一种快速有效分析烟草花冠中花青素苷的方法

花青素苷（anthocyanins）成分和含量的分析是花色研究的重要内容之一。该文利用高效液相色谱-电喷雾离子化-质谱技术（HPLC-ESI-MS）, 建立了一种快速有效地分析烟草（Nicotiana tabacum）花冠中花青素苷成分及含量的方法。在保证良好分离效果的基础上, 将分析时间缩短至15分钟, 大大提高了分析速度, 降低了成本, 并成功应用于转基因烟草花冠中花青素苷成分的分析。为不具备高效分析仪器（如超高效液相色谱-串联质谱（UPLC-MS/MS））的一般实验室研究花青素苷合成相关基因在烟草中的异源表达提供了一种有效且实用的分析方法。     

Biochemical synthesis of novel,self-assembling glycolipids from ricinoleic acid by a recombinant α-glucosidase from <Emphasis Type="Italic">Geobacillus</Emphasis> sp.

Purpose of work  

To explore a novel glycolipid, we performed biochemical reactions using a recombinant α-glucosidase from Geobacillus sp. which shows excellent transglycosylation reaction to hydroxyl groups in a variety of compounds.     

人和动物胃肠道微生物丁酸合成途径相关酶基因的研究进展

人和动物胃肠道中都栖息了大量的产丁酸微生物。研究表明,参与丁酸合成中心途径的酶基因成簇存在,其相关基因的排列形式多样并具有属种特异性。参与丁酸合成最后一步的(丁酰辅酶A/乙酸)辅酶A(CoA)转移酶在胃肠道产丁酸微生物中广泛存在,并在丁酸合成中发挥重要作用。本文结合我们的研究工作,综述了国内外丁酸合成相关酶基因和基因簇的最新研究进展。     

羊水直接PCR法快速诊断脊肌萎缩症的实验研究

目的：建立一种快速、简便的产前诊断脊肌萎缩症的检测方法。方法：基于运动神经元生存基因SMNc和SMNt的两个同源拷贝碱基的差异,对SMNt基因第7、8外显子进行缺失分析,抽取孕妇羊水后用作者自行研制的“HpH—Buffer”,以离心后羊水沉淀中的胎儿细胞为模板直接进行PCR扩增,扩增产物进行酶切限制性片断长度多态性分析,对2例有脊肌萎缩症患儿生育史的孕妇怀有的胎儿进行产前基因诊断。同时,为考察羊水直接扩增方法的效率,对羊水样本直接扩增和对羊水样本中提取的基因组DNA扩增进行了平行实验。结果：2例胎儿均有SMNt基因第7、8外显子缺失;以羊水为模板和以基因组DNA为模板进行扩增的效率相似。结论：应用“HpHBuffer”直接对羊水样本中SMN基因上外显子7、8进行扩增,结合限制性片断长度多态性分析SMNt上是否发生缺失,可缩短诊断时间,节约诊断成本,减少检测过程中可能出现的样本交叉污染,有望成为临床上产前诊断脊肌萎缩症的新方法。     

O‐GlcNAcylation of protein kinase A catalytic subunits enhances its activity: a mechanism linked to learning and memory deficits in Alzheimer's disease

Alzheimer's disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)‐CREB signaling plays a critical role in learning and memory. It is known that glucose uptake and O‐GlcNAcylation are reduced in AD brain. In this study, we found that PKA catalytic subunits (PKAcs) were posttranslationally modified by O‐linked N‐acetylglucosamine (O‐GlcNAc). O‐GlcNAcylation regulated the subcellular location of PKAcα and PKAcβ and enhanced their kinase activity. Upregulation of O‐GlcNAcylation in metabolically active rat brain slices by O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidenamino) N‐phenylcarbamate (PUGNAc), an inhibitor of N‐acetylglucosaminidase, increased the phosphorylation of tau at the PKA site, Ser214, but not at the non‐PKA site, Thr205. In contrast, in rat and mouse brains, downregulation of O‐GlcNAcylation caused decreases in the phosphorylation of CREB at Ser133 and of tau at Ser214, but not at Thr205. Reduction in O‐GlcNAcylation through intracerebroventricular injection of 6‐diazo‐5‐oxo‐l ‐norleucine (DON), the inhibitor of glutamine fructose‐6‐phosphate amidotransferase, suppressed PKA‐CREB signaling and impaired learning and memory in mice. These results indicate that in addition to cAMP and phosphorylation, O‐GlcNAcylation is a novel mechanism that regulates PKA‐CREB signaling. Downregulation of O‐GlcNAcylation suppresses PKA‐CREB signaling and consequently causes learning and memory deficits in AD.     

大鼠初级感觉神经元P2X3受体的表达及其与SP的关系

目的研究在大鼠初级感觉神经元细胞上P2X3受体的表达情况及其与P物质的关系。方法取SD大鼠背根神经节(DRG)和三叉神经节(TG)固定后切片;用抗P2X3受体抗体和抗SP抗体进行免疫组织化学反应,并通过两种不同的显色方法同时进行P2X3受体和SP的双标。结果P2X3免疫反应阳性细胞主要集中在小细胞和中等细胞(其中在TG,P2X3-ir阳性神经元约占整个细胞的24.8%;在DRG约31.7%的神经元是P2X3-ir阳性),并且在DRG和TG细胞上均存在有P2X3受体和SP共存(TG上的双标细胞占P2X3-ir阳性细胞总数的36.26%,DRG上占46.81%)。结论由于ATP门控阳离子通道受体P2X3本身就与伤害性感受的初级传入有关,而它与SP的共存可提示当组织中的ATP释放时可以通过P2X3受体作用于含SP的伤害性感觉神经末梢上,促使SP释放引起痛觉过敏。     

干扰小RNA序列非依赖性地影响胰腺癌细胞的基因表达

干扰小RNA(small interference RNA, siRNA)是基因敲减的常用工具,广泛用于基因沉默技术和基因功能研究,在临床疾病治疗等方面也有潜在的应用。一般认为,达到一定长度（比如大于27 bp）的双链RNA可以诱导干扰素反应,降低相关基因的表达。目前,siRNA对基因表达的非特异性作用尚不完全清楚。为研究siRNA干扰的非特异基因表达,本研究以胰腺癌细胞HPAC 和BxPC3 为模型,采用高通量测序技术对6种不同干扰小RNA处理及未作处理的HPAC和BxPC3细胞进行转录组测序分析,筛选出干扰小RNA处理后表达量共同下调的基因进行研究。通过生物信息学方法对表达下调基因的功能进行研究,并利用实时荧光定量PCR(qRT-PCR)技术对部分下调基因进行验证。结果表明,短片段双链小RNA能够显著改变细胞的基因表达,而这些基因表达谱的变化是有规律的,特定功能的基因优先发生变化。在表达下调的基因中,某些特定类型的基因变化非常显著,包括氨基酸代谢相关基因、Hedgehog信号途径基因和多巴胺受体D5基因等。这些结果表明,在使用siRNA时需要考虑其序列非依赖性地基因表达调控作用。     

Recent amplification of the kangaroo endogenous retrovirus, KERV, limited to the centromere

Mammalian retrotransposons, transposable elements that are processed through an RNA intermediate, are categorized as short interspersed elements (SINEs), long interspersed elements (LINEs), and long terminal repeat (LTR) retroelements, which include endogenous retroviruses. The ability of transposable elements to autonomously amplify led to their initial characterization as selfish or junk DNA; however, it is now known that they may acquire specific cellular functions in a genome and are implicated in host defense mechanisms as well as in genome evolution. Interactions between classes of transposable elements may exert a markedly different and potentially more significant effect on a genome than interactions between members of a single class of transposable elements. We examined the genomic structure and evolution of the kangaroo endogenous retrovirus (KERV) in the marsupial genus Macropus. The complete proviral structure of the kangaroo endogenous retrovirus, phylogenetic relationship among relative retroviruses, and expression of this virus in both Macropus rufogriseus and M. eugenii are presented for the first time. In addition, we show the relative copy number and distribution of the kangaroo endogenous retrovirus in the Macropus genus. Our data indicate that amplification of the kangaroo endogenous retrovirus occurred in a lineage-specific fashion, is restricted to the centromeres, and is not correlated with LINE depletion. Finally, analysis of KERV long terminal repeat sequences using massively parallel sequencing indicates that the recent amplification in M. rufogriseus is likely due to duplications and concerted evolution rather than a high number of independent insertion events.     

Effects of rare earth elements on telomerase activity and apoptosis of human peripheral blood mononuclear cells

To study the effects of rare earth exposure on human telomerase and apoptosis of mononuclear cells from human peripheral blood (PBMNCs). The blood contents of 15 rare earth elements, including La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and Y, were measured by inductively coupled plasma-mass spectrometry. Telomeric repeat amplification protocol assay and flow cytometer analysis were carried out to analyze the telomerase activity and apoptosis of PBMNCs, respectively. The total content of rare earth elements in the blood showed significant differences between the exposed group and the control group. The rare earth exposure increased the telomerase activity and the percentages of cells in the S-phase and the G₂/M phase in PBMNCs, but it had no effect on the apoptotic rate of PBMNCs. Under the exposure to lower concentrations of rare earth elements, the telomerase activity of PBMNCs in the exposed group was higher than that of the control group, and there was no effect on the apoptotic rate of PBMNCs, but promoted the diploid DNA replication and increased the percentages of G₂/M- and S-phase cells.