高温致金黄地鼠神经管畸形差异表达基因的筛选、鉴定及功能研究
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摘要
神经管形成是胚胎早期发育中的重要事件，是指从神经板出现、卷折形成神经褶到左右神经褶在背侧中线靠拢愈合形成神经管的连续生物学过程。神经管是中枢神经系统发生的原基，神经管的发生和分化是脑和脊髓正常发育的前提。在神经管发育过程中，多种环境致畸因素可对其产生影响，引发神经管畸形(neural tube defects，NTDs)。神经管畸形是发生率最高、危害也最为严重的一类先天畸形，不仅危及患儿的生命和健康，而且给社会和家庭造成沉重的经济负担。
从上个世纪开始，科学家开始检测诱发神经管畸形的各种致畸因子，其中高温是较常见且难以预防和避免的一种环境致畸因素。因此，探讨高温致神经管畸形的发生机理成为实验畸形学中的一个研究热点。近年来，人们从细胞、亚细胞和分子水平对高温致神经管畸形的机理进行了大量的动物实验和细胞培养研究，观察了高温对细胞增殖、凋亡、分化、粘着、类聚、组织诱导和器官发生等方面的影响。研究发现，在神经管形成和分化过程中，某一或某些相关基因的特异表达或特异不表达、上调表达或下调表达都会导致神经管发育异常，引发神经管畸形。高温致神经管畸形的发生是一个多基因参与的复杂过程，这其中有些基因上调表达，有些基因下调表达，而关于这些差异表达基因的系统研究至今仍未见报道。
抑制性消减杂交(suppression subtractive hybridization，SSH)是一种寻找差异表达基因的技术，可以克隆出与驱动子相比在检测子中特异表达和上调表达的基因。应用抑制性消减杂交方法构建的消减cDNA文库具有特异性和均等化两大突出优点。在本项实验中，我们应用抑制性消减杂交技术构建了高温致金黄地鼠神经管畸形的双向消减cDNA文库，从中筛选到了高温致神经管畸形过程中上调表达和下调表达的基因。通过序列测定和同源性比较对这些差异表达基因进行了分析，然后通过Northern杂交方法进一步证实这些基因在高温致神经管畸形过程中的差异表达情况。随后从这些差异表达基因中，挑选在胚胎发育中起重要作用的下调表达基因——Npm1，对其功能进行了体外研究。
第一部分高温致金黄地鼠神经管畸形双向消减cDNA文库的构建及鉴定
为筛选高温致神经管畸形过程中的差异表达基因，我们首先构建了高温致金黄地鼠神经管畸形双向消减cDNA文库。实验中，我们分别提取高温组和对照组金黄地鼠胚胎神经管组织总RNA，通过SMART~(TM) cDNA合成的方法反转录得到双链cDNA(ds cDNA)。将酚氯仿纯化后的ds cDNA用RsaI消化，消化后的ds cDNA再次经酚氯仿纯化，并用双蒸水调整浓度至300ng/μl。取纯化后的ds cDNA用于抑制性消减杂交。按PCR-select~(TM) cDNA subtraction kit说明进行正反两个方向的消减杂交，以高温组ds cDNA做检测子、对照组ds cDNA做驱动子进行消减杂交，构建高温致金黄地鼠神经管畸形正向消减cDNA文库，可从中筛选高温致神经管畸形过程中上调表达的基因。反之，以对照组ds cDNA做检测子、高温组ds cDNA做驱动子进行消减杂交，构建高温致金黄地鼠神经管畸形反向消减cDNA文库，可从中筛选高温致神经管畸形过程中下调表达的基因。将看家基因Gapdh设立为消减杂交实验的对照以检测消减效率。结果，Gapdh的表达丰度在消减杂交结束后明显降低，表明我们的消减效率很高。将消减杂交得到的产物纯化，然后通过T-A克隆的方法将其与T载体连接，转化感受态大肠杆菌DH5α，铺含氨苄青霉素的LB/X-gal/IPTG平板，构建消减cDNA文库。经蓝白斑初步筛选后，再利用菌落PCR方法进一步鉴定。结果显示，构建的消减cDNA文库中包含150 bp-1 kb的长短不一的差异表达基因片段。这说明我们构建的高温致金黄地鼠神经管畸形双向消减cDNA文库是成功的，可用于筛选差异表达基因。
第二部分高温致金黄地鼠神经管畸形差异表达基因的筛选、序列分析及鉴定
由于消减cDNA文库中包含的基因信息较多，一般采用随机法挑选文库中的克隆进行测序。本实验中，我们选取经菌落PCR证实插入片段较长的克隆进行测序，并将测序结果通过blastn软件与Genbank数据库中的已知基因进行序列比对和同源性分析。在高温致金黄地鼠神经管畸形反向消减cDNA文库中，我们共筛选到14个下调表达基因，经分析均与已知基因有较高的同源性。这些基因编码的蛋白有核糖体蛋白，参与代谢的酶，翻译及转录因子和其他。随后通过Northern杂交证实了这些基因在高温致畸胚胎神经管中表达水平均明显降低。而在高温致金黄地鼠神经管畸形正向消减cDNA文库中，由于扩增得到的片段普遍较短，测序和同源性分析后仅发现了一个有意义的差异表达基因片段。此片段与小鼠磷酸甘油酸酯激酶(Pgk1)同源且同源性高达92％。Northern杂交证实该基因在高温致畸胚胎神经管中表达水平明显升高。从高温致金黄地鼠神经管畸形双向消减cDNA文库中筛选得到的基因，它们的差异表达情况均得到Northern杂交验证，证实这些基因在高温致神经管畸形过程中的表达发生了异常变化，同时也说明这些基因的差异表达与高温致神经管畸形的发生密切相关。
第三部分Npm1基因功能的体外研究
Npm1(nucleophosmin)是我们从高温致金黄地鼠神经管畸形反向消减cDNA文库中筛选到的一个基因。以往的研究显示，Npm1在胚胎发育过程中扮演着重要的角色。Npm1~(-/-)的小鼠胚胎较正常胚胎发育迟缓，前脑发育缺陷，眼睛缺失。说明Npm1对神经系统的正常发育是必需的。因此我们对Npm1在高温致畸中的作用机制进行了研究。由于在高温致神经管畸形过程中Npm1基因呈下调表达，我们在神经干细胞(neural stem cells，NSCs)的体外培养中，应用RNA干扰(RNA interference，RNAi)技术，对该基因的功能进行了比较深入的研究。实验结果显示，RNAi技术成功地在mRNA和蛋白水平上降低了Npm1的表达，干扰效果得到了RT-PCR和Western blot证实。随后我们从细胞增殖、凋亡和分化三个方面着手，研究Npm1基因表达降低对神经干细胞的影响。结果表明，Npm1基因表达降低后，神经干细胞的增殖速度明显减慢，凋亡的发生率明显增加，而神经干细胞向神经元和神经胶质细胞方向分化的比例未受到影响。此外，Npm1基因表达降低后，p53蛋白表达升高，说明Npm1基因干扰后引发的神经干细胞凋亡与p53信号通路密切相关。
本项研究成功地构建了高温致金黄地鼠神经管畸形双向消减cDNA文库，并从中筛选到了与高温致神经管畸形密切相关的下调表达基因和上调表达基因；还以体外培养的神经干细胞为研究模型，用RNAi技术，对在胚胎发育中起重要作用的下调表达基因Npm1在高温致神经管畸形中的作用机制进行了比较深入的研究，从而为高温致神经管畸形在基因水平上的机理和防治研究提供了重要的理论和技术支持。
Formation of neural tube is an important event during early embryogenesis and involves formation of neural plate, neural plate folding into neural ridge and neural ridge merging into neural tube. Neural tube is the primordium of central nervous system (CNS). The genesis and differentiation of neural tube are precondition of normal development of brain and spinal cord. During neural tube development, many environmental teratogens may have adverse effects and thus induce neural tube defects (NTDs). NTDs are amongst the most prevalent and serious of all human congenital anomalies and place an enormous emotional and economic burden on the population.
Since last century, scientists have been working on exploring various environmental teratogens that may induce NTDs. They found that hyperthermia is a prevalent and unavoidable factor. This has motivated extensive research about mechanism of hyperthermia induced NTDs. There is now an extensive literature about the effects of hyperthermia on cell proliferation, apoptosis, differentiation, migration, adhesion and aggregation as well as on tissue induction and organogenesis. Previous studies revealed that many genes participate in the course of hyperthermia induced NTDs. None or insufficent expression of some genes and specific or excessive expression of some other genes may induce NTDs. Hyperthermia induced NTDs are complicated because genes including both upregulated and downregulated are involved. However, there is still no detailed research about the differentially expressed genes in hyperthermia induced NTDs.
Recently, several PCR-based techniques for differential genes screening have been established. A technique named suppression subtractive hybridization (SSH), owing to its high specificity and parity, has been widely applied to many molecular cloning studies for identification of differentially expressed genes in disease, development and different tissues. Here, we constructed subtractive cDNA libraries of hyperthermia induced NTDs of golden hamster in two directions using SSH. Then we identified both upregulated and downregulated genes in hyperthermia induced NTDs from these two subtractive cDNA libraries. Sequence and homology analysis were made to these differentially expressed genes and Northern blot were performed to verify differential expression levels of these genes. Then we selected Npm1, an important gene for embryogenesis as our study target and explored its function in vitro.
PART ONE Construction and Validation of Forward and Reverse Subtractive cDNA Libraries of Hyperthermia Induced NTDs of Golden Hamster
To identify differentially expressed genes in hyperthermia induced NTDs of golden hamster, we first constructed subtractive cDNA libraries in two directions. Total RNA were isolated from both control and hyperthermia groups and then were reverse transcripted into double-strand cDNA (ds cDNA) using SMART~(TM) cDNA synthesis method. We applied method of phenol and chloroform extraction to purify ds cDNA. Then, the purified cDNA were digested by RsaI to generate shorter, blunt-ended ds cDNA fragments and purified again. The final concentration of cDNA were adjusted to 300 ng/μl. Then SSH was carried out in two directions: forward and reverse, with PCR-Select cDNA Subtraction Kit. For the forward SSH, ds cDNA derived from control group was considered the 'driver' pool, ds cDNA derived from hyperthermia group the 'tester' pool. From the forward SSH, we could identify upregulated genes in hyperthermia induced NTDs. For the reverse SSH, ds cDNA from hyperthermia group was regarded as the 'driver' pool, ds cDNA from control group the 'tester' pool. From the reverse SSH, we could identify downregulated genes in hyperthermia induced NTDs. The housekeeping gene—Gapdh was used as control in SSH to evaluate subtractive efficiency. The significant decrease of Gapdh after SSH suggested high subtractive efficiency in our experiment. Then the purifed subtracted cDNA were ligated with pGEM-T Easy vector and transformed into E. Coli DH5αusing CaCl_2 method. The two subtractive cDNA libraries were separately plated on LB agar plates containing ampicillin (100μg/ml) and IPTG/X-gal. The results of blue-white selection and clony PCR showed that the two subtractive cDNA libraries included differentially expressed gene fragments with length from 150 bp to 1 kb. This suggests that our subtractive cDNA libraries were successfully constructed and suitable for identification of differentially expressed genes in hyperthermia induced NTDs.
PART TWO Screening, Sequencing and Verification of Differentially Expressed Genes in Hyperthermia Induced NTDs of Golden Hamster
Generally, a subtractive cDNA library contains so many differentially expressed genes that researchers cannot sequence them all. So we just selected clones from the two subtractive cDNA libraries randomly. Then we sequenced these clones and compared the sequencing results with Genbank database by blastn software. From the reverse subtractive cDNA library, we identified 14 downregulated genes in all and found them all homologous to known genes with high homology. The proteins encoded by these genes included ribosomal proteins, metabolic enzymes, transcription and translation factors and others. Northern blot showed that expression of all these 14 genes decreased significantly in defected neural tubes induced by hyperthermia. However, from the forward subtractive cDNA library, most of the inserts were short and only two meaningful fragments were identified after sequence and homology analysis. The two inserts were of the same sequence and homologous to mouse Pgk1 with homology of 92%. The increased expression of Pgk1 in defected neural tubes induced by hyperthermia was also confirmed by Northern blot. In this part, we verified these differentially expressed genes by Northern blot, showed differential expression of these genes are strongly related to hyperthermia induced NTDs. PART THREE Functional Study of Npm1 in vitro Npm1 was a gene identified from the reverse subtractive cDNA library. Previous studies showed that Npm1 plays an important role in embryogenesis. Npm1~(-/-) embryos were consistently smaller in size compared with normal ones. The Npm1~(-/-) mutants also showed distinct developmental abnormalities. Most noticeably, they displayed deficient anterior brain organogenesis, with complete absence of eyes. This suggested that Npm1 is essential for neurogenesis. So we selected Npm1 to explore its role in NTDs induced by hyperthermia. Neural stem cells (NSCs) were used as our study model in vitro. As Npm1 was downregulated in hyperthermia induced NTDs, we suppressed expression of Npm1 in NSCs by RNA interference (RNAi). After RNAi, Npm1 was downregulated at both mRNA and protein levels. Then we studied cell proliferation, apoptosis and differentiation in NSCs after Npm1 suppression. As a result, suppression of Npm1 decreased proliferation rate of NSCs, increased apoptosis occurrence but did not seem to make any significant difference in cell differentiation. As there was an increase of p53 protein after Npm1 was suppressed, we speculated that apoptosis of NSCs induced by downregulation of Npm1 was strongly related to p53 pathway.
In our work, we successfully constructed forward and reverse subtractive cDNA libraries of hyperthermia induced NTDs of golden hamster. And we identified several downregulated and upregulated genes involved in hyperthermia induced NTDs. In addition, we employed RNAi with NSCs in vitro to study the function of Npm1 in hyperthermia induced NTDs. Our work provides important theoretical and technical support for mechanism and prevention of hyperthermia induced NTDs.
从上个世纪开始，科学家开始检测诱发神经管畸形的各种致畸因子，其中高温是较常见且难以预防和避免的一种环境致畸因素。因此，探讨高温致神经管畸形的发生机理成为实验畸形学中的一个研究热点。近年来，人们从细胞、亚细胞和分子水平对高温致神经管畸形的机理进行了大量的动物实验和细胞培养研究，观察了高温对细胞增殖、凋亡、分化、粘着、类聚、组织诱导和器官发生等方面的影响。研究发现，在神经管形成和分化过程中，某一或某些相关基因的特异表达或特异不表达、上调表达或下调表达都会导致神经管发育异常，引发神经管畸形。高温致神经管畸形的发生是一个多基因参与的复杂过程，这其中有些基因上调表达，有些基因下调表达，而关于这些差异表达基因的系统研究至今仍未见报道。
抑制性消减杂交(suppression subtractive hybridization，SSH)是一种寻找差异表达基因的技术，可以克隆出与驱动子相比在检测子中特异表达和上调表达的基因。应用抑制性消减杂交方法构建的消减cDNA文库具有特异性和均等化两大突出优点。在本项实验中，我们应用抑制性消减杂交技术构建了高温致金黄地鼠神经管畸形的双向消减cDNA文库，从中筛选到了高温致神经管畸形过程中上调表达和下调表达的基因。通过序列测定和同源性比较对这些差异表达基因进行了分析，然后通过Northern杂交方法进一步证实这些基因在高温致神经管畸形过程中的差异表达情况。随后从这些差异表达基因中，挑选在胚胎发育中起重要作用的下调表达基因——Npm1，对其功能进行了体外研究。
第一部分高温致金黄地鼠神经管畸形双向消减cDNA文库的构建及鉴定
为筛选高温致神经管畸形过程中的差异表达基因，我们首先构建了高温致金黄地鼠神经管畸形双向消减cDNA文库。实验中，我们分别提取高温组和对照组金黄地鼠胚胎神经管组织总RNA，通过SMART~(TM) cDNA合成的方法反转录得到双链cDNA(ds cDNA)。将酚氯仿纯化后的ds cDNA用RsaI消化，消化后的ds cDNA再次经酚氯仿纯化，并用双蒸水调整浓度至300ng/μl。取纯化后的ds cDNA用于抑制性消减杂交。按PCR-select~(TM) cDNA subtraction kit说明进行正反两个方向的消减杂交，以高温组ds cDNA做检测子、对照组ds cDNA做驱动子进行消减杂交，构建高温致金黄地鼠神经管畸形正向消减cDNA文库，可从中筛选高温致神经管畸形过程中上调表达的基因。反之，以对照组ds cDNA做检测子、高温组ds cDNA做驱动子进行消减杂交，构建高温致金黄地鼠神经管畸形反向消减cDNA文库，可从中筛选高温致神经管畸形过程中下调表达的基因。将看家基因Gapdh设立为消减杂交实验的对照以检测消减效率。结果，Gapdh的表达丰度在消减杂交结束后明显降低，表明我们的消减效率很高。将消减杂交得到的产物纯化，然后通过T-A克隆的方法将其与T载体连接，转化感受态大肠杆菌DH5α，铺含氨苄青霉素的LB/X-gal/IPTG平板，构建消减cDNA文库。经蓝白斑初步筛选后，再利用菌落PCR方法进一步鉴定。结果显示，构建的消减cDNA文库中包含150 bp-1 kb的长短不一的差异表达基因片段。这说明我们构建的高温致金黄地鼠神经管畸形双向消减cDNA文库是成功的，可用于筛选差异表达基因。
第二部分高温致金黄地鼠神经管畸形差异表达基因的筛选、序列分析及鉴定
由于消减cDNA文库中包含的基因信息较多，一般采用随机法挑选文库中的克隆进行测序。本实验中，我们选取经菌落PCR证实插入片段较长的克隆进行测序，并将测序结果通过blastn软件与Genbank数据库中的已知基因进行序列比对和同源性分析。在高温致金黄地鼠神经管畸形反向消减cDNA文库中，我们共筛选到14个下调表达基因，经分析均与已知基因有较高的同源性。这些基因编码的蛋白有核糖体蛋白，参与代谢的酶，翻译及转录因子和其他。随后通过Northern杂交证实了这些基因在高温致畸胚胎神经管中表达水平均明显降低。而在高温致金黄地鼠神经管畸形正向消减cDNA文库中，由于扩增得到的片段普遍较短，测序和同源性分析后仅发现了一个有意义的差异表达基因片段。此片段与小鼠磷酸甘油酸酯激酶(Pgk1)同源且同源性高达92％。Northern杂交证实该基因在高温致畸胚胎神经管中表达水平明显升高。从高温致金黄地鼠神经管畸形双向消减cDNA文库中筛选得到的基因，它们的差异表达情况均得到Northern杂交验证，证实这些基因在高温致神经管畸形过程中的表达发生了异常变化，同时也说明这些基因的差异表达与高温致神经管畸形的发生密切相关。
第三部分Npm1基因功能的体外研究
Npm1(nucleophosmin)是我们从高温致金黄地鼠神经管畸形反向消减cDNA文库中筛选到的一个基因。以往的研究显示，Npm1在胚胎发育过程中扮演着重要的角色。Npm1~(-/-)的小鼠胚胎较正常胚胎发育迟缓，前脑发育缺陷，眼睛缺失。说明Npm1对神经系统的正常发育是必需的。因此我们对Npm1在高温致畸中的作用机制进行了研究。由于在高温致神经管畸形过程中Npm1基因呈下调表达，我们在神经干细胞(neural stem cells，NSCs)的体外培养中，应用RNA干扰(RNA interference，RNAi)技术，对该基因的功能进行了比较深入的研究。实验结果显示，RNAi技术成功地在mRNA和蛋白水平上降低了Npm1的表达，干扰效果得到了RT-PCR和Western blot证实。随后我们从细胞增殖、凋亡和分化三个方面着手，研究Npm1基因表达降低对神经干细胞的影响。结果表明，Npm1基因表达降低后，神经干细胞的增殖速度明显减慢，凋亡的发生率明显增加，而神经干细胞向神经元和神经胶质细胞方向分化的比例未受到影响。此外，Npm1基因表达降低后，p53蛋白表达升高，说明Npm1基因干扰后引发的神经干细胞凋亡与p53信号通路密切相关。
本项研究成功地构建了高温致金黄地鼠神经管畸形双向消减cDNA文库，并从中筛选到了与高温致神经管畸形密切相关的下调表达基因和上调表达基因；还以体外培养的神经干细胞为研究模型，用RNAi技术，对在胚胎发育中起重要作用的下调表达基因Npm1在高温致神经管畸形中的作用机制进行了比较深入的研究，从而为高温致神经管畸形在基因水平上的机理和防治研究提供了重要的理论和技术支持。
Formation of neural tube is an important event during early embryogenesis and involves formation of neural plate, neural plate folding into neural ridge and neural ridge merging into neural tube. Neural tube is the primordium of central nervous system (CNS). The genesis and differentiation of neural tube are precondition of normal development of brain and spinal cord. During neural tube development, many environmental teratogens may have adverse effects and thus induce neural tube defects (NTDs). NTDs are amongst the most prevalent and serious of all human congenital anomalies and place an enormous emotional and economic burden on the population.
Since last century, scientists have been working on exploring various environmental teratogens that may induce NTDs. They found that hyperthermia is a prevalent and unavoidable factor. This has motivated extensive research about mechanism of hyperthermia induced NTDs. There is now an extensive literature about the effects of hyperthermia on cell proliferation, apoptosis, differentiation, migration, adhesion and aggregation as well as on tissue induction and organogenesis. Previous studies revealed that many genes participate in the course of hyperthermia induced NTDs. None or insufficent expression of some genes and specific or excessive expression of some other genes may induce NTDs. Hyperthermia induced NTDs are complicated because genes including both upregulated and downregulated are involved. However, there is still no detailed research about the differentially expressed genes in hyperthermia induced NTDs.
Recently, several PCR-based techniques for differential genes screening have been established. A technique named suppression subtractive hybridization (SSH), owing to its high specificity and parity, has been widely applied to many molecular cloning studies for identification of differentially expressed genes in disease, development and different tissues. Here, we constructed subtractive cDNA libraries of hyperthermia induced NTDs of golden hamster in two directions using SSH. Then we identified both upregulated and downregulated genes in hyperthermia induced NTDs from these two subtractive cDNA libraries. Sequence and homology analysis were made to these differentially expressed genes and Northern blot were performed to verify differential expression levels of these genes. Then we selected Npm1, an important gene for embryogenesis as our study target and explored its function in vitro.
PART ONE Construction and Validation of Forward and Reverse Subtractive cDNA Libraries of Hyperthermia Induced NTDs of Golden Hamster
To identify differentially expressed genes in hyperthermia induced NTDs of golden hamster, we first constructed subtractive cDNA libraries in two directions. Total RNA were isolated from both control and hyperthermia groups and then were reverse transcripted into double-strand cDNA (ds cDNA) using SMART~(TM) cDNA synthesis method. We applied method of phenol and chloroform extraction to purify ds cDNA. Then, the purified cDNA were digested by RsaI to generate shorter, blunt-ended ds cDNA fragments and purified again. The final concentration of cDNA were adjusted to 300 ng/μl. Then SSH was carried out in two directions: forward and reverse, with PCR-Select cDNA Subtraction Kit. For the forward SSH, ds cDNA derived from control group was considered the 'driver' pool, ds cDNA derived from hyperthermia group the 'tester' pool. From the forward SSH, we could identify upregulated genes in hyperthermia induced NTDs. For the reverse SSH, ds cDNA from hyperthermia group was regarded as the 'driver' pool, ds cDNA from control group the 'tester' pool. From the reverse SSH, we could identify downregulated genes in hyperthermia induced NTDs. The housekeeping gene—Gapdh was used as control in SSH to evaluate subtractive efficiency. The significant decrease of Gapdh after SSH suggested high subtractive efficiency in our experiment. Then the purifed subtracted cDNA were ligated with pGEM-T Easy vector and transformed into E. Coli DH5αusing CaCl_2 method. The two subtractive cDNA libraries were separately plated on LB agar plates containing ampicillin (100μg/ml) and IPTG/X-gal. The results of blue-white selection and clony PCR showed that the two subtractive cDNA libraries included differentially expressed gene fragments with length from 150 bp to 1 kb. This suggests that our subtractive cDNA libraries were successfully constructed and suitable for identification of differentially expressed genes in hyperthermia induced NTDs.
PART TWO Screening, Sequencing and Verification of Differentially Expressed Genes in Hyperthermia Induced NTDs of Golden Hamster
Generally, a subtractive cDNA library contains so many differentially expressed genes that researchers cannot sequence them all. So we just selected clones from the two subtractive cDNA libraries randomly. Then we sequenced these clones and compared the sequencing results with Genbank database by blastn software. From the reverse subtractive cDNA library, we identified 14 downregulated genes in all and found them all homologous to known genes with high homology. The proteins encoded by these genes included ribosomal proteins, metabolic enzymes, transcription and translation factors and others. Northern blot showed that expression of all these 14 genes decreased significantly in defected neural tubes induced by hyperthermia. However, from the forward subtractive cDNA library, most of the inserts were short and only two meaningful fragments were identified after sequence and homology analysis. The two inserts were of the same sequence and homologous to mouse Pgk1 with homology of 92%. The increased expression of Pgk1 in defected neural tubes induced by hyperthermia was also confirmed by Northern blot. In this part, we verified these differentially expressed genes by Northern blot, showed differential expression of these genes are strongly related to hyperthermia induced NTDs. PART THREE Functional Study of Npm1 in vitro Npm1 was a gene identified from the reverse subtractive cDNA library. Previous studies showed that Npm1 plays an important role in embryogenesis. Npm1~(-/-) embryos were consistently smaller in size compared with normal ones. The Npm1~(-/-) mutants also showed distinct developmental abnormalities. Most noticeably, they displayed deficient anterior brain organogenesis, with complete absence of eyes. This suggested that Npm1 is essential for neurogenesis. So we selected Npm1 to explore its role in NTDs induced by hyperthermia. Neural stem cells (NSCs) were used as our study model in vitro. As Npm1 was downregulated in hyperthermia induced NTDs, we suppressed expression of Npm1 in NSCs by RNA interference (RNAi). After RNAi, Npm1 was downregulated at both mRNA and protein levels. Then we studied cell proliferation, apoptosis and differentiation in NSCs after Npm1 suppression. As a result, suppression of Npm1 decreased proliferation rate of NSCs, increased apoptosis occurrence but did not seem to make any significant difference in cell differentiation. As there was an increase of p53 protein after Npm1 was suppressed, we speculated that apoptosis of NSCs induced by downregulation of Npm1 was strongly related to p53 pathway.
In our work, we successfully constructed forward and reverse subtractive cDNA libraries of hyperthermia induced NTDs of golden hamster. And we identified several downregulated and upregulated genes involved in hyperthermia induced NTDs. In addition, we employed RNAi with NSCs in vitro to study the function of Npm1 in hyperthermia induced NTDs. Our work provides important theoretical and technical support for mechanism and prevention of hyperthermia induced NTDs.
引文
1 Wallingford JB. Neural tube closure and neural tube defects: studies in animal models reveal known knowns and known unknowns. Am J Med Genet C Semin Med Genet. 2005, 135(1): 59-68.
2 Li Z, Ren A, Liu J, Pei L, Zhang L, Guo Z, Li Z. Maternal flu or fever, medication use, and neural tube defects: A population-based case-control study in northern China. Birth Defects Res A Clin Mol Teratol. 2007 Jan 10; [Epub ahead of print]
3 Lary JM, Edmonds LD. Prevalence of spina bifida at birth—United States, 1983-1990: a comparison of two surveillance systems. MMWR CDC Surveill Summ. 1996, 45(2): 15-26.
4 Zlotogora J, Amitai Y, Kaluski DN, Leventhal A. Surveillance of neural tube defects in Israel. Isr Med Assoc J. 2002, 4(12): 1111-1114.
5 Yuskiv N, Andelin CO, Polischuk S, Shevchuk O, Sosynyuk Z, Vihovska T, Yevtushok L, Oakley GP Jr, Wertelecki W. High rates of neural tube defects in Ukraine. Birth Defects Res A Clin Mol Teratol. 2004, 70(6): 400-402.
6 Graham JM, Edwards MJ. Teratogenic effects of maternal hyperthermia, Annu Rep Res Inst Environ Med Nagoya Univ. 1988, 40: 365-374.
7 Lundberg YW, Wing MJ, Xiong W, Zhao J, Finnell RH. Genetic dissection of hyperthermia-induced neural tube defects in mice. Birth Defects Res A Clin Mol Teratol. 2003, 67(6): 409-413.
8 Wilson RS. Synchronies in mental development: an epigenetic perspective. Science. 1978, 202(4371): 939-948.
9 Welsch F. In vitro approaches to the elucidation of mechanisms of chemical teratogenesis. Teratology. 1992, 46(1): 3-14.
10 Cabrera RM, Hill DS, Etheredge A J, Finnell RH. Investigations into the etiology of neural tube defects. Birth Defects Res C Embryo Today. 2004, 72(4): 330-344.
11 Miller MW, Nvborg WL, Dewey WC, Edwards M J, Abramowicz JS, Bravman AA. Hyperthermic teratogenicity, thermal dose and diagnostic ultrasound during pregnancy: implications of new standards on tissue heating. Int J Hyperthermia. 2002, 18(5): 361-384.
12 Edwards MJ. Review: hyperthermia and fever during pregnancy. Birth Defects Res A Clin Mol Teratol. 2006, 76(7): 507-516.
13 Kimmel GL, Cuff JM, Kimmel CA, Heredia D J, Tudor N, Silvennan PM. Embryonic development in vitro following short-duration exposure to heat. Teratology. 1993, 47(3): 243-251.
14 Edwards MJ. Congenital defects in guinea pig: following induced hyperthermia during gestration. Arch Pathol. 1967, 84(1): 42-48.
15 Edwards MJ. Congenital malformations in the rat following induced hyperthermia during gestation. Teratology. 1968, 1(2): 173-177.
16 Edwards MJ. Hyperthermia as a teratogen: a review of experimental studies and their clinical significance. Teratog Carcinog Mutaggen. 1986, 6(6): 563-582.
17 Chambers CD, Johnson KA, Dick LM, Felix RJ, Jones KL. Maternal fever and birth outcome: a prospective study. Teratology. 1998, 58(6): 251-257.
18 Milunsky A, Ulcickas M, Rothman KJ, Willett W, Jick SS, Jick H. Maternal heat exposure and neural tube defects. JAMA. 1992, 268(7): 882-885.
19 Moretti ME, Bar-Oz B, Fried S, Koren G.Maternal hyperthermia and the risk for neural tube defects in offspring: systematic review and meta-analysis. Epidemiology. 2005, 16(2): 216-219.
20 Chamber CD. Risks of hyperthermia associated with hot tub or spa use by pregnant women. Birth Defects Res A Clin Mol Teratol. 2006, 76(8): 569-573.
21 Edwards MJ. Apoptosis, the heat shock response, hyperthermia, birth defects, disease and cancer. Where are the common links? Cell Stress Chaperones. 1998, 3(4): 213-220.
22 Walsh D, Li Z, Nagata K. Heat shock and the role of the HSPs during neural palate induction in early mammalian CNS and brain development. CMLS. 1993, 53: 198-211.
23 Mirkes PE, Cornel LM, Park HW, Cunnigham ML. Induction of thermotolerance in early postimplantation rat embryos is associated with increased resistance to hyperthermia-induced apoptosis. Teratology. 1997, 56(3): 210-219.
24 Graham JM Jr. Marshall J Edwards discoverer of maternal hyperthermia as a human teratogen. Birth Defects Res A Clin Mol Teratol. 2005, 73(11): 857-864.
25 Padmanabhan R, Al-Menhali NM, Tariq S, Shafiullah M. Mitochondrial dysmorphology in the neuroepithelium of rat embryos following a single dose of maternal hyperthermia during gestation. Exp Brain Res. 2006, 173(2): 298-308.
26 Soleman D, Cornel L, Little SA, Mirkes PE. Teratogen-induced activation of the mitochondrial apoptotic pathway in the yolk sac of day 9 mouse embryos. Birth Defects Res A Clin Mol Teratol. 2003, 67(2): 98-107.
27 Hinoue A, Fushiki S, Nishimura Y, Shiota K. In utero exposure to brief hyperthermia interferes with the production and migration of neocortical neurons and induces apoptotic neuronal death in the fetal mouse brain. Brain Res Dev Brain Res. 2001, 132(1): 59-67.
28 Kim WK, Mirkes PE. Alterations in mitochondrial morphology are associated with hyperthermia-induced apoptosis in early postimplantation mouse embryos. Birth Defects Res A Clin Mol Teratol. 2003, 67(11): 929-940.
29 Chen H, Chan DC. Mitochondrial dynamics in mammals. Curr Top Dev Biol. 2004, 59: 119-144.
30 Paula-Lopes FF, Hansen PJ. Heat shock-induced apoptosis in preimplantation bovine embryos is a developmentally regulated phenomenon. Biol Reprod. 2002, 66(4): 1169-1177.
31 Arechiga CF, Ealy AD, Hansen PJ. Evidence that glutathione is involved in thermotolerance of preimplantation murine embryos. Biol Reprod.1995, 52(6): 1296-1301.
32 Bennett GD, Mohl VK, Finnell RH. Embryonic and maternal heat shock responses to a teratogenic hyperthermic insult. Reprod Toxicol. 1990, 4(2): 113-119.
33 Edwards MJ, Saunders RD, Shiota K. Effects of heat on embryos and foetuses. Int J Hyperthermia. 2003, 19(3): 295-324.
34 Breen JG, Claggett TW, Kimmel GL, Kimmel CA. Heat shock during rat embryo development in vitro results in decreased mitosis and abundant cell death. Reprod Toxicol. 1999, 13(1): 31-39.
35 Finnell RH, Moon SP, Abbott LC, Golden JA, Chemoff GF. Strain differences in heat-induced neural tube defects in mice. Teratology. 1986, 33(2): 247-252.
36 Fleming A, Copp AJ. A genetic risk factor for mouse neural tube defects: defining the embryonic basis. Hum Mol Genet. 2000, 9(4): 575-581.
37 Vacha SJ, Bennett GD, Mackler SA, Koebbe MJ, Finnell RH. Identification of a growth arrest specific (gas5) gene by differential display as a candidate gene for determining susceptibility to hyperthermia-induced exencephaly in mice. Dev Genet. 1997, 21(3): 212-222.
38 Harris MJ. Why are the genes that cause risk of human neural tube defects so hard to find? Teratology. 2001, 63(5): 165-166.
39 MRC. Prevention of neural tube defects: results of the Medical Research Council Vitamin Study. MRC Vitamin Study Research Group. Lancet. 1991, 338(8760): 131-137.
40 Czeizel AE, Dudas I. Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation. N Engl J Med. 1992, 327(26): 1832-1835.
41 Shin JH, Shiota K. Folic acid supplementation of pregnant mice suppresses heat-induced neural tube defects in the offspring. J Nutr. 1999, 129(11): 2070-2073.
42 Botto LD, Erickson JD, Mulinare J, Lynberg MC, Liu Y. Maternal fever, multivitamin use, and selected birth defects: evidence of interaction? Epidemiology. 2002, 13(4): 485-488.
43 Suarez L, Felkner M, Hendricks K. The effect of fever, febrile illnesses, and heat exposures on the risk of neural tube defects in a Texas-Mexico border population. Birth Defects Res A Clin Mol Teratol. 2004, 70(10): 815-819.
44 Acs N, Banhidy F, Puho E, Czeizel AE. Maternal influenza during pregnancy and risk of congenital abnormalities in offspring. Birth Defects Res A Clin Mol Teratol. 2005, 73(12): 989-996.
45 Rozen R. Molecular genetics of methylenetetrahydrofolate reductase deficiency. J Inherit Metab Dis. 1996, 19(5): 589-594.
46 van der Put NM, Gabreels F, Stevens EM, Smeitink JA, Trijbels FJ, Eskes TK, van den Heuvel LP, Blom HJ. A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am J Hum Genet. 1998, 62(5): 1044-1051.
47 Weisberg I, Tran P, Christensen B, Sibani S, Rozen R. A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. Mol Genet Metab. 1998, 64(3): 169-172.
48 Koch MC, Stegmann K, Ziegler A, Schroter B, Ermert A. Evaluation of the MTHFR C677T allele and the MTHFR gene locus in a German spina bifida population. Eur J Pediatr. 1998, 157(6): 487-492.
49 Melnick M, Marazita ML. Neural tube defects, methylenetetrahydrofolate reductase mutation, and north/south dietary differences in China. J Craniofac Genet Dev Biol. 1998, 18(4): 233-235.
50 Shaw GM, Rozen R, Finnell RH, Todoroff K, Lammer EJ. Infant C677T mutation in MTHFR, maternal periconceptional vitamin use, and cleft lip. Am J Med Genet. 1998, 80(3): 196-198.
51 Padmanabhan R. Etiology, pathogenesis and prevention of neural tube defects. Congenit Anom (Kyoto). 2006, 46(2): 55-67.
52 Maddox DM, Manlapat A, Roon P, Prasad P, Ganapathy V, Smith SB. Reduced-folate carrier (RFC) is expressed in placenta and yolk sac, as well as in cells of the developing forebrain, hindbrain, neural tube, craniofacial region, eye, limb buds and heart. BMC Dev Biol. 2003, 3: 6.
53 Zhao R, Russell RG, Wang Y, Liu L, Gao F, Kneitz B, Edelmann W, Goldman ID. Rescue of embryonic lethality in reduced folate carrier-deficient mice by maternal folic acid supplementation reveals early neonatal failure of hematopoietic organs. J Biol Chem. 2001, 276(13): 10224-10228.
54 Barber RC, Shaw GM, Lammer EJ, Greer KA, Biela TA, Lacey SW, Wasserman CR, Finnell RH. Lack of association between mutations in the folate receptor-alpha gene and spina bifida. Am J Med Genet. 1998, 76(4): 310-317.
55 Heil SG, van der Put NM, Trijbels FJ, Gabreels FJ, Blom HJ. Molecular genetic analysis of human folate receptors in neural tube defects. Eur J Hum Genet. 1999, 7(3): 393-396.
56 O'Leary VB, Mills JL, Kirke PN, Parle-McDermott A, Swanson DA, Weiler A, Pangilinan F, Conley M, Molloy AM, Lynch M, Cox C, Scott JM, Brody LC. Analysis of the human folate receptor beta gene for an association with neural tube defects. Mol Genet Metab. 2003, 79(2): 129-133.
57 Homanics GE, Maeda N, Traber MG, Kayden HJ, Dehart DB, Sulik KK. Exencephaly and hydrocephaly in mice with targeted modification of the apolipoprotein B (Apob) gene. Teratology. 1995, 51(1): 1-10.
58 Huang LS, Voyiaziakis E, Markenson DF, Sokol KA, Hayek T, Breslow JL. apo B gene knockout in mice results in embryonic lethality in homozygotes and neural tube defects, male infertility, and reduced HDL cholesterol ester and apo A-I transport rates in heterozygotes. J Clin Invest. 1995, 96(50): 2152-2161.
59 Bamforth SD, Braganca J, Eloranta JJ, Murdoch JN, Marques FI, Kranc KR, Farza H, Henderson DJ, Hurst HC, Bhattacharya S. Cardiac malformations, adrenal agenesis, neural crest defects and exencephaly in mice lacking Cited2, a new Tfap-2 co-activator. Nat Genet. 2001, 29(4): 469-474.
60 Juriloff DM, Gunn TM, Harris MJ, Mah DG, Wu MK, Dewell SL. Multifactorial genetics of exencephaly in SELH/Bc mice. Teratology. 2001, 64(4): 189-200.
61 Ishii M, Han J, Yen HY, Sucov HM, Chai Y, Maxson RE Jr. Combined deficiencies of Msx1 and Msx2 cause impaired patterning and survival of the cranial neural crest. Development. 2005, 132(22): 4937-4950.
62 Stegmann K, Boecker J, Richter B, Capra V, Finnell RH, Ngo ET, Strehl E, Ermert A, Koch MC. A screen for mutations in human homologues of mice exencephaly genes Tfap2alpha and Msx2 in patients with neural tube defects. Teratology. 2001, 63(5): 167-175.
63 Pani L, Horal M, Loeken MR. Rescue of neural tube defects in Pax-3-deficient by p53 loss of function: implications for Pax-3-dependent development and tumorigenesis. Genes Dev. 2002, 16(6): 676-680.
64 Lu W, Zhu H, Wen S, Laurent C, Shaw GM, Lammer EJ, Finnell RH. Screening for novel PAX3 polymorphisms and risks of spina bifida. Birth Defects Res A Clin Mol Teratol. 2007, 79(1): 45-49.
65 Gos M, Szpecht-Potocka A. Genetic basis of neural tube defects. I. Regulatory genes for the neurulation process. J Appl Genet. 2002, 43(3): 343-350.
66 Aruga J. The role of Zic genes in neural development. Mol Cell Neurosci. 2004, 26(2): 205-221.
67 Grinberg I, Millen KJ. The ZIC gene family in development and disease. Clin Genet. 2005, 67(4): 290-296.
68 Morrison K, Papapetrou C, Attwood J, Hol F, Lynch SA, Sampath A, Hamel B, Burn J, Sowden J, Stott D, Mariman E, Edwards YH. Genetic mapping of the human homologue (T) of mouse T (Brachyury) and a search for allele association between human T and spina bifida. Hum Mol Genet. 1996, 5(5): 669-674.
69 Buckiova D, Brown NA. Mechanism of hyperthermia effects on CNS development: rostral gene expression domains remain, despite severe head truncation; and the hindbrain/otocyst relationship is altered. Teratology. 1999, 59(3): 139-147.
70 Gebicke-Haerter P. Expression profiling methods used in drug abuse research. Addict Biol. 2005, 10(1): 37-46.
71 Rosok O, Sioud M. Discovery of differentially expressed genes: technical considerations. Methods Mol Biol. 2007, 360:115-129.
72 Diachenko L, Lau YC, Campbell AP, Chenchik A, Mogadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD. Suppression subtractive hybridization: A method for generating differential regulated or tissue-specifical cDNA probes and libraries. Proc Natl Acad Sci USA. 1996, 93(12): 6025-6030.
73 Gurskaya NG, Diatchenko L, Chenchik A, Siebert PD, Khaspekov GL, Lukyanov KA, Vagner LL, Ermolaeva OD, Lukyanov SA, and Sverdlov ED. Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction: Cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate. Anal Biochem. 1996, 240(1): 90-97.
74 von Stein OD, Thies WG, Hofmann M. A high throughput screening for rarely transcribed differentially expressed genes. Nucleic Acids Res. 1997, 25(13): 2598-2602.
75 Xiao W, Pacyna-Gengelbach M, Sehluns K, An Q, Gao Y, Cheng S, Petersen I. Differentially expressed genes associated with human lung cancer. Oncol Rep. 2005, 14(1): 229-234.
76 Rho J, Altmann CR, Soeei ND, et al. Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis. DNA Cell Biol. 2002, 21(8): 541-549.
77 Wallner EI, Wada J, Lin S, Pan X, Reddy JK, Chugh SS, Kanwar YS. Renal gene expression in embryonic and newborn diabetic mice. Exp Nephrol. 2002, 10(2): 130-138.
78 Wang X, Feuerstein GZ. Suppression subtractive hybridization: application in the discovery of novel pharmacological targets. Pharmacogenomics. 2000, 1(1): 101-108.
79 Chen ZB, Yang J, Wang H, Sun OJ, Wang SQ. Screening and identification of differentially expressed genes from influenza virus infected-host cells by suppression subtractive hybridization. Chinese J Biochem Mol Bio. 2003, 19(2): 156-161.
1 Edwards MJ. Congenital defects in guinea pig: following induced hyperthermia during gestration. Arch Pathol. 1967, 84(1): 42-48.
2 Edwards MJ. Hyperthermia as a teratogen: a review of experimental studies and their clinical significance. Teratog Carcinog Mutaggen. 1986, 6(6): 563-582.
3 Moretti ME, Bar-Oz B, Fried S, Koren G. Maternal hyperthermia and the risk for neural tube defects in offspring: systematic review and meta-analysis. Epidemiology. 2005, 16(2): 216-219.
4 Chamber CD. Risks of hyperthermia associated with hot tub or spa use by pregnant women. Birth Defects Res A Clin Mol Teratol. 2006, 76(8): 569-573.
5 吕锋，高英茂，管英俊．高温致神经管畸形动物模型的建立及其形态发生的研究．解剖学报，1996，27(3)：273-276．
6 Diatchenko L, Lau YFC, Campbell AP, et al. Suppression subtractive hybridization:a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93(18): 6025-6030.
7 Gurskaya NG, Diatchenko L, Cbenchik A, Siebert PD, Khaspekov GL, Lukyanov KA, Vagner LL, Ermolaeva OD, Lukyanov SA, and Sverdlov ED. Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction: Cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate. Anal Biochem. 1996, 240(1): 90-97.
8 Von SOD, Thies WG, Hofmann M, et al. A high throughput screening for rarely transcribed differentially expressed genes. Nucleic Acid Res. 1997, 25(13): 2598-2602.
9 Chenchik A, Zhu YY, Diatchenko L, Li R, Hill J&Siebert PD. (1998) Generation and use of high-quality cDNA from small amounts of total RNA by SMART PCR. In Gene Cloning and Analysis by RT-PCR. BioTechniques Books, MA. I998 pp305-319.
10 Wellenreuther R, Schupp I, Poustka A, Wiemann S. SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones. BMC Genomics. 2004, 5(1): 36.
11 Wilfinger WW, Mackey K, Chomczynski P. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques. 1997, 22(3): 474-476, 478-481.
1 Copp AJ, Greene ND, Murdonch JN. The genetic basis of mammalian neurulation. Nat Rev Genet. 2003, 4(10): 784-793.
2 Harris MJ. Why are the genes that cause risk of human neural tube defects so hard to find? Teratology. 2001, 63(5): 165-166.
3 German J. Embryonic stress hypothesis of teratogenesis. Am J Med. 1984, 76(2): 293-301.
4 Cabrera RM, Hill DS, Etheredge AJ, Finnell RH. Investigations into the etiology of neural tube defects. Birth Defects Res. 2005, 72(4): 330-344.
5 Boyles AL, Hammock P, Speer MC. Candidate gene analysis in human neural tube defects. Am J Med Genet C Semin Med Genet, 2005, 135(1): 9-23.
6 Wool IG, Chan YL, Gluck A. Structure and evolution of mammalian ribosomal proteins. Biochem Cell Biol. 1995, 73(11-12): 933-947.
7 Vilardell J, Chartrand P, Singer RH, Warner JR.The odyssey of a regulated transcript. RNA. 2000, 6(12): 1773-1780.
8 Watanabe M, Zinn AR, Page DC, Nishimoto T. Functional equivalence of human X- and Y-encoded isoforms of ribosomal protein S4 consistent with a role in Turner syndrome. Nature Genetics. 1993, 4(3): 268-271.
9 Watanabe M, Furuno N, Goebl M, Go M, Miyauchi K, Sekiguchi T, Basilico C, Nishimito T. Molecular cloning of the human gene, CCG2, that complements the BHK-derived temperature-sensitive cell cycle mutant tsBN63: identity of CCG2 with the human X chromosomal SCAR/RPS4X gene. Journal of Cell Science. 1991, 100(Pt 1): 35-43.
10 Balakirev MY, Tcherniuk SO, Jaquinod M, Chrobocaek J. Otubains: a new family of cysteine proteases in the ubiquitin pathway. EMBO Reports. 2003, 4(5): 517-522.
11 Soares L, Seroogy C, Skrenta H, Anandasabapathy N, Lovelace P, Chung CD, Engleman E, Fathrrlan CG. Two isoforms of otubain 1 regulate T cell anergy via GRAIL. Nature Immunology. 2004, 5(1): 45-54.
12 Strayhorn WD, Wadzinski BE. A novel in vitro assay for deubiquitination of IκBα. Arch Biocem Biophys. 2002, 400(1): 76-84.
13 Balakirey MY, Tcherniuk SO, Jaquinod M, Chroboczek J. Otubains: a new family of cysteine proteases in the ubiquitin pathway. EMBO Rep. 2003, 4(5):517-522.
14 Ernst H, Duncan RF, Hershey JW. Cloning and sequencing of complementary DNAs encoding the alpha-subunit of translational initiation factor Eif-2. Characterization of the protein and its messenger RNA. Journal of Biological Chemistry. 1987, 262(3): 1206-1212.
15 Scheuner D, Song B, McEwen E, Liu C, Laybutt R, Gillespie P, Saunders T, Bonner-Weir S, Kaufrnan RJ. Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Molecular Cell. 2001, 7(6): 1165-1176.
16 Boyce M, Bryant KF, Jousse C, Long K, Harding HP, Scheuner D, Kaufman RJ, Ma D, Coen DM, Ron D, Yuan J. A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress. Science. 2005, 307(5711): 935-939.
17 Komar AA, Gross SR, Barth-Baus D, Strachan R, Hensold JO, Goss Kinzy T, Merrick WC. Novel characteristics of the biological properties of the yeast saccharomyces cerevisiae eukaryotic initiation factor 2A. J Biol Chem. 2005, 280(16): 15601-15611.
18 Zoll WL, Horton LE, Komar AA, Hensold JO, Merrick WC. Characterization of mammalian eIF2A and identification of the yeast homolog. J Biol Chem. 2002, 277(40): 37079-37087.
19 Page AB, Owen CR, Kumar R, Miller JM, Rafols JA, White BC, DeGracia DJ, Krause GS. Persistent eIF2alpha(P) is colocalized with cytoplasmic cytochrome c in vulnerable hippocampal neurons after 4 hours of reperfusion following 10-minute complete brain ischemia. Acta Neuropathologica. 2003, 106(1): 8-16.
20 Suske G The Sp-family of transcription factors. Gene. 1999, 238(2): 291-300.
21 Kingsley C, Winoto A. Cloning of GT box-binding proteins: a novel Sp 1 multigene family regulating T-cell receptor gene expression. Molecular Cell Biology. 1992, 12(10): 4251-4261.
22 Hagen G, Muller S, Beato M, Suske G. Spl-mediated transcriptional activation is repressed by Sp3. EMBO Journal. 1994, 13(16): 3843-3851.
23 Lou Z, Maher VM, McCormick JJ. Identification of the promoter of human transcription factor Sp3 and evidence of the role of factors Sp1 and Sp3 in the expression of Sp3 protein. Gene. 2005, 351:51-59.
24 Dobson MJ, Tuite MF, Roberts NA, Kingsman AJ, Kingsman SM, Perkins RE, Conroy SC, Fothergill LA. Conversation of high efficiency promotor sequences in Saccharomyces cerevisiae. Nucleic Acids Res. 1982, 10(8): 2625-2637.
25 Rathjen J, Mellor J. Characterization of sequences required for RNA initiation from the PGK promoter of Saccharomyces cerevisiae. Nucleic Acids Res. 1990, 18(11): 3219-3125.
26 de Almeida JR, de Moraes LM, Torres FA. Molecular characterization of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris. Yeast. 2005, 22(9): 725-737.
27 Gao L, Mejias R, Echevarria M, Lopez-Barneo J. Induction of the glucose-6-phosphate dehydrogenase gene expression by chronic hypoxia in PC 12 cells. FEBS Lett. 2004, 569(1-3): 256-260.
1 Schmidt-Zachmann MS, Franke WW. DNA cloning and amino acid sequence determination of a major constituent protein of mammalian nucleoli. Correspondence of the nucleoplasmin-related protein NO38 to mammalian protein B23. Chromosoma. 1988, 96(6): 417-426.
2 Grisendi S, Pandolfi PP. NPM mutations in acute myelogenous leukemia. N Engl J Med. 2005, 352(3): 291-292.
3 Eirin-Lopez JM, Frehlick LJ, Ausio J. Long-term evolution and functional diversification in the members of the nucleophosmin/nueleoplasmin family of nuclear chaperones. Genetics. 2006, 173(4): 1835-1850.
4 Dingwall C, Laskey RA. Nucleoplasmin: the archetypal molecular chaperone. Semin Cell Biol. 1990, 1(1): 11-17.
5 Szebeni A, Herrera JE, Olson MO. Interaction of nuclear protein B23 with peptides related to nuclear localization signals. Biochemistry. 1995, 34(25): 8037-8042.
6 Borer RA, Lehner CF, Eppenberger HM, Nigg EA. Major nueleolar proteins shuttle between nucleus and cytoplasm. Cell. 1989, 56(3): 379-390.
7 Grisendi S, Bernardi R, Rossi M, Cheng K, Khandker L, Manova K, Pandolfi PP. Role of nucleophosmin in embryonic development and tumorigenesis. Nature. 2005, 437(7055): 147-153.
8 Chan WY, Liu QR, Borjiqin J, Busch H, Rennert OM, Tease LA, Chan PK. Characterization of the cDNA encoding human nucleophosmin and studies of its role in normal and abnormal growth. Biochemistry. 1989, 28(3): 1033-1039.
9 Herrera JE, Savkur R, Olson MO. The ribonuclease activity of nucleolar protein B23. Nucleic Acids Res. 1995, 23(19): 3974-3979.
10 Nozawa, Y, Van Belzen N, Van der Made AC, Dinjens WN, Bosman FT. Expression of nucleophosmin/B23 in normal and neoplastic colorectal mucosa. J Pathol. 1996, 178(1): 48-52.
11 Subong EN, Shue MJ, Epstein JI, Briggrnan JV, Chan PK, Partin AW. Monoclonal antibody to prostate cancer nuclear matrix protein (PRO:4-216) recognizes nucleophosmin/B23. Prostate. 1999, 39(4): 298-304.
12 Herrera JE, Savkur R, Olson MO. The ribonuelease activity of nueleolar protein B23. Nucleic Acids Res. 1995, 23(19): 3974-3979.
13 Wang BB, Lu R, Wang WC, Jin Y. Inducible and reversible suppression of Npm1 gene expression using stably integrated small interfering RNA vector in mouse embryonic stem cells. Biochem Biophys Res Commun. 2006, 347(4): 1129-1137.
14 He J, Chang LJ. Functional characterization of hepatoma-specific stem cell antigen-2. Molecular Carcinogenesis. 2004, 40(2): 90-103.
15 Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998, 391(6669): 806-811.
16 Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001, 411(6836): 494-498.
17 Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs the ATP-dependent cleavage ofmRNA at 21 to 23 nucleotide intervals. Cell. 2000, 101(1): 25-33.
18 Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev. 2001, 15(2): 188-200.
19 Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature. 2001, 409(6818): 363-366.
20 Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell. 2003, 115(2): 199-208.
21 Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell. 2003, 115(2): 209-216.
22 Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature. 2000, 404(6775): 293-296.
23 Nykanen A, Haley B, Zamore PD. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell. 2001, 107(3): 309-321.
24 Martinez J, Patkaniowska A, Urlaub H, Luhrmann R, Tuschl T. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell. 2002, 110(5): 563-574.
25 Mosmann T. Rapid colorimetric assay for cellular growth and survival: applications to proliferation and cytotoxicity assays. J Immunol Methods. 1983, 65(1-2): 55-63.
26 Berridge MV, Tan AS. Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch Biochem Biophys. 1993,303(2): 474-482.
27 Hayon T, Dvilansky A, Shpilberg O, Nathan I. Appraisal of the MTT-based assay as a useful tool for predicting drug chemosensitivity in leukemia. Leuk Lymphoma. 2003, 44(11): 1957-1962.
28 Tchounwou PB, Yedjou CG, Foxx D, Ishaque A, Shen E. Lead-induced cytotoxity and transcriptional activation of stress genes in human liver carcinoma cells. Mol Cell Biochem. 2004, 255(1-2): 161-170.
29 Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Armu Rev. 2005, 11: 127-152.
30 Zeller KI, Haggerty TJ, Barrett JF, Guo Q, Wonsey DR, Dang CV. Characterization ofnucleophosmin (B23) as a Myc target by scanning chromatin immunoprecipitation. J Biol Chem. 2001, 276(51): 48285-48291.
31 Lim MJ, Wang XW. Nucleophosmin and human cancer. Cancer Detect Prev. 2006, 30(6): 481-490.
32 Hsu CY, Yung BY. Down-regulation of nucleophosmin/B23 during retinoic acid-induced differentiation of human promyelocytic leukemia HL-60 cells. Oncogene. 1998, 16(7): 915-923.
33 Liu WH, Yung BY. Mortalization of human promyelocytic leukemia HL-60 cells to be more susceptible to sodium butyrate-induced apoptosis and inhibition of telomerase activity by down-regulation of nucleophosmin/B23. Oncogene. 1998, 17(23): 3055-3064.
34 Patterson SD, Grossman JS, D'Andrea P, Latter GI. Reduced numatrin/B23/nucleophosmin labeling in apoptotic Jurkat T-lymphoblasts. J Biol Chem. 1995, 270(16): 9429-9436.
35 Feuerstein N, Mond JJ. Identification of a prominent nuclear protein associated with proliferation of normal and malignant B cells. J Immunol. 1987, 139(6): 1818-1822.
36 Gerke V, Moss SE. Annexins: from structure to function. Physiol Rev. 2002, 82(2): 331-371.
37 Williamson P, van den Eijnde S, Schlegel RA. Phosphatidylserine exposure and phagocytosis of apoptotic cells. Methods Cell Biol. 2001, 66: 339-364.
38 van Engeland M, Nieland LJ, Ramaekers FC, Schutte B. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry. 1998, 31(1): 1-9.
39 Plasier B, Lloyd DR, Paul GC, Thomas CR, Al-Rubeai M. Automatic image analysis for quantification of apoptosis in animal cell culture by annexin-V affinity assay. J Immunol Methods. 1999, 229(1-2): 81-95.
40 Hanshaw RG, Smith BD. New reagents for phosphatidylserine recognition and detection of apoptosis. Bioorg Med Chem. 2005, 13(17): 5035-5042.
41 Kanagaraj P, Vijayababu MR, Ravisankar B, Anbalagan J, Aruldhas MM, Arunakaran J. Effect of lycopene on insulin-like growth factor-Ⅰ, IGF binding protein-3 and IGF type-Ⅰ receptor in prostate cancer cells. J Cancer Res Clin Oncol. 2007 Jan 12; [Epub ahead of print]
42 Kuida K, Haydar TF, Kuan CY, et al. Reduced apoptpsis and cytochrome c-mediated caspase activation in mice lacking caspase-9. Cell. 1998, 94(3): 325-337.
43 Kuida K, Zheng TS, Na S, Kuan C,et al. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature. 1996, 384(6607): 368-372.
44 Jin Z, El-Deiry WS. Overview of cell death signaling pathways. Cancer Biol Ther. 2005, 4(2): 139-163.
45 Creagh EM, Conroy H, Martin SJ. Caspase-activation pathways in apoptosis and immunity. Immunol Rev. 2003, 193: 10-21.
46 Nunez G, Benedict MA, Hu Y, Inohara N. Caspase: the proteases of the apoptotic pathway. Oncogene. 1998, 17(25): 3237-3245.
47 Fan TJ, Han LH, Cong RS, Liang J. Caspase family proteases and apoptosis. Acta Biochim Biophys Sin (Shanghai). 2005, 37(11): 719-727.
48 Kuribayashi K, Mayes PA, El-Deiry WS. What are caspase 3 and 7 doing upstream of the mitochondria? Cancer Biol Ther. 2006, 5(7): 763-765.
49 Itahana K, Bhat KP, Jin A, Itahana Y, Hawke D, Kobayashi R, Zhang Y. Tumor suppressor ARF degrades B23, a nucleolar protein involved in ribosome biogenesis and cell proliferation. Mol Cell. 2003, 12(5): 1151-1164.
50 Grisendi S, Pandolfi PP. NPM mutations in acute myelogenous leukemia. N Engl J Med. 2005, 352(3): 291-292.
51 Colombo E, Bonetti P, Lazzerini Denchi E, Martinelli P, Zamponi R, Marine JC, Helin K, Falini B, Pelicci PG. Nucleophosmin is required for DNA integrity and p 19Arfprotein stability. Mol Cell Biol. 2005, 25(20): 8874-8886.
52 Colombo E, Marine JC, Danovi D, Falini B, Pelicci PG. Nucleophosmin regulates the stability and transcriptional activity of p53. Nat Cell Biol. 2002, 4(7): 529-533.
53 Chan HJ, Weng JJ, Yung BY. Nucleophosmin/B23-binding peptide inhibits tumor growth and up-regulates transcriptional activity of p53. Biochem Biophys Res Commun. 2005, 333(2): 396-403.
54 Lambert B, Buckle M. Characterisation of the interface between nucleophosmin (NPM) and p53: potential role in p53 stabilisation. FEBS Lett. 2006, 580(3): 345-350.
55 Chou CC, Yung BY. Increased stability of nucleophosmin/B23 in anti-apoptotic effect ofras during serum deprivation. Mol Pharmacol. 2001, 59(1): 38-45.
56 Li J, Zhang XL, Sejas DP, Pang Q. Negative regulation of p53 by nucleophosmin antagonizes stess-induced apoptosis in human normal and malignant hematopoietic cells. Leukemia Research. 2005, 29(12): 1415-1423.
57 Li J, Sejas DP, Rani R, Koretsky T, Bagby GC, Pang Q. Nucleophosmin regulates cell cycle progression and stress response in hematopoietic stem/progenitor cells. J Biol Chem. 2006, 281(24): 16536-16545.
2 Li Z, Ren A, Liu J, Pei L, Zhang L, Guo Z, Li Z. Maternal flu or fever, medication use, and neural tube defects: A population-based case-control study in northern China. Birth Defects Res A Clin Mol Teratol. 2007 Jan 10; [Epub ahead of print]
3 Lary JM, Edmonds LD. Prevalence of spina bifida at birth—United States, 1983-1990: a comparison of two surveillance systems. MMWR CDC Surveill Summ. 1996, 45(2): 15-26.
4 Zlotogora J, Amitai Y, Kaluski DN, Leventhal A. Surveillance of neural tube defects in Israel. Isr Med Assoc J. 2002, 4(12): 1111-1114.
5 Yuskiv N, Andelin CO, Polischuk S, Shevchuk O, Sosynyuk Z, Vihovska T, Yevtushok L, Oakley GP Jr, Wertelecki W. High rates of neural tube defects in Ukraine. Birth Defects Res A Clin Mol Teratol. 2004, 70(6): 400-402.
6 Graham JM, Edwards MJ. Teratogenic effects of maternal hyperthermia, Annu Rep Res Inst Environ Med Nagoya Univ. 1988, 40: 365-374.
7 Lundberg YW, Wing MJ, Xiong W, Zhao J, Finnell RH. Genetic dissection of hyperthermia-induced neural tube defects in mice. Birth Defects Res A Clin Mol Teratol. 2003, 67(6): 409-413.
8 Wilson RS. Synchronies in mental development: an epigenetic perspective. Science. 1978, 202(4371): 939-948.
9 Welsch F. In vitro approaches to the elucidation of mechanisms of chemical teratogenesis. Teratology. 1992, 46(1): 3-14.
10 Cabrera RM, Hill DS, Etheredge A J, Finnell RH. Investigations into the etiology of neural tube defects. Birth Defects Res C Embryo Today. 2004, 72(4): 330-344.
11 Miller MW, Nvborg WL, Dewey WC, Edwards M J, Abramowicz JS, Bravman AA. Hyperthermic teratogenicity, thermal dose and diagnostic ultrasound during pregnancy: implications of new standards on tissue heating. Int J Hyperthermia. 2002, 18(5): 361-384.
12 Edwards MJ. Review: hyperthermia and fever during pregnancy. Birth Defects Res A Clin Mol Teratol. 2006, 76(7): 507-516.
13 Kimmel GL, Cuff JM, Kimmel CA, Heredia D J, Tudor N, Silvennan PM. Embryonic development in vitro following short-duration exposure to heat. Teratology. 1993, 47(3): 243-251.
14 Edwards MJ. Congenital defects in guinea pig: following induced hyperthermia during gestration. Arch Pathol. 1967, 84(1): 42-48.
15 Edwards MJ. Congenital malformations in the rat following induced hyperthermia during gestation. Teratology. 1968, 1(2): 173-177.
16 Edwards MJ. Hyperthermia as a teratogen: a review of experimental studies and their clinical significance. Teratog Carcinog Mutaggen. 1986, 6(6): 563-582.
17 Chambers CD, Johnson KA, Dick LM, Felix RJ, Jones KL. Maternal fever and birth outcome: a prospective study. Teratology. 1998, 58(6): 251-257.
18 Milunsky A, Ulcickas M, Rothman KJ, Willett W, Jick SS, Jick H. Maternal heat exposure and neural tube defects. JAMA. 1992, 268(7): 882-885.
19 Moretti ME, Bar-Oz B, Fried S, Koren G.Maternal hyperthermia and the risk for neural tube defects in offspring: systematic review and meta-analysis. Epidemiology. 2005, 16(2): 216-219.
20 Chamber CD. Risks of hyperthermia associated with hot tub or spa use by pregnant women. Birth Defects Res A Clin Mol Teratol. 2006, 76(8): 569-573.
21 Edwards MJ. Apoptosis, the heat shock response, hyperthermia, birth defects, disease and cancer. Where are the common links? Cell Stress Chaperones. 1998, 3(4): 213-220.
22 Walsh D, Li Z, Nagata K. Heat shock and the role of the HSPs during neural palate induction in early mammalian CNS and brain development. CMLS. 1993, 53: 198-211.
23 Mirkes PE, Cornel LM, Park HW, Cunnigham ML. Induction of thermotolerance in early postimplantation rat embryos is associated with increased resistance to hyperthermia-induced apoptosis. Teratology. 1997, 56(3): 210-219.
24 Graham JM Jr. Marshall J Edwards discoverer of maternal hyperthermia as a human teratogen. Birth Defects Res A Clin Mol Teratol. 2005, 73(11): 857-864.
25 Padmanabhan R, Al-Menhali NM, Tariq S, Shafiullah M. Mitochondrial dysmorphology in the neuroepithelium of rat embryos following a single dose of maternal hyperthermia during gestation. Exp Brain Res. 2006, 173(2): 298-308.
26 Soleman D, Cornel L, Little SA, Mirkes PE. Teratogen-induced activation of the mitochondrial apoptotic pathway in the yolk sac of day 9 mouse embryos. Birth Defects Res A Clin Mol Teratol. 2003, 67(2): 98-107.
27 Hinoue A, Fushiki S, Nishimura Y, Shiota K. In utero exposure to brief hyperthermia interferes with the production and migration of neocortical neurons and induces apoptotic neuronal death in the fetal mouse brain. Brain Res Dev Brain Res. 2001, 132(1): 59-67.
28 Kim WK, Mirkes PE. Alterations in mitochondrial morphology are associated with hyperthermia-induced apoptosis in early postimplantation mouse embryos. Birth Defects Res A Clin Mol Teratol. 2003, 67(11): 929-940.
29 Chen H, Chan DC. Mitochondrial dynamics in mammals. Curr Top Dev Biol. 2004, 59: 119-144.
30 Paula-Lopes FF, Hansen PJ. Heat shock-induced apoptosis in preimplantation bovine embryos is a developmentally regulated phenomenon. Biol Reprod. 2002, 66(4): 1169-1177.
31 Arechiga CF, Ealy AD, Hansen PJ. Evidence that glutathione is involved in thermotolerance of preimplantation murine embryos. Biol Reprod.1995, 52(6): 1296-1301.
32 Bennett GD, Mohl VK, Finnell RH. Embryonic and maternal heat shock responses to a teratogenic hyperthermic insult. Reprod Toxicol. 1990, 4(2): 113-119.
33 Edwards MJ, Saunders RD, Shiota K. Effects of heat on embryos and foetuses. Int J Hyperthermia. 2003, 19(3): 295-324.
34 Breen JG, Claggett TW, Kimmel GL, Kimmel CA. Heat shock during rat embryo development in vitro results in decreased mitosis and abundant cell death. Reprod Toxicol. 1999, 13(1): 31-39.
35 Finnell RH, Moon SP, Abbott LC, Golden JA, Chemoff GF. Strain differences in heat-induced neural tube defects in mice. Teratology. 1986, 33(2): 247-252.
36 Fleming A, Copp AJ. A genetic risk factor for mouse neural tube defects: defining the embryonic basis. Hum Mol Genet. 2000, 9(4): 575-581.
37 Vacha SJ, Bennett GD, Mackler SA, Koebbe MJ, Finnell RH. Identification of a growth arrest specific (gas5) gene by differential display as a candidate gene for determining susceptibility to hyperthermia-induced exencephaly in mice. Dev Genet. 1997, 21(3): 212-222.
38 Harris MJ. Why are the genes that cause risk of human neural tube defects so hard to find? Teratology. 2001, 63(5): 165-166.
39 MRC. Prevention of neural tube defects: results of the Medical Research Council Vitamin Study. MRC Vitamin Study Research Group. Lancet. 1991, 338(8760): 131-137.
40 Czeizel AE, Dudas I. Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation. N Engl J Med. 1992, 327(26): 1832-1835.
41 Shin JH, Shiota K. Folic acid supplementation of pregnant mice suppresses heat-induced neural tube defects in the offspring. J Nutr. 1999, 129(11): 2070-2073.
42 Botto LD, Erickson JD, Mulinare J, Lynberg MC, Liu Y. Maternal fever, multivitamin use, and selected birth defects: evidence of interaction? Epidemiology. 2002, 13(4): 485-488.
43 Suarez L, Felkner M, Hendricks K. The effect of fever, febrile illnesses, and heat exposures on the risk of neural tube defects in a Texas-Mexico border population. Birth Defects Res A Clin Mol Teratol. 2004, 70(10): 815-819.
44 Acs N, Banhidy F, Puho E, Czeizel AE. Maternal influenza during pregnancy and risk of congenital abnormalities in offspring. Birth Defects Res A Clin Mol Teratol. 2005, 73(12): 989-996.
45 Rozen R. Molecular genetics of methylenetetrahydrofolate reductase deficiency. J Inherit Metab Dis. 1996, 19(5): 589-594.
46 van der Put NM, Gabreels F, Stevens EM, Smeitink JA, Trijbels FJ, Eskes TK, van den Heuvel LP, Blom HJ. A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am J Hum Genet. 1998, 62(5): 1044-1051.
47 Weisberg I, Tran P, Christensen B, Sibani S, Rozen R. A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. Mol Genet Metab. 1998, 64(3): 169-172.
48 Koch MC, Stegmann K, Ziegler A, Schroter B, Ermert A. Evaluation of the MTHFR C677T allele and the MTHFR gene locus in a German spina bifida population. Eur J Pediatr. 1998, 157(6): 487-492.
49 Melnick M, Marazita ML. Neural tube defects, methylenetetrahydrofolate reductase mutation, and north/south dietary differences in China. J Craniofac Genet Dev Biol. 1998, 18(4): 233-235.
50 Shaw GM, Rozen R, Finnell RH, Todoroff K, Lammer EJ. Infant C677T mutation in MTHFR, maternal periconceptional vitamin use, and cleft lip. Am J Med Genet. 1998, 80(3): 196-198.
51 Padmanabhan R. Etiology, pathogenesis and prevention of neural tube defects. Congenit Anom (Kyoto). 2006, 46(2): 55-67.
52 Maddox DM, Manlapat A, Roon P, Prasad P, Ganapathy V, Smith SB. Reduced-folate carrier (RFC) is expressed in placenta and yolk sac, as well as in cells of the developing forebrain, hindbrain, neural tube, craniofacial region, eye, limb buds and heart. BMC Dev Biol. 2003, 3: 6.
53 Zhao R, Russell RG, Wang Y, Liu L, Gao F, Kneitz B, Edelmann W, Goldman ID. Rescue of embryonic lethality in reduced folate carrier-deficient mice by maternal folic acid supplementation reveals early neonatal failure of hematopoietic organs. J Biol Chem. 2001, 276(13): 10224-10228.
54 Barber RC, Shaw GM, Lammer EJ, Greer KA, Biela TA, Lacey SW, Wasserman CR, Finnell RH. Lack of association between mutations in the folate receptor-alpha gene and spina bifida. Am J Med Genet. 1998, 76(4): 310-317.
55 Heil SG, van der Put NM, Trijbels FJ, Gabreels FJ, Blom HJ. Molecular genetic analysis of human folate receptors in neural tube defects. Eur J Hum Genet. 1999, 7(3): 393-396.
56 O'Leary VB, Mills JL, Kirke PN, Parle-McDermott A, Swanson DA, Weiler A, Pangilinan F, Conley M, Molloy AM, Lynch M, Cox C, Scott JM, Brody LC. Analysis of the human folate receptor beta gene for an association with neural tube defects. Mol Genet Metab. 2003, 79(2): 129-133.
57 Homanics GE, Maeda N, Traber MG, Kayden HJ, Dehart DB, Sulik KK. Exencephaly and hydrocephaly in mice with targeted modification of the apolipoprotein B (Apob) gene. Teratology. 1995, 51(1): 1-10.
58 Huang LS, Voyiaziakis E, Markenson DF, Sokol KA, Hayek T, Breslow JL. apo B gene knockout in mice results in embryonic lethality in homozygotes and neural tube defects, male infertility, and reduced HDL cholesterol ester and apo A-I transport rates in heterozygotes. J Clin Invest. 1995, 96(50): 2152-2161.
59 Bamforth SD, Braganca J, Eloranta JJ, Murdoch JN, Marques FI, Kranc KR, Farza H, Henderson DJ, Hurst HC, Bhattacharya S. Cardiac malformations, adrenal agenesis, neural crest defects and exencephaly in mice lacking Cited2, a new Tfap-2 co-activator. Nat Genet. 2001, 29(4): 469-474.
60 Juriloff DM, Gunn TM, Harris MJ, Mah DG, Wu MK, Dewell SL. Multifactorial genetics of exencephaly in SELH/Bc mice. Teratology. 2001, 64(4): 189-200.
61 Ishii M, Han J, Yen HY, Sucov HM, Chai Y, Maxson RE Jr. Combined deficiencies of Msx1 and Msx2 cause impaired patterning and survival of the cranial neural crest. Development. 2005, 132(22): 4937-4950.
62 Stegmann K, Boecker J, Richter B, Capra V, Finnell RH, Ngo ET, Strehl E, Ermert A, Koch MC. A screen for mutations in human homologues of mice exencephaly genes Tfap2alpha and Msx2 in patients with neural tube defects. Teratology. 2001, 63(5): 167-175.
63 Pani L, Horal M, Loeken MR. Rescue of neural tube defects in Pax-3-deficient by p53 loss of function: implications for Pax-3-dependent development and tumorigenesis. Genes Dev. 2002, 16(6): 676-680.
64 Lu W, Zhu H, Wen S, Laurent C, Shaw GM, Lammer EJ, Finnell RH. Screening for novel PAX3 polymorphisms and risks of spina bifida. Birth Defects Res A Clin Mol Teratol. 2007, 79(1): 45-49.
65 Gos M, Szpecht-Potocka A. Genetic basis of neural tube defects. I. Regulatory genes for the neurulation process. J Appl Genet. 2002, 43(3): 343-350.
66 Aruga J. The role of Zic genes in neural development. Mol Cell Neurosci. 2004, 26(2): 205-221.
67 Grinberg I, Millen KJ. The ZIC gene family in development and disease. Clin Genet. 2005, 67(4): 290-296.
68 Morrison K, Papapetrou C, Attwood J, Hol F, Lynch SA, Sampath A, Hamel B, Burn J, Sowden J, Stott D, Mariman E, Edwards YH. Genetic mapping of the human homologue (T) of mouse T (Brachyury) and a search for allele association between human T and spina bifida. Hum Mol Genet. 1996, 5(5): 669-674.
69 Buckiova D, Brown NA. Mechanism of hyperthermia effects on CNS development: rostral gene expression domains remain, despite severe head truncation; and the hindbrain/otocyst relationship is altered. Teratology. 1999, 59(3): 139-147.
70 Gebicke-Haerter P. Expression profiling methods used in drug abuse research. Addict Biol. 2005, 10(1): 37-46.
71 Rosok O, Sioud M. Discovery of differentially expressed genes: technical considerations. Methods Mol Biol. 2007, 360:115-129.
72 Diachenko L, Lau YC, Campbell AP, Chenchik A, Mogadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD. Suppression subtractive hybridization: A method for generating differential regulated or tissue-specifical cDNA probes and libraries. Proc Natl Acad Sci USA. 1996, 93(12): 6025-6030.
73 Gurskaya NG, Diatchenko L, Chenchik A, Siebert PD, Khaspekov GL, Lukyanov KA, Vagner LL, Ermolaeva OD, Lukyanov SA, and Sverdlov ED. Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction: Cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate. Anal Biochem. 1996, 240(1): 90-97.
74 von Stein OD, Thies WG, Hofmann M. A high throughput screening for rarely transcribed differentially expressed genes. Nucleic Acids Res. 1997, 25(13): 2598-2602.
75 Xiao W, Pacyna-Gengelbach M, Sehluns K, An Q, Gao Y, Cheng S, Petersen I. Differentially expressed genes associated with human lung cancer. Oncol Rep. 2005, 14(1): 229-234.
76 Rho J, Altmann CR, Soeei ND, et al. Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis. DNA Cell Biol. 2002, 21(8): 541-549.
77 Wallner EI, Wada J, Lin S, Pan X, Reddy JK, Chugh SS, Kanwar YS. Renal gene expression in embryonic and newborn diabetic mice. Exp Nephrol. 2002, 10(2): 130-138.
78 Wang X, Feuerstein GZ. Suppression subtractive hybridization: application in the discovery of novel pharmacological targets. Pharmacogenomics. 2000, 1(1): 101-108.
79 Chen ZB, Yang J, Wang H, Sun OJ, Wang SQ. Screening and identification of differentially expressed genes from influenza virus infected-host cells by suppression subtractive hybridization. Chinese J Biochem Mol Bio. 2003, 19(2): 156-161.
1 Edwards MJ. Congenital defects in guinea pig: following induced hyperthermia during gestration. Arch Pathol. 1967, 84(1): 42-48.
2 Edwards MJ. Hyperthermia as a teratogen: a review of experimental studies and their clinical significance. Teratog Carcinog Mutaggen. 1986, 6(6): 563-582.
3 Moretti ME, Bar-Oz B, Fried S, Koren G. Maternal hyperthermia and the risk for neural tube defects in offspring: systematic review and meta-analysis. Epidemiology. 2005, 16(2): 216-219.
4 Chamber CD. Risks of hyperthermia associated with hot tub or spa use by pregnant women. Birth Defects Res A Clin Mol Teratol. 2006, 76(8): 569-573.
5 吕锋，高英茂，管英俊．高温致神经管畸形动物模型的建立及其形态发生的研究．解剖学报，1996，27(3)：273-276．
6 Diatchenko L, Lau YFC, Campbell AP, et al. Suppression subtractive hybridization:a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93(18): 6025-6030.
7 Gurskaya NG, Diatchenko L, Cbenchik A, Siebert PD, Khaspekov GL, Lukyanov KA, Vagner LL, Ermolaeva OD, Lukyanov SA, and Sverdlov ED. Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction: Cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate. Anal Biochem. 1996, 240(1): 90-97.
8 Von SOD, Thies WG, Hofmann M, et al. A high throughput screening for rarely transcribed differentially expressed genes. Nucleic Acid Res. 1997, 25(13): 2598-2602.
9 Chenchik A, Zhu YY, Diatchenko L, Li R, Hill J&Siebert PD. (1998) Generation and use of high-quality cDNA from small amounts of total RNA by SMART PCR. In Gene Cloning and Analysis by RT-PCR. BioTechniques Books, MA. I998 pp305-319.
10 Wellenreuther R, Schupp I, Poustka A, Wiemann S. SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones. BMC Genomics. 2004, 5(1): 36.
11 Wilfinger WW, Mackey K, Chomczynski P. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques. 1997, 22(3): 474-476, 478-481.
1 Copp AJ, Greene ND, Murdonch JN. The genetic basis of mammalian neurulation. Nat Rev Genet. 2003, 4(10): 784-793.
2 Harris MJ. Why are the genes that cause risk of human neural tube defects so hard to find? Teratology. 2001, 63(5): 165-166.
3 German J. Embryonic stress hypothesis of teratogenesis. Am J Med. 1984, 76(2): 293-301.
4 Cabrera RM, Hill DS, Etheredge AJ, Finnell RH. Investigations into the etiology of neural tube defects. Birth Defects Res. 2005, 72(4): 330-344.
5 Boyles AL, Hammock P, Speer MC. Candidate gene analysis in human neural tube defects. Am J Med Genet C Semin Med Genet, 2005, 135(1): 9-23.
6 Wool IG, Chan YL, Gluck A. Structure and evolution of mammalian ribosomal proteins. Biochem Cell Biol. 1995, 73(11-12): 933-947.
7 Vilardell J, Chartrand P, Singer RH, Warner JR.The odyssey of a regulated transcript. RNA. 2000, 6(12): 1773-1780.
8 Watanabe M, Zinn AR, Page DC, Nishimoto T. Functional equivalence of human X- and Y-encoded isoforms of ribosomal protein S4 consistent with a role in Turner syndrome. Nature Genetics. 1993, 4(3): 268-271.
9 Watanabe M, Furuno N, Goebl M, Go M, Miyauchi K, Sekiguchi T, Basilico C, Nishimito T. Molecular cloning of the human gene, CCG2, that complements the BHK-derived temperature-sensitive cell cycle mutant tsBN63: identity of CCG2 with the human X chromosomal SCAR/RPS4X gene. Journal of Cell Science. 1991, 100(Pt 1): 35-43.
10 Balakirev MY, Tcherniuk SO, Jaquinod M, Chrobocaek J. Otubains: a new family of cysteine proteases in the ubiquitin pathway. EMBO Reports. 2003, 4(5): 517-522.
11 Soares L, Seroogy C, Skrenta H, Anandasabapathy N, Lovelace P, Chung CD, Engleman E, Fathrrlan CG. Two isoforms of otubain 1 regulate T cell anergy via GRAIL. Nature Immunology. 2004, 5(1): 45-54.
12 Strayhorn WD, Wadzinski BE. A novel in vitro assay for deubiquitination of IκBα. Arch Biocem Biophys. 2002, 400(1): 76-84.
13 Balakirey MY, Tcherniuk SO, Jaquinod M, Chroboczek J. Otubains: a new family of cysteine proteases in the ubiquitin pathway. EMBO Rep. 2003, 4(5):517-522.
14 Ernst H, Duncan RF, Hershey JW. Cloning and sequencing of complementary DNAs encoding the alpha-subunit of translational initiation factor Eif-2. Characterization of the protein and its messenger RNA. Journal of Biological Chemistry. 1987, 262(3): 1206-1212.
15 Scheuner D, Song B, McEwen E, Liu C, Laybutt R, Gillespie P, Saunders T, Bonner-Weir S, Kaufrnan RJ. Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Molecular Cell. 2001, 7(6): 1165-1176.
16 Boyce M, Bryant KF, Jousse C, Long K, Harding HP, Scheuner D, Kaufman RJ, Ma D, Coen DM, Ron D, Yuan J. A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress. Science. 2005, 307(5711): 935-939.
17 Komar AA, Gross SR, Barth-Baus D, Strachan R, Hensold JO, Goss Kinzy T, Merrick WC. Novel characteristics of the biological properties of the yeast saccharomyces cerevisiae eukaryotic initiation factor 2A. J Biol Chem. 2005, 280(16): 15601-15611.
18 Zoll WL, Horton LE, Komar AA, Hensold JO, Merrick WC. Characterization of mammalian eIF2A and identification of the yeast homolog. J Biol Chem. 2002, 277(40): 37079-37087.
19 Page AB, Owen CR, Kumar R, Miller JM, Rafols JA, White BC, DeGracia DJ, Krause GS. Persistent eIF2alpha(P) is colocalized with cytoplasmic cytochrome c in vulnerable hippocampal neurons after 4 hours of reperfusion following 10-minute complete brain ischemia. Acta Neuropathologica. 2003, 106(1): 8-16.
20 Suske G The Sp-family of transcription factors. Gene. 1999, 238(2): 291-300.
21 Kingsley C, Winoto A. Cloning of GT box-binding proteins: a novel Sp 1 multigene family regulating T-cell receptor gene expression. Molecular Cell Biology. 1992, 12(10): 4251-4261.
22 Hagen G, Muller S, Beato M, Suske G. Spl-mediated transcriptional activation is repressed by Sp3. EMBO Journal. 1994, 13(16): 3843-3851.
23 Lou Z, Maher VM, McCormick JJ. Identification of the promoter of human transcription factor Sp3 and evidence of the role of factors Sp1 and Sp3 in the expression of Sp3 protein. Gene. 2005, 351:51-59.
24 Dobson MJ, Tuite MF, Roberts NA, Kingsman AJ, Kingsman SM, Perkins RE, Conroy SC, Fothergill LA. Conversation of high efficiency promotor sequences in Saccharomyces cerevisiae. Nucleic Acids Res. 1982, 10(8): 2625-2637.
25 Rathjen J, Mellor J. Characterization of sequences required for RNA initiation from the PGK promoter of Saccharomyces cerevisiae. Nucleic Acids Res. 1990, 18(11): 3219-3125.
26 de Almeida JR, de Moraes LM, Torres FA. Molecular characterization of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris. Yeast. 2005, 22(9): 725-737.
27 Gao L, Mejias R, Echevarria M, Lopez-Barneo J. Induction of the glucose-6-phosphate dehydrogenase gene expression by chronic hypoxia in PC 12 cells. FEBS Lett. 2004, 569(1-3): 256-260.
1 Schmidt-Zachmann MS, Franke WW. DNA cloning and amino acid sequence determination of a major constituent protein of mammalian nucleoli. Correspondence of the nucleoplasmin-related protein NO38 to mammalian protein B23. Chromosoma. 1988, 96(6): 417-426.
2 Grisendi S, Pandolfi PP. NPM mutations in acute myelogenous leukemia. N Engl J Med. 2005, 352(3): 291-292.
3 Eirin-Lopez JM, Frehlick LJ, Ausio J. Long-term evolution and functional diversification in the members of the nucleophosmin/nueleoplasmin family of nuclear chaperones. Genetics. 2006, 173(4): 1835-1850.
4 Dingwall C, Laskey RA. Nucleoplasmin: the archetypal molecular chaperone. Semin Cell Biol. 1990, 1(1): 11-17.
5 Szebeni A, Herrera JE, Olson MO. Interaction of nuclear protein B23 with peptides related to nuclear localization signals. Biochemistry. 1995, 34(25): 8037-8042.
6 Borer RA, Lehner CF, Eppenberger HM, Nigg EA. Major nueleolar proteins shuttle between nucleus and cytoplasm. Cell. 1989, 56(3): 379-390.
7 Grisendi S, Bernardi R, Rossi M, Cheng K, Khandker L, Manova K, Pandolfi PP. Role of nucleophosmin in embryonic development and tumorigenesis. Nature. 2005, 437(7055): 147-153.
8 Chan WY, Liu QR, Borjiqin J, Busch H, Rennert OM, Tease LA, Chan PK. Characterization of the cDNA encoding human nucleophosmin and studies of its role in normal and abnormal growth. Biochemistry. 1989, 28(3): 1033-1039.
9 Herrera JE, Savkur R, Olson MO. The ribonuclease activity of nucleolar protein B23. Nucleic Acids Res. 1995, 23(19): 3974-3979.
10 Nozawa, Y, Van Belzen N, Van der Made AC, Dinjens WN, Bosman FT. Expression of nucleophosmin/B23 in normal and neoplastic colorectal mucosa. J Pathol. 1996, 178(1): 48-52.
11 Subong EN, Shue MJ, Epstein JI, Briggrnan JV, Chan PK, Partin AW. Monoclonal antibody to prostate cancer nuclear matrix protein (PRO:4-216) recognizes nucleophosmin/B23. Prostate. 1999, 39(4): 298-304.
12 Herrera JE, Savkur R, Olson MO. The ribonuelease activity of nueleolar protein B23. Nucleic Acids Res. 1995, 23(19): 3974-3979.
13 Wang BB, Lu R, Wang WC, Jin Y. Inducible and reversible suppression of Npm1 gene expression using stably integrated small interfering RNA vector in mouse embryonic stem cells. Biochem Biophys Res Commun. 2006, 347(4): 1129-1137.
14 He J, Chang LJ. Functional characterization of hepatoma-specific stem cell antigen-2. Molecular Carcinogenesis. 2004, 40(2): 90-103.
15 Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998, 391(6669): 806-811.
16 Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001, 411(6836): 494-498.
17 Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs the ATP-dependent cleavage ofmRNA at 21 to 23 nucleotide intervals. Cell. 2000, 101(1): 25-33.
18 Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev. 2001, 15(2): 188-200.
19 Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature. 2001, 409(6818): 363-366.
20 Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell. 2003, 115(2): 199-208.
21 Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell. 2003, 115(2): 209-216.
22 Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature. 2000, 404(6775): 293-296.
23 Nykanen A, Haley B, Zamore PD. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell. 2001, 107(3): 309-321.
24 Martinez J, Patkaniowska A, Urlaub H, Luhrmann R, Tuschl T. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell. 2002, 110(5): 563-574.
25 Mosmann T. Rapid colorimetric assay for cellular growth and survival: applications to proliferation and cytotoxicity assays. J Immunol Methods. 1983, 65(1-2): 55-63.
26 Berridge MV, Tan AS. Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch Biochem Biophys. 1993,303(2): 474-482.
27 Hayon T, Dvilansky A, Shpilberg O, Nathan I. Appraisal of the MTT-based assay as a useful tool for predicting drug chemosensitivity in leukemia. Leuk Lymphoma. 2003, 44(11): 1957-1962.
28 Tchounwou PB, Yedjou CG, Foxx D, Ishaque A, Shen E. Lead-induced cytotoxity and transcriptional activation of stress genes in human liver carcinoma cells. Mol Cell Biochem. 2004, 255(1-2): 161-170.
29 Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Armu Rev. 2005, 11: 127-152.
30 Zeller KI, Haggerty TJ, Barrett JF, Guo Q, Wonsey DR, Dang CV. Characterization ofnucleophosmin (B23) as a Myc target by scanning chromatin immunoprecipitation. J Biol Chem. 2001, 276(51): 48285-48291.
31 Lim MJ, Wang XW. Nucleophosmin and human cancer. Cancer Detect Prev. 2006, 30(6): 481-490.
32 Hsu CY, Yung BY. Down-regulation of nucleophosmin/B23 during retinoic acid-induced differentiation of human promyelocytic leukemia HL-60 cells. Oncogene. 1998, 16(7): 915-923.
33 Liu WH, Yung BY. Mortalization of human promyelocytic leukemia HL-60 cells to be more susceptible to sodium butyrate-induced apoptosis and inhibition of telomerase activity by down-regulation of nucleophosmin/B23. Oncogene. 1998, 17(23): 3055-3064.
34 Patterson SD, Grossman JS, D'Andrea P, Latter GI. Reduced numatrin/B23/nucleophosmin labeling in apoptotic Jurkat T-lymphoblasts. J Biol Chem. 1995, 270(16): 9429-9436.
35 Feuerstein N, Mond JJ. Identification of a prominent nuclear protein associated with proliferation of normal and malignant B cells. J Immunol. 1987, 139(6): 1818-1822.
36 Gerke V, Moss SE. Annexins: from structure to function. Physiol Rev. 2002, 82(2): 331-371.
37 Williamson P, van den Eijnde S, Schlegel RA. Phosphatidylserine exposure and phagocytosis of apoptotic cells. Methods Cell Biol. 2001, 66: 339-364.
38 van Engeland M, Nieland LJ, Ramaekers FC, Schutte B. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry. 1998, 31(1): 1-9.
39 Plasier B, Lloyd DR, Paul GC, Thomas CR, Al-Rubeai M. Automatic image analysis for quantification of apoptosis in animal cell culture by annexin-V affinity assay. J Immunol Methods. 1999, 229(1-2): 81-95.
40 Hanshaw RG, Smith BD. New reagents for phosphatidylserine recognition and detection of apoptosis. Bioorg Med Chem. 2005, 13(17): 5035-5042.
41 Kanagaraj P, Vijayababu MR, Ravisankar B, Anbalagan J, Aruldhas MM, Arunakaran J. Effect of lycopene on insulin-like growth factor-Ⅰ, IGF binding protein-3 and IGF type-Ⅰ receptor in prostate cancer cells. J Cancer Res Clin Oncol. 2007 Jan 12; [Epub ahead of print]
42 Kuida K, Haydar TF, Kuan CY, et al. Reduced apoptpsis and cytochrome c-mediated caspase activation in mice lacking caspase-9. Cell. 1998, 94(3): 325-337.
43 Kuida K, Zheng TS, Na S, Kuan C,et al. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature. 1996, 384(6607): 368-372.
44 Jin Z, El-Deiry WS. Overview of cell death signaling pathways. Cancer Biol Ther. 2005, 4(2): 139-163.
45 Creagh EM, Conroy H, Martin SJ. Caspase-activation pathways in apoptosis and immunity. Immunol Rev. 2003, 193: 10-21.
46 Nunez G, Benedict MA, Hu Y, Inohara N. Caspase: the proteases of the apoptotic pathway. Oncogene. 1998, 17(25): 3237-3245.
47 Fan TJ, Han LH, Cong RS, Liang J. Caspase family proteases and apoptosis. Acta Biochim Biophys Sin (Shanghai). 2005, 37(11): 719-727.
48 Kuribayashi K, Mayes PA, El-Deiry WS. What are caspase 3 and 7 doing upstream of the mitochondria? Cancer Biol Ther. 2006, 5(7): 763-765.
49 Itahana K, Bhat KP, Jin A, Itahana Y, Hawke D, Kobayashi R, Zhang Y. Tumor suppressor ARF degrades B23, a nucleolar protein involved in ribosome biogenesis and cell proliferation. Mol Cell. 2003, 12(5): 1151-1164.
50 Grisendi S, Pandolfi PP. NPM mutations in acute myelogenous leukemia. N Engl J Med. 2005, 352(3): 291-292.
51 Colombo E, Bonetti P, Lazzerini Denchi E, Martinelli P, Zamponi R, Marine JC, Helin K, Falini B, Pelicci PG. Nucleophosmin is required for DNA integrity and p 19Arfprotein stability. Mol Cell Biol. 2005, 25(20): 8874-8886.
52 Colombo E, Marine JC, Danovi D, Falini B, Pelicci PG. Nucleophosmin regulates the stability and transcriptional activity of p53. Nat Cell Biol. 2002, 4(7): 529-533.
53 Chan HJ, Weng JJ, Yung BY. Nucleophosmin/B23-binding peptide inhibits tumor growth and up-regulates transcriptional activity of p53. Biochem Biophys Res Commun. 2005, 333(2): 396-403.
54 Lambert B, Buckle M. Characterisation of the interface between nucleophosmin (NPM) and p53: potential role in p53 stabilisation. FEBS Lett. 2006, 580(3): 345-350.
55 Chou CC, Yung BY. Increased stability of nucleophosmin/B23 in anti-apoptotic effect ofras during serum deprivation. Mol Pharmacol. 2001, 59(1): 38-45.
56 Li J, Zhang XL, Sejas DP, Pang Q. Negative regulation of p53 by nucleophosmin antagonizes stess-induced apoptosis in human normal and malignant hematopoietic cells. Leukemia Research. 2005, 29(12): 1415-1423.
57 Li J, Sejas DP, Rani R, Koretsky T, Bagby GC, Pang Q. Nucleophosmin regulates cell cycle progression and stress response in hematopoietic stem/progenitor cells. J Biol Chem. 2006, 281(24): 16536-16545.