栉孔扇贝(Chlamys farreri)Toll样受体及其信号传递相关基因的克隆与表达

扇贝是我国海水养殖的重要品种，但自1994年以来，养殖扇贝陆续爆发的大规模死亡，不但造成了巨大的经济损失，而且直接威胁到现有产业的生存和发展。引起扇贝大规模死亡原因是多方面的，其主要原因是养殖环境恶化、扇贝种质衰退和抗病力下降。因此，深入研究扇贝免疫防御机制，探讨提高机体抗病力的有效途径和方法，改良种质和培育抗病品系，无疑是解决目前困扰扇贝养殖业健康可持续发展的必经之路。 Toll样受体（TLRs）家族是新近发现的模式识别受体（PRRs），参与识别病原体相关的分子模式（PAMPs），在天然免疫系统中起着非常重要的作用。哺乳动物中Toll样受体信号通路还参与诱导树枝状细胞成熟、参与免疫耐受、参与凋亡发生发展、介导非感染性因素的识别等，被视为联系天然免疫和获得性免疫的桥梁。同时果蝇的Toll信号通路也是不具备获得性免疫的果蝇赖以抵御病毒、细菌和真菌感染，介导天然免疫反应的重要信号通路。 本研究采用大规模EST测序方法，结合Genome Walker库的构建和cDNA末端快速扩增技术，从栉孔扇贝克隆得到CfToll-1、CfMyd88、CfTRAF6和CfCactus这四个Toll样受体信号通路基因的全长cDNA，同时用荧光实时定量PCR技术检测了这些基因的组织分布及在脂多糖（LPS）和肽聚糖（PGN）刺激下的表达规律。 栉孔扇贝Toll样受体（CfToll-1）的cDNA序列全长4308 bp，包含5’非翻译区（UTR）211 bp，3597 bp的开放阅读框，500 bp的3’UTR，最后为18个腺嘌呤的ploy A 尾巴。开放阅读框编码1198个氨基酸的多肽，该多肽的估计分子量为137.41kd，估计的等电点为5.62，该多肽有信号肽，具有一个预测的跨膜区，因此是一种跨膜蛋白。经BLAST比对，CfToll-1基因与节肢动物多种Toll蛋白高度的相似性。SMART（Simple Modular Architecture Research Tool）软件分析，CfToll-1包含典型的Toll样受体的结构：富含亮氨酸的重复序列的胞外区（leucine-rich repeats, LRR），一段跨膜结构域，以及胞内区的TIR结构域（Toll/IL-1 receptor homologous region）。利用Real-time RT-PCR发现CfToll-1mRNA在扇贝体内普遍存在于血细胞、肌肉、外套膜、心、性腺和鳃组织中。利用体外培养的原代血细胞系研究不同浓度LPS刺激后CfToll-1的表达变化，结果显示低剂量（100ng.mL-1 ）LPS 使CfToll-1 mRNA表达量减小，该变化在1.5h、3h 和9h组差异显著，虽然在6h组表达量稍有恢复，但尚未达到对照水平；用1μg.mL-1LPS处理细胞时， 6h组CfToll-1表达量明显上调，约为对照水平的2倍。证实细菌结构脂多糖对CfToll-1基因的表达有影响，且这种影响有剂量依赖效应。 栉孔扇贝Myd88同源基因（CfMyd88）的cDNA序列全长1554bp，包含5’UTR 427 bp，1101bp的开放阅读框，最后为18个腺嘌呤的ploy A 尾。CfMyd88的开放阅读框可编码367个氨基酸的多肽，该多肽的估计分子量为42.37kD，估计的等电点为5.71。利用SMART程序分析发现CfMyd88编码了Death和TIR结构域, 这两个结构域是Myd88特征结构。BLAST程序发现扇贝的序列与数据库哺乳动物的Myd88基因高度同源。原代培养的扇贝血细胞在受到PGN刺激后，CfMyd88 mRNA表达在1.5小时开始下调，直到9小时下调至对照表达量的1/10，证实肽聚糖结构对CfMyd88基因的表达有影响。 栉孔扇贝TRAF6同源基因（CfTRAF6）的cDNA序列全长2510bp，包含5’UTR 337 bp，1965bp的开放阅读框，3’UTR 208bp，最后为21 个腺嘌呤的ploy A 尾巴。CfTRAF6开放阅读框编码655个氨基酸的多肽，该多肽的估计分子量为74.09kD，估计的等电点为6.01。InterPro Scan在线分析发现CfTRAF6有典型的TRAF蛋白家族的特征结构，包括的一个指环结构，两个锌指结构，一个MATH （the meprin and TRAF homology）结构域以及Coiled-coil区域。CfTRAF6的序列与数据库多物种的TRAF6高度同源，同源性最高的是乌贼序列（Identity＝68）和鼠类（Identity＝45%）。利用Real-time RT-PCR，发现CfTRAF6在各组织普遍存在，在性腺中的表达最高。原代培养的扇贝血细胞在受到不同浓度PGN刺激后，与CfMyd88的情况一样，CfTRAF6的表达量变化减少，且这种变化随剂量的增加更加明显。 栉孔扇贝Cactus同源基因（CfCactus）的cDNA序列全长2488bp，包含5’UTR 181 bp，840bp的开放阅读框， 3’UTR 1467bp，最后为19个腺嘌呤的ploy A 尾巴。CfCactus的开放阅读框编码279个氨基酸的多肽，该多肽的估计分子量为31.37 kD；估计的等电点为4.74，与果蝇的Cactus基因的等电点相近（4.5）。利用SMART程序分析发现CfCactus主要编码了ANK结构域（ankyrin repeats）。Cactus基因为哺乳动物NF-κB抑制蛋白IκB的同源分子，BLAST 程序发现扇贝的序列与数据库多物种的Cactus或IκB基因高度同源。同源性最高的是太平洋牡蛎（Identity=35%）和圆尾鲎（Identities = 44%）。对CfTCactus mRNA在扇贝的血细胞、性腺、 肠的组织表达进行分析，并同时与CfTRAF6和CfMyd88的表达量进行了对比，发现CfCactus的表达水平明显高于这两个基因，而且CfTRAF6的基因表达量也高于CfMyd88，表现出级联放大效应。正常情况下，三个基因在性腺的表达量最高，推测这条通路可能和发育等功能密切相关。 通过本研究我们首次在双壳类软体动物找得到与果蝇Toll蛋白家族高度同源的CfToll-1基因，同时发现其他三个在Toll样受体信号传递过程中起重要作用的基因，其中包括在软体动物中获得的第一个Toll样受体的接头分子－CfMyd88基因，该结果直接证明软体动物具有与哺乳动物和节肢动物高度类似Myd88依赖的Toll样受体信号通路。同时通过这些基因组织分布的研究以及细菌结构LPS和PGN对这条通路上基因表达的影响，证明扇贝Toll信号通路可能与在果蝇中一样，参与扇贝的发育和免疫防御等多种功能。

中国明对虾病原检测新方法及酚氧化酶通路相关基因的研究

对虾病害在世界范围内肆虐，给水产养殖和沿海农村经济造成了重大损失。在水产养殖的实践中快速检测水产动物的病害并及时采取隔离等措施对于控制病害尤为重要，其中关键的环节就是快速检测出病害，并在对虾免疫机制上寻找对虾疾病防治的有效方法。研究表明当对虾等甲壳动物受到外界病原刺激时，极微量的微生物多糖就可以激活proPO系统。激活过程中涉及和产生一系列活性物质，如黑色素、酚氧化酶原激活因子（PPA）、模式识别蛋白（BGBP、PGBP、LGBP、LBP）及其膜上受体和A2巨球蛋白等，它们可通过多种方式参与防御反应，包括提供调理素，促进血细胞吞噬作用，形成结节或包囊以及介导凝集和凝固，产生杀菌物质并且黑色素化。黑色素常常在节肢动物的体表形成黑色斑点，形成的色素沉着对机体起到保护作用。所以，酚氧化酶原激活的级联反应是节肢动物免疫的关键因素。本论文研究开发了以环等温介导技术（LAMP）为基础的检测对虾白斑病毒（WSSV）和鳗弧菌（V. anguillarum）的快速检测方法。并从对虾对病害的免疫机制为切入点，从中国明对虾体内克隆了酚氧化酶原（PrpPO）和丝氨酸蛋白酶FcSP3这两个免疫系统中重要的基因，分析了它们的分子结构特征，组织分布及应答鳗弧菌病原刺激的表达变化模式。 建立的对虾常见病害对虾白班病毒（WSSV）和鳗弧菌（V. anguillarum）的LAMP检测方法，经过实验比对和Blast检索，发现本研究中使用的引物，比已经报导的LAMP方法或者PCR方法具有更宽的检测范围（更低的假阴性）。检测WSSV的LAMP方法使用病毒的VP28基因设计引物，而鳗弧菌的检测方法使用empA基因设计引物。在方法中，首次提出加入UNG酶和dUTP的措施来预防污染，在实际检测中非常有效。LAMP方法与PCR检测方法的灵敏性比较也进行了研究，二者灵敏性相当。 依据中国明对虾血液cDNA文库提供的部分片段信息，结合SMART-RACE技术，克隆了酚氧化酶原（PrpPO）基因，通过序列比对分析发现，PrpPO基因cDNA全长为3040 bp，其中开放阅读框2061 bp，编码686个氨基酸，其中推测的信号肽为12个氨基酸。推测的序列与斑节对虾（P. monodon）同源性为93%,与短钩对虾（P. semisulcatus.）同源性为92%。real time RT-PCR实验结果表明， ProPO在血细胞中的相对表达量最高，肝胰脏中表达量最低。弧菌刺激实验中注射弧菌，刺激了血细胞和淋巴器官中的ProPO mRNA显著增加，说明在血细胞和淋巴器官中存在快速反应的ProPO通路。而ProPO mRNA量在淋巴器官中在时间上早于血液中升至最高，说明该动物在在病原刚开始入侵的时候先有淋巴器官发挥主要的免疫作用，随着时间推移血细胞便变成主要的免疫器官。 根据中国明对虾肝胰脏cDNA文库提供EST信息，经过SMART-RACE克隆了一个丝氨酸蛋白酶FcSP3基因，通过序列比对分析发现，该丝氨酸蛋白酶基因cDNA全长为1622 bp，其中开放阅读框1431 bp，编码477个氨基酸，其中推测的信号肽为22个氨基酸。推测的序列与疟蚊的丝氨酸蛋白酶（A. gambiae）同源性为33%,与丽蝇蛹集金小蜂的酚氧化酶原激活因子（N. vitripennis）同源性为32%，与东北大黑鳃金龟的酚氧化酶原激活因子（H. diomphalia）同源性为34%。淋巴器官中PPAⅡ表达量约为血液中表达量的47560倍，肝胰脏中的FCSP3表达量为血细胞表达量的6226倍。鳗弧菌注射对虾后，淋巴器官中刺激组和对照组FcSP3的mRNA量在刺激后6小时显著降低，但是刺激组的表达量明显高于对照组。刺激组的血细胞与肝胰脏中FcSP3 mRNA的相对表达量增高。而病原刺激后的血液与肝胰脏中的FcSP3 mRNA的增长趋势也在时间上先与ProPO mRNA。这说明FcSP3对ProPO有正调控的作用，但这个调控有一个时间差，并且在不同组织中有不同的调控效率。

中国明对虾重要分子伴侣蛋白基因的克隆及表达研究

对虾养殖业的可持续发展面临着种质退化、病害严重和养殖环境恶化等问题的严重挑战。养殖环境恶化造成的环境胁迫，不但影响对虾的生长性状，而且导致对虾的抵抗力下降，更容易引发病害的发生。养殖环境恶化已经严重影响了对虾养殖业的健康可持续发展。本论文针对环境恶化造成的环境胁迫对对虾影响的分子机理进行了研究。 克隆了中国明对虾对环境胁迫应答的重要伴侣蛋白基因，包括钙网蛋白（FcCRT）、葡萄糖调节蛋白78 （FcGrp78）、热休克蛋白70（FcHsp70）和热休克蛋白90（FcHsp90）的全长cDNA，研究了这些基因的组织表达特征，并对这些基因在不同胁迫条件下的转录表达特征进行了分析。 钙网蛋白是一种多功能的内质网钙结合蛋白，负责蛋白折叠和糖蛋白修饰。本论文首次在中国明对虾报道了钙网蛋白FcCRT基因的全长cDNA序列，编码406个氨基酸，具有保守的N-，P-和C-功能域，以及信号肽和保守的HDEL内质网回收标签。FcCRT基因与其它物种的钙网蛋白具有高度的相似性，系统进化分析表明，FcCRT在亲缘关系上更接近昆虫的钙网蛋白。Northern blot和原位杂交结果显示，FcCRT基因在中国明对虾各组织中均有表达，且在卵巢中发育早起的卵母细胞中表达量最高，说明FcCRT很可能参与了卵母细胞的成熟。FcCRT基因在不同胁迫条件下，其转录表达均呈现明显的变化。在WSSV感染实验中，中国明对虾肝胰脏和淋巴器官中FcCRT转录表达均明显上调；热休克、重金属处理均可引起FcCRT基因转录表达的变化，但不同重金属处理引起FcCRT转录表达变化的模式不同。铜离子处理6小时，会引起FcCRT基因的下调表达，但在12小时之后出现明显上调；镉离子处理12小时后引起FcCRT基因的下调表达，但在24小时又出现明显的上调表达。 葡萄糖调节蛋白78（GRP78）是内质网重要的伴侣蛋白。中国明对虾的Grp78基因（FcGrp78）的cDNA全长为2325bp，编码665个氨基酸。FcGrp78具有三个Hsp70蛋白家族标签，并含有KDEL内质网回收标签。FcGrp78基因与中国明对虾已有的Hsc70和Hsp70具有高度相似性。Northern blot杂交结果显示FcGrp78基因在中国明对虾各组织中均有表达。FcGrp78基因在WSSV感染的中国明对虾肝胰脏中呈上调表达，在血细胞中下调表达，说明FcGrp78可能与对虾的免疫应答有关。热休克处理会诱导FcGrp78基因转录的上调。不同重金属离子胁迫引起的FcGrp78转录表达有所不同：铜离子处理可以诱导FcGrp78基因在处理后24小时的上调表达；镉离子的处理导致FcGrp78基因处理后12小时的下调以及处理后24小时的上调表达变化。短期低氧胁迫则抑制对虾FcGrp78基因的转录表达。 本论文报道的中国明对虾FcHsp90基因 cDNA全长2552bp，编码726个氨基酸，具有保守的N端功能域、中间功能域和C端功能域，具有五个保守的Hsp90蛋白家族标签，序列上与其他物种Hsp90相似性高。Real-time RT-PCR结果显示FcHsp90基因在发育的卵巢中表达量较高，说明Hsp90可能参与了对虾卵母细胞成熟过程中的蛋白合成和卵黄蛋白原的分泌。WSSV感染引起中国明对虾肝胰脏的FcHsp90的转录表达明显上调，说明FcHsp90很可能与对虾的免疫相关。热休克处理诱导FcHsp90基因转录表达的迅速上调。铜离子处理也可诱导FcHsp90基因转录的上调表达，而镉离子处理首先引起FcHsp90基因的下调表达，24小时后开始上调。低氧胁迫也会抑制FcHsp90基因在对虾体内的转录表达。 诱导型FcHsp70基因cDNA全长2511bp，编码629个氨基酸，具有三个保守的Hsp70蛋白家族标签和C末端EEVD序列。与其他物种的Hsp70蛋白具有高度的相似性。FcHsp70基因转录表达对于热休克处理和铜离子的处理非常敏感：热休克处理2小时后FcHsp70基因的转录水平是对照组的80倍；铜离子处理12小时FcHsp70基因转录表达达到对照组的15倍。而镉离子处理后没有诱导FcHsp70显著的上调表达。 以上研究结果为阐明对虾对环境胁迫应答的机制奠定了重要基础，并可为抗逆对虾的培育提供依据。

青海家牦牛HIF-1α基因组织特异性表达

[目的]探究青海家牦牛HIF-1α基因组织的特异性表达。[方法]应用半定量反转录PCR和实时定量反转录PCR（SYBRGreen）技术对青海家牦牛HIF-1α基因的组织特异性表达进行检测。通过提取不同组织总RNA，经DNase I消化后，用随机引物进行反转录合成cDNA，采用特异性引物分别对HIF-1α和β-actin基因进行RT-PCR和Real Time RT-PCR扩增。[结果]结果表明，HIF-1α基因在心、肝、脾、肺、肾、脑、肌肉、睾丸组织中均有表达，其中以睾丸和脾中HIF-1α基因表达量最高，肌肉的表达量最低。[结论]该研究为进一步揭示HIF-1α在高原土著动物低氧适应过程中的分子机制有着重要的意义。

Effects of Aging and Moderate Calorie Restriction on the Reproductive Axis of the Male Rhesus Macaque (Macaca mulatta)

Calorie restriction (CR) has been established as the only non-genetic method of altering longevity and attenuating biological changes associated with aging. This nutritional paradigm has been effective in nematodes, flies, rodents, dogs and possibly non-human primates. Its long history notwithstanding, little is known regarding the exact mechanism(s) of CR action or its potential impact on the hypothalamic-pituitary-gonadal (HPG) axis. The objectives of this project were to: 1) analyze neuroendocrine changes to the HPG axis that occur with aging and 2) evaluate the effects of moderate CR on reproductive function in male rhesus macaques. Pituitary gene expression profiling, semi-quantitative RT-PCR (sqRT-PCR) and immunohistochemistry showed circadian clock mechanism components present in three age categories of macaques, demonstrated age differences in expression for Per2, indicated differential expression of Per2 and Bmal1 at opposing time points and revealed daily rhythmic expression of REV-ERBα protein. These data indicate the ability of the macaque pituitary to express core-clock genes, their protein products, and to do so in a 24-hour rhythm. Young Adult CON and CR pituitary gene expression profiles detected potential differential expression in <150 probesets. A decline in>TSHR and CGA was detected in CR macaques as measured by sqRT-PCR. Other genes investigated showed no diet-induced changes. Young Adult CON and CR testicular gene expression profiles detected potential differential expression in <300 probesets although mRNA expression was not altered based on sqRT-PCR and real-time RT-PCR. Age-related>and/or diet-induced changes in HSD17β3, INSL3, CSNK1E and CGA were observed in a separate experiment with CGA in Old Adult CR subjects returning to youthful levels. Semen samples were collected from Young Adult CON and CR macaques. Normal spermiogram measures, ZP-binding, AR assay and SCSA^® were conducted and indicated no differences between CON and CR-treated animals. Both groups exhibited similar daily testosterone profiles with no differences in mean or maximum levels; however, daily minimum testosterone levels were lower in CON animals. It appears that moderate CR had limited impact on neuroendocrine or reproductive function in male rhesus macaques based on our selected endpoints. Thus, advantageous CR health benefits can be achieved without obvious negative consequences to the HPG axis.

The genomic analysis of erythrocyte microRNA expression in sickle cell diseases.

BACKGROUND: Since mature erythrocytes are terminally differentiated cells without nuclei and organelles, it is commonly thought that they do not contain nucleic acids. In this study, we have re-examined this issue by analyzing the transcriptome of a purified population of human mature erythrocytes from individuals with normal hemoglobin (HbAA) and homozygous sickle cell disease (HbSS). METHODS AND FINDINGS: Using a combination of microarray analysis, real-time RT-PCR and Northern blots, we found that mature erythrocytes, while lacking ribosomal and large-sized RNAs, contain abundant and diverse microRNAs. MicroRNA expression of erythrocytes was different from that of reticulocytes and leukocytes, and contributed the majority of the microRNA expression in whole blood. When we used microRNA microarrays to analyze erythrocytes from HbAA and HbSS individuals, we noted a dramatic difference in their microRNA expression pattern. We found that miR-320 played an important role for the down-regulation of its target gene, CD71 during reticulocyte terminal differentiation. Further investigation revealed that poor expression of miR-320 in HbSS cells was associated with their defective downregulation CD71 during terminal differentiation. CONCLUSIONS: In summary, we have discovered significant microRNA expression in human mature erythrocytes, which is dramatically altered in HbSS erythrocytes and their defect in terminal differentiation. Thus, the global analysis of microRNA expression in circulating erythrocytes can provide mechanistic insights into the disease phenotypes of erythrocyte diseases.

Differentiation of mouse induced pluripotent stem cells (iPSCs) into nucleus pulposus-like cells in vitro.

A large percentage of the population may be expected to experience painful symptoms or disability associated with intervertebral disc (IVD) degeneration - a condition characterized by diminished integrity of tissue components. Great interest exists in the use of autologous or allogeneic cells delivered to the degenerated IVD to promote matrix regeneration. Induced pluripotent stem cells (iPSCs), derived from a patient's own somatic cells, have demonstrated their capacity to differentiate into various cell types although their potential to differentiate into an IVD cell has not yet been demonstrated. The overall objective of this study was to assess the possibility of generating iPSC-derived nucleus pulposus (NP) cells in a mouse model, a cell population that is entirely derived from notochord. This study employed magnetic activated cell sorting (MACS) to isolate a CD24(+) iPSC subpopulation. Notochordal cell-related gene expression was analyzed in this CD24(+) cell fraction via real time RT-PCR. CD24(+) iPSCs were then cultured in a laminin-rich culture system for up to 28 days, and the mouse NP phenotype was assessed by immunostaining. This study also focused on producing a more conducive environment for NP differentiation of mouse iPSCs with addition of low oxygen tension and notochordal cell conditioned medium (NCCM) to the culture platform. iPSCs were evaluated for an ability to adopt an NP-like phenotype through a combination of immunostaining and biochemical assays. Results demonstrated that a CD24(+) fraction of mouse iPSCs could be retrieved and differentiated into a population that could synthesize matrix components similar to that in native NP. Likewise, the addition of a hypoxic environment and NCCM induced a similar phenotypic result. In conclusion, this study suggests that mouse iPSCs have the potential to differentiate into NP-like cells and suggests the possibility that they may be used as a novel cell source for cellular therapy in the IVD.

COVALENT LIPOPROTEIN(A) ASSEMBLY: Characterization of Oxidase Activity Responsible for Catalyzing Covalent Lipoprotein(a) Assembly

Lipoprotein(a) (Lp(a)) has been identified as an emerging risk factor for the development of vascular diseases. The Lp(a) particle is assembled in a 2-step process upon secretion of the LDL and apo(a) components from hepatocytes. Work done by the Koschinsky group has identified an oxidase-like activity present in the conditioned medium (CM) harvested from human hepatoma (HepG2), as well as HEK 293 (human endothelian kidney) cells that catalyzes the rate of covalent Lp(a) formation. We have taken a candidate enzyme approach to identifying this oxidase activity. Specifically, we have proposed that the QSOX (Quiescin/sulfhydryl oxidase) is responsible for catalysis of covalent Lp(a) assembly. An oxidase activity assay developed by Dr. Thorpe (University of Delaware) was used to detect QSOX1 in CM harvested from cultured cell lines that catalyze covalent Lp(a) assembly. In addition, the QSOX1 transcript was identified in each cell line and quantified with the use of Real-Time RT-PCR. Quantitative assays of covalent Lp(a) assembly were performed to study some characteristics of the unkwown oxidase activity. First, conditioned medium was dialyzed through a 5 kDa cutoff, as this has previously been shown to reduce the aforementioned oxidase activity. Purified QSOX was then added back to the reaction and the rate of catalysis was observed. The addition of QSOX appeared to enhance the rate of covalent Lp(a) assembly in a dose-dependent manner. Additional covalent Lp(a) assembly assays were performed where various chemicals were added to determine whether Lp(a) assembly was affected. The addition of EDTA did not affect covalent assembly, suggesting that the oxidase activity may not be metallo-dependent. Moreover, dose-dependent addition of Calcium, DTT, Copper and glutathione to dialyzed medium also did not affect the rate of Lp(a) assembly. Taken together, these studies will aid in identifying the nature of the oxidase activity that catalyzes covalent Lp(a) assembly. This will provide us with valuable information on how Lp(a) particles are assembled, and may lead to the development of drugs inhibiting Lp(a) formation.

A novel method for isolation of human lung T cells from lung resection tissue reveals increased expression of GAPDH and CXCR6

Lung T lymphocytes are important in pulmonary immunity and inflammation. it has been difficult to study these cells due to contamination with other cell types, mainly alveolar macrophages. We have developed a novel method for isolating lung T cells from lung resection tissue, using a combination of approaches. Firstly the lung tissue was finely chopped and filtered through a nylon mesh. Lymphocytic cells were enriched by Percoll density centrifugation and the T cells purified using human CD3 microbeads, resulting in 90.5% +/- 1.9% (n = 11) pure lymphocytes. The T cell yield from the crude cell preparation was 10.8 +/- 2.1% and viability, calculated using propidium iodide (PI) staining and trypan blue, was typically over 95%. The purification process did not affect expression of CD69 or CD103, nor was there a difference in the proportion of CD4 and CD8 cells between the starting population and the purified cells. Microarray analysis and real time RT-PCR revealed upregulation of GAPDH and CXCR6 of the lung T cells as compared to blood-derived T cells. This technique highly enriches lung T cells to allow detailed investigation of the biology of these cells. (C) 2008 Elsevier B.V. All rights reserved.

Inhibition of Advanced Glycation and Absence of Galectin-3 Prevent Blood-Retinal Barrier Dysfunction during Short-Term Diabetes

Breakdown of the inner blood-retinal barrier (iBRB) occurs early in diabetes and is central to the development of sight-threatening diabetic macular edema (DME) as retinopathy progresses. In the current study, we examined how advanced glycation end products (AGEs) forming early in diabetes could modulate vasopermeability factor expression in the diabetic retina and alter inter-endothelial cell tight junction (TJ) integrity leading to iBRB dysfunction. We also investigated the potential for an AGE inhibitor to prevent this acute pathology and examined a role of the AGE-binding protein galectin-3 (Gal-3) in AGE-mediated cell retinal pathophysiology. Diabetes was induced in C57/BL6 wild-type (WT) mice and in Gal-3(-/-) transgenic mice. Blood glucose was monitored and AGE levels were quantified by ELISA and immunohistochemistry. The diabetic groups were subdivided, and one group was treated with the AGE-inhibitor pyridoxamine (PM) while separate groups of WT and Gal-3(-/-) mice were maintained as nondiabetic controls. iBRB integrity was assessed by Evans blue assay alongside visualisation of TJ protein complexes via occludin-1 immunolocalization in retinal flat mounts. Retinal expression levels of the vasopermeability factor VEGF were quantified using real-time RT-PCR and ELISA. WT diabetic mice showed significant AGE -immunoreactivity in the retinal microvasculature and also showed significant iBRB breakdown (P < .005). These diabetics had higher VEGF mRNA and protein expression in comparison to controls (P < .01). PM-treated diabetics had normal iBRB function and significantly reduced diabetes-mediated VEGF expression. Diabetic retinal vessels showed disrupted TJ integrity when compared to controls, while PM-treated diabetics demonstrated near-normal configuration. Gal-3(-/-) mice showed significantly less diabetes-mediated iBRB dysfunction, junctional disruption, and VEGF expression changes than their WT counterparts. The data suggests an AGE-mediated disruption of iBRB via upregulation of VEGF in the diabetic retina, possibly modulating disruption of TJ integrity, even after acute diabetes. Prevention of AGE formation or genetic deletion of Gal-3 can effectively prevent these acute diabetic retinopathy changes.

Calbindin 2 (CALB2) Regulates 5-Fluorouracil Sensitivity in Colorectal Cancer by Modulating the Intrinsic Apoptotic Pathway

The role of the calcium binding protein, Calbindin 2 (CALB2), in regulating the response of colorectal cancer (CRC) cells to 5-Fluorouracil (5-FU) was investigated. Real-time RT-PCR and Western blot analysis revealed that CALB2 mRNA and protein expression were down-regulated in p53 wild-type and p53 null isogenic HCT116 CRC cell lines following 48 h and 72 h 5-FU treatment. Moreover, 5-FU-induced apoptosis was significantly reduced in HCT116 and LS174T CRC cell lines in which CALB2 expression had been silenced. Further investigation revealed that CALB2 translocated to the mitochondria following 5-FU treatment and that 5-FU-induced loss of mitochondrial membrane potential (Delta psi(m)) was abrogated in CALB2-silenced cells. Furthermore, CALB2 silencing decreased 5-FU-induced cytochrome c and smac release from the mitochondria and also decreased 5-FU-induced activation of caspases 9 and 3/7. Of note, co-silencing of XIAP overcame 5-FU resistance in CALB2-silenced cells. Collectively, these results suggest that following 5-FU treatment in CRC cell lines, CALB2 is involved in apoptosis induction through the intrinsic mitochondrial pathway. This indicates that CALB2 may be an important mediator of 5-FU-induced cell death. Moreover, down-regulation of CALB2 in response to 5-FU may represent an intrinsic mechanism of resistance to this anti-cancer drug.

Intervention With an Erythropoietin-Derived Peptide Protects Against Neuroglial and Vascular Degeneration During Diabetic Retinopathy.

OBJECTIVE:
Erythropoietin (EPO) may be protective for early stage diabetic retinopathy, although there are concerns that it could exacerbate retinal angiogenesis and thrombosis. A peptide based on the EPO helix-B domain (helix B-surface peptide [pHBSP]) is nonerythrogenic but retains tissue-protective properties, and this study evaluates its therapeutic potential in diabetic retinopathy.
RESEARCH DESIGN AND METHODS:
After 6 months of streptozotocin-induced diabetes, rats (n = 12) and age-matched nondiabetic controls (n = 12) were evenly split into pHBSP and scrambled peptide groups and injected daily (10 µg/kg per day) for 1 month. The retina was investigated for glial dysfunction, microglial activation, and neuronal DNA damage. The vasculature was dual stained with isolectin and collagen IV. Retinal cytokine expression was quantified using real-time RT-PCR. In parallel, oxygen-induced retinopathy (OIR) was used to evaluate the effects of pHBSP on retinal ischemia and neovascularization (1-30 µg/kg pHBSP or control peptide).
RESULTS:
pHBSP or scrambled peptide treatment did not alter hematocrit. In the diabetic retina, Müller glial expression of glial fibrillary acidic protein was increased when compared with nondiabetic controls, but pHBSP significantly reduced this stress-related response (P < 0.001). CD11b+ microglia and proinflammatory cytokines were elevated in diabetic retina responses, and some of these responses were attenuated by pHBSP (P < 0.01-0.001). pHBSP significantly reduced diabetes-linked DNA damage as determined by 8-hydroxydeoxyguanosine and transferase-mediated dUTP nick-end labeling positivity and also prevented acellular capillary formation (P < 0.05). In OIR, pHBSP had no effect on preretinal neovascularization at any dose.
CONCLUSIONS:
Treatment with an EPO-derived peptide after diabetes is fully established can significantly protect against neuroglial and vascular degenerative pathology without altering hematocrit or exacerbating neovascularization. These findings have therapeutic implications for disorders such as diabetic retinopathy.

Pan-serotypic detection of foot-and-mouth disease virus using a minor groove binder probe reverse transcription polymerase chain reaction assay

A novel assay for the pan-serotypic detection of foot-and-mouth disease virus (FMDV) was designed using a 5' conjugated minor groove binder (MGB) probe real-time RT-PCR system. This assay targets the 3D region of the FMDV genome and is capable of detecting 20 copies of a transcribed RNA standard. The linear range of the test was eight logs from 2 x 10(1) to 2 x 10(8) copies and amplification time was approximately 2 h. Using a panel of 83 RNA samples from representative FMDV isolates, the diagnostic sensitivity of this test was shown to be equivalent to a TaqMan real-time RT-PCR that targets the 5' untranslated region of FMDV. Furthermore, the assay does not detect viruses causing similar clinical diseases in pigs such as swine vesicular disease virus and vesicular stomatitis virus, nor does it detect marine caliciviruses causing vesicular exanthema. The development of this assay provides a useful tool for the differential diagnosis of FMD, potentially for use in statutory or emergency testing programmes, or for detection of FMDV RNA in research applications. (C) 2011 Elsevier B.V. All rights reserved.

O-antigen modal chain length in Shigella flexneri 2a is growth-regulated through RfaH-mediated transcriptional control of the wzy gene

Shigella flexneri 2a 2457T produces lipopolysaccharide (LPS) with two O-antigen (OAg) chain lengths: a short (S-OAg) controlled by WzzB and a very long (VL-OAg) determined by Wzz(pHS-2). This study demonstrates that the synthesis and length distribution of the S. flexneri OAg are under growth-phase-dependent regulation. Quantitative electrophoretic analysis showed that the VL-OAg increased during growth while the S-OAg distribution remained constant. Increased production of VL-OAg correlated with the growth-phase-regulated expression of the transcription elongation factor RfaH, and was severely impaired in a DeltarfaH mutant, which synthesized only low-molecular-mass OAg molecules and a small amount of S-OAg. Real-time RT-PCR revealed a drastic reduction of wzy polymerase gene expression in the DeltarfaH mutant. Complementation of this mutant with the wzy gene cloned into a high-copy-number plasmid restored the bimodal OAg distribution, suggesting that cellular levels of Wzy influence not only OAg polymerization but also chain-length distribution. Accordingly, overexpression of wzy in the wild-type strain resulted in production of a large amount of high-molecular-mass OAg molecules. An increased dosage of either wzzB or wzz(pHS-2) also altered OAg chain-length distribution. Transcription of wzzB and wzz(pHS-2) genes was regulated during bacterial growth but in an RfaH-independent manner. Overall, these findings indicate that expression of the wzy, wzzB and wzz(pHS-2) genes is finely regulated to determine an appropriate balance between the proteins responsible for polymerization and chain-length distribution of S. flexneri OAg.

Genetic characterization of tigecycline resistance in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes

OBJECTIVES:
The intrinsically encoded ramA gene has been linked to tigecycline resistance through the up-regulation of efflux pump AcrAB in Enterobacter cloacae. The molecular basis for increased ramA expression in E. cloacae and Enterobacter aerogenes, as well as the role of AraC regulator rarA, has not yet been shown. To ascertain the intrinsic molecular mechanism(s) involved in tigecycline resistance in Enterobacter spp., we analysed the expression levels of ramA and rarA and corresponding efflux pump genes acrAB and oqxAB in Enterobacter spp. clinical isolates.

METHODS:
The expression levels of ramA, rarA, oqxA and acrA were tested by quantitative real-time RT-PCR. The ramR open reading frames of the ramA-overexpressing strains were sequenced; strains harbouring mutations were transformed with wild-type ramR to study altered ramA expression and tigecycline susceptibility.

RESULTS:
Tigecycline resistance was mediated primarily by increased ramA expression in E. cloacae and E. aerogenes. Only the ramA-overexpressing E. cloacae isolates showed increased rarA and oqxA expression. Upon complementation with wild-type ramR, all Enterobacter spp. containing ramR mutations exhibited decreased ramA and acrA expression and increased tigecycline susceptibility. Exceptions were one E. cloacae strain and one E. aerogenes strain, where a decrease in ramA levels was not accompanied by lower acrA expression.

CONCLUSIONS:
Increased ramA expression due to ramR deregulation is the primary mediator of tigecycline resistance in clinical isolates of E. cloacae and E. aerogenes. However, some ramA-overexpressing isolates do not show changes in ramR, suggesting alternate pathways of ramA regulation; the rarA regulator and the oqxAB efflux pump may also play a role in tigecycline resistance in E. cloacae.