor cell motility. We report here that Src utilizes Cas to induce podoplanin (Pdpn) expression to promote tumor cell migration. Pdpn is a membrane-bound extracellular glycoprotein that associates with endogenous ligands to promote tumor cell migration leading to cancer invasion and metastasis. In fact, Pdpn expression accounted for a major part of the increased migration seen in Src-transformed cells. Moreover, nontransformed cells suppressed Pdpn expression in adjacent Src-transformed cells. Of >39,000 genes, Pdpn was one of only 23 genes found to be induced by transforming Src activity and suppressed by contact normalization of Src-transformed cells. In addition, we found 16 genes suppressed by Src and induced by contact normalization. These genes encode growth factor receptors, adaptor proteins, and products that have not yet been annotated and may play important roles in tumor cell growth and migration.     

Histone Deacetylase Inhibition Impairs Normal Intestinal Cell Proliferation and Promotes Specific Gene Expression

Mechanisms that maintain proliferation and delay cell differentiation in the intestinal crypt are not yet fully understood. We have previously shown the implication of histone methylation in the regulation of enterocytic differentiation. In this study, we investigated the role of histone deacetylation as an important epigenetic mechanism that controls proliferation and differentiation of intestinal cells using the histone deacetylase inhibitor suberanilohydroxamic acid (SAHA) on the proliferation and differentiation of human and mouse intestinal cells. Treatment of newly confluent Caco‐2/15 cells with SAHA resulted in growth arrest, increased histone acetylation and up‐regulation of the expression of intestine‐specific genes such as those encoding sucrase‐isomaltase, villin and the ion exchanger SLC26A3. Although SAHA has been recently used in clinical trials for cancer treatment, its effect on normal intestinal cells has not been documented. Analyses of small and large intestines of mice treated with SAHA revealed a repression of crypt cell proliferation and a higher expression of sucrase‐isomaltase in both segments compared to control mice. Expression of SLC26A3 was also significantly up‐regulated in the colons of mice after SAHA administration. Finally, SAHA was also found to strongly inhibit normal human intestinal crypt cell proliferation in vitro. These results demonstrate the important implication of epigenetic mechanisms such as histone acetylation/deacetylation in the regulation of normal intestinal cell fate and proliferation. J. Cell. Biochem. 116: 2695–2708, 2015. © 2015 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.     

Lumican Binds ALK5 to Promote Epithelium Wound Healing

Lumican (Lum), a small leucine-rich proteoglycan (SLRP) family member, has multiple matricellular functions both as an extracellular matrix component and as a matrikine regulating cell proliferation, gene expression and wound healing. To date, no cell surface receptor has been identified to mediate the matrikine functions of Lum. This study aimed to identify a perspective receptor that mediates Lum effects on promoting wound healing. Transforming growth factor-β receptor 1 (ALK5) was identified as a potential Lum-interacting protein through in silico molecular docking and molecular dynamics. This finding was verified by biochemical pull-down assays. Moreover, the Lum function on wound healing was abrogated by an ALK5-specific chemical inhibitor as well as by ALK5 shRNAi. Finally, we demonstrated that eukaryote-specific post-translational modifications are not required for the wound healing activity of Lum, as recombinant GST-Lum fusion proteins purified from E. coli and a chemically synthesized LumC₁₃ peptide (the last C-terminal 13 amino acids of Lum) have similar effects on wound healing in vitro and in vivo.     

美国有机农业发展及对我国的启示

美国有机农业起步较早、发展迅速,已经成为全球有机农产品第一大消费市场,分析美国有机农业起源、发展、标识管理和财政支持对发展我国有机农业、提高我国农产品质量安全具有重要借鉴意义。     

Kaiso, a New Protein of the BTB/POZ Family, Specifically Binds to Methylated DNA Sequences

A protein specifically binding to a symmetrically methylated DNA fragment of the first intron of the mts1gene was studied. The protein was purified by gel filtration and affinity chromatography. Mass spectrometry showed that the protein is Kaiso, a new member of the BTB/POZ family. To study the association with methylated DNA sequences in vivo, the location of Kaiso in NIH 3T3 cells was analyzed. Immunofluorescent staining with polyclonal antibodies against Kaiso showed that the protein is predominantly associated with the nucleoli. The causes of its distribution awaits further investigation. The zinc-finger domains of Kaiso were for the first time demonstrated to specifically recognize symmetrically methylated DNA sequences in vitro.     

Genome-Wide Gene Expression Profiling Revealed a Critical Role for GATA3 in the Maintenance of the Th2 Cell Identity

Functionally polarized CD4+ T helper (Th) cells such as Th1, Th2 and Th17 cells are central to the regulation of acquired immunity. However, the molecular mechanisms governing the maintenance of the polarized functions of Th cells remain unclear. GATA3, a master regulator of Th2 cell differentiation, initiates the expressions of Th2 cytokine genes and other Th2-specific genes. GATA3 also plays important roles in maintaining Th2 cell function and in continuous chromatin remodeling of Th2 cytokine gene loci. However, it is unclear whether continuous expression of GATA3 is required to maintain the expression of various other Th2-specific genes. In this report, genome-wide DNA gene expression profiling revealed that GATA3 expression is critical for the expression of a certain set of Th2-specific genes. We demonstrated that GATA3 dependency is reduced for some Th2-specific genes in fully developed Th2 cells compared to that observed in effector Th2 cells, whereas it is unchanged for other genes. Moreover, effects of a loss of GATA3 expression in Th2 cells on the expression of cytokine and cytokine receptor genes were examined in detail. A critical role of GATA3 in the regulation of Th2-specific gene expression is confirmed in in vivo generated antigen-specific memory Th2 cells. Therefore, GATA3 is required for the continuous expression of the majority of Th2-specific genes involved in maintaining the Th2 cell identity.     

DNA甲基化抑制鼻咽癌细胞系膜联蛋白A1基因表达

为了研究不同分化程度和转移潜能鼻咽癌(NPC)细胞系膜联蛋白A1(ANXA1)mRNA和蛋白质表达情况及其与基因甲基化的关系．培养NPC细胞系CNE1、CNE2、5-8F、6-10B和永生化非癌性人鼻咽黏膜上皮细胞NP69细胞用于实验,用甲基化特异性聚合酶链反应(MSP)方法检测ANXA1基因甲基化状态,同时利用逆转录-聚合酶链反应(RT-PCR)方法检测ANXA1基因的mRNA表达水平．然后用不同浓度的5-杂氮-2′-脱氧胞苷(5-aza-2dC)对NPC细胞进行去甲基化处理,MSP和RT-PCR方法检测处理组和对照组细胞ANXA1基因甲基化状况和mRNA表达水平,并用Western-blotting方法检测ANXA1基因蛋白质表达水平．结果发现,NP69细胞ANXA1基因无甲基化,4株NPC细胞系ANXA1基因都存在不同程度的甲基化,甲基化程度与细胞的分化程度和转移潜能相关．NPC细胞ANXA1基因mRNA表达水平降低,低于NP69细胞,其降低的程度与基因的甲基化程度相关．5-aza-2dC能够剂量依赖性地引起ANXA1基因去甲基化,经去甲基化处理后,NPC细胞系ANXA1基因的mRNA和蛋白质的表达水平相应提高．研究证明,NPC细胞系ANXA1基因的mRNA和蛋白质表达水平出现下调,甲基化是导致表达下调的主要原因,5-aza-2dC去甲基化处理能够恢复ANXA1基因的表达水平．     

Fibronectin Affects Transient MMP2 Gene Expression through DNA Demethylation Changes in Non-Invasive Breast Cancer Cell Lines

Metastasis accounts for more than 90% of cancer deaths. Cells from primary solid tumors may invade adjacent tissues and migrate to distant sites where they establish new colonies. The tumor microenvironment is now recognized as an important participant in the signaling that induces cancer cell migration. An essential process for metastasis is extracellular matrix (ECM) degradation by metalloproteases (MMPs), which allows tumor cells to invade local tissues and to reach blood vessels. The members of this protein family include gelatinase A, or MMP-2, which is responsible for the degradation of type IV collagen, the most abundant component of the basal membrane, that separates epithelial cells in the stroma. It is known that fibronectin is capable of promoting the expression of MMP-2 in MCF7 breast cancer cells in culture. In addition, it was already shown that the MMP2 gene expression is regulated by epigenetic mechanisms. In this work, we showed that fibronectin was able to induce MMP2 expression by 30% decrease in its promoter methylation. In addition, a histone marker for an open chromatin conformation was significantly increased. These results indicate a new role for fibronectin in the communication between cancer cells and the ECM, promoting epigenetic modifications.