河南省HIV21 新近感染者耐药基因研究

目的　了解河南省艾滋病病毒Ⅰ型( HIV21) 新近感染者的耐药情况。方法　2006 年8 月- 2007 年6 月,在河南省艾滋病自愿咨询(VCT) 检测点发现的未进行抗病毒治疗的HIV21 感染者,以酶联免疫吸附试验初筛、 蛋白印迹试验确认HIV21 感染,BED2CEIA 方法检测新近感染。检出的新近感染样品进行基因型耐药检测,提取 血浆中RNA ,套式聚合酶链反应(Nested2PCR) 扩增HIV21 pol 基因区,PCR 产物双脱氧法测序,所得序列与Los Alamos HIV 标准株序列比对,构建系统进化树分析亚型;利用Stanford HIVdb Drug Resistance Database 分析耐 药相关突变(DRM) 和耐药情况。结果　共检出HIV21 新近感染39 例,扩增测序有34 例新近感染样品分析成功。 亚型分析结果为B′亚型32 例(9411 %) ,CRF01_AE 重组亚型1 例(219 %) ,C 亚型1 例(219 %) 。未发现蛋白酶抑 制剂( PI) 主要DRM ,检测到10 例(2914 %) 存在PI 次要DRM;未发现核苷类逆转录酶抑制剂(NRTI) 的DRM;3 例(818 %) 存在非核苷类逆转录酶抑制剂(NNRTI) 的DRM。耐药分析显示,有2 例(519 %) 对NNRTI 类药物耐 药。结论　目前河南省HIV21 新近感染人群中耐药状况处于中等水平,有必要加强HIV21 的耐药监测。

中国部分地区未经治疗HIV-1 感染者耐药基因研究

高效抗逆转录病毒治疗(HAART)的应用，极大的降低了AIDS发病率和死亡 率，延长了HIV感染者的生命。但HIV耐药在很大程度上影响了HAART的疗效， 耐药株的产生成为影响抗病毒治疗效果的主要因素。欧洲、美国的耐药监测技术 规范均推荐在新感染未经抗病毒药物治疗的患者中进行原发耐药检测。我国政府 于2003年底出台了艾滋病治疗的“四免一关怀”政策，陆续在全国范围内开展了大 规模的免费抗病毒治疗，监测我国未经抗病毒药物治疗HIV-1感染者中的耐药情 况可以为制定合理的用药方案和减少耐药毒株出现提供科学依据。 根据世界卫生组织(WHO)的“HIV 耐药监测指南”，无偿献血者中的HIV-1 感染者，可以认定为HIV 新诊断未治疗人群。分析了云南无偿献血者的血浆和 外周血单核细胞(PBMC)，研究云南无偿献血人群的耐药状况。 已有实验室血清学方法识别HIV-1 新近感染和长期感染，用BED-CEIA 方 法，在河南、安徽、山西自愿咨询检测(VCT)人群中检出新近感染人群，进行耐 药基因研究, 对照研究了部分长期感染人群。 样品提取核酸后，巢式聚合酶链反应(nested-PCR)扩增pol 基因区(含蛋白酶 区1～99 氨基酸全长和逆转录酶区1～242 氨基酸)。PCR 产物双脱氧法测序，所 得序列与洛斯阿拉莫斯HIV 核酸序列库(Los Alamos HIV Database)标准株构建系 统进化树分析亚型；用斯坦福大学耐药数据库(Standford HIV Drug Resistance Database)分析耐药。 研究发现，云南省2005～2006 年无偿献血者中，有52 例为HIV-1 阳性，其 中49 例血浆和相应的PBMC 样品病毒基因扩增成功。序列分析表明，HIV 病毒 的亚型分布为CRF08_BC (51.0%), CRF07_BC (24.5%), CRF01_AE (20.4%)和B (4.1%)；所有样品均未发现蛋白酶抑制剂（PI）耐药基因位点主要突变，只在6 例(11.7%)样品中发现7 例次PI 次要耐药位点突变；另外，在9 例(18.4%)样品中发现10 例次核苷类逆转录酶抑制剂(NRTI) 耐药突变，1 例(2.0%)发生非核苷类 逆转录酶抑制剂(NNRTI) 耐药突变；针对具体药物PI/NRTI/NNRTI 均只有1 例 有潜在的低度耐药，临床仍对药物敏感。PBMC 和血浆的病毒耐药没有显著差异。 从河南、安徽、山西27 个VCT 检测点2006～2007 年采集的10310 例样品 中，通过WB 和BED-CEIA 检测出新近感染人群63 例，分析成功50 例血浆样 品；河南VCT 长期感染样品中随机抽样，分析成功19 例样品。分析成功的69 例VCT 样品中，HIV 病毒株的亚型分布分别为B’ (95.7%)，CRF01_AE(2.9%)和 C(1.4%)。上诉样品均未检出PI 主要耐药相关突变，只在26 例(37.7%)样品中存 在27 例次PI 次要耐药相关突变；3 例(4.3%)样品出现6 例次NRTI 耐药相关突 变，7 例(10.1%)样品出现8 例次NNRTI 耐药相关突变。通过与斯坦福大学耐药 数据库比对，没有发现针对PI 类药物的临床耐药；但有2 例(2.8%)针对NRTI 类 药物耐药，1 例有M184V 突变导致对拉米夫定(3TC)和氟代拉米夫定(FTC)高度 耐药；1 例样品存在T215Y、M41L、L210W 三重突变位点，对阿巴卡韦(ABC)、 去羟肌苷(ddI)和坦那夫韦(TDF)中度耐药，对齐多夫定(AZT)和司他夫定(d4T)高 度耐药；针对NNRTI 类药物，有3 例(4.3%)毒株有耐药，1 例有K103N 突变导 致对奈韦拉平(NVP)、地拉韦啶(DLV)和依菲韦伦(EFV)的高度耐药；1 例有Y188L 突变导致对NVP 和EFV 的高度耐药；1 例存在K101E 和G190A 双重突变，导 致对NVP 的高度耐药，对DLV、EFV 和依曲韦林(ETR)中度耐药。 比较长期感染和新近感染者之间的亚型和耐药，未发现显著差异。 研究结果表明，云南、河南和安徽未经治疗HIV-1 感染者中耐药处于低流行 状态。亚型分布云南无偿献血者以CRF_BC 为主，河南、安徽VCT 人群以B’ 为主。应持续在未经治疗人群中进行耐药监测。

来自易变山羊草抗禾谷孢囊线虫基因的克隆与序列分析

禾谷孢囊线虫（Heterodera avenae）是严重危害禾谷类作物的病原线虫之一,它广泛分布于澳大利亚、欧洲、北美、印度和中国等世界主要小麦产区,使作物严重减产,造成巨大的经济损失。目前最有效的防治措施之一是将外源抗性基因导入栽培小麦(Triticum aestivum L.)，培育抗禾谷孢囊线虫的新品种。但迄今为止抗禾谷孢囊线虫基因克隆研究的相关报道却很少。 本实验根据此前从抗禾谷孢囊线虫材料E-10扩增得到的与来自节节麦（Aegilops tauschii）的抗禾谷孢囊线虫基因Cre3高度同源的序列Rccn4,设计出三条嵌套引物,采用SON-PCR(single oligonucleotide nested PCR)方法,从E-10基因组DNA中得到一个长为1264 bp的扩增产物(命名为Rccn-L),测序比对结果显示,这一序列将Rccn4的3’端延伸了1209 bp,与抗禾谷孢囊线虫Cre3基因核苷酸同源性为86﹪，核苷酸编码区长1026 bp，含一个不完整的开放阅读框,一个终止密码子，没有起始密码子和内含子结构,编码一个342个氨基酸残基的蛋白质。该蛋白质等电点为5.19，分子量为38112.6Da。从序列的第113位开始到第332位是NBS-LRR类抗病性基因LRR区,呈现XXLXXLXXL重复。LRR编码区内亮氨酸残基的含量达17﹪，与抗禾谷孢囊线虫Cre3基因LRR编码区的核苷酸和氨基酸同源性分别为89﹪和78﹪。本实验首次将SON-PCR成功地运用于植物基因克隆，为植物基因克隆提供了又一有效方法。 此外,还根据Cre3基因及其他的NBS-LRR类植物抗性基因的NBS和LRR区保守序列设计了两对特异性引物，从禾谷孢囊线虫抗性材料易变山羊草基因组DNA中扩增到两个相应的目标条带。测序分析结果表明，它们的长度分别为532bp和1175bp，构成了一个有32bp的共同序列的NBS-LRR编码区。其序列总长为1675bp（命名为RCCN），含有一个不完整的开放阅读框，没有起始密码子、终止密码子和内含子结构。其中编码序列为1673bp，可编码一个557个氨基酸的蛋白质，等电点（pI）为5.39，分子量为63537.5Da。与Cre3的核苷酸和氨基酸同源性分别为87.8﹪和77﹪。RCCN氨基酸序列中含有已知抗病基因NBS区域的几个保守模体：kinase2区的ILDD、kinase3的（ⅰ）ESKILVTTRSK，（ⅱ）KGSPLAARTVGG，（ⅲ）RRCFAYCS及EGF。RCCN NBS区与Cre3 NBS区的核苷酸和氨基酸的同源性分别为96.4﹪和94﹪。从氨基酸序列的274位到548位为LRR保守区，呈现不规则的aXXLXXLXXL（其中a代表I，V，L，F或M）重复，其中亮氨酸的含量为15.6﹪。该区域与Cre3的LRR区的核苷酸和氨基酸同源性分别为80.8﹪和74﹪。推测该序列可能为一个抗禾谷孢囊线虫的新基因。 本文对抗禾谷孢囊线虫基因的克隆研究，为进一步克隆基因全序列，探索其结构与功能，和研究该基因表达与调控提供了关键信息。同时也为通过基因工程途径将抗性基因向优良小麦品种高效、定向转移，最终培育出小麦抗禾谷孢囊线虫新品种奠定了基础。 Cereal cyst nematode (CCN) is a damaging pathogen of broad acre cereal crops in Australia, Europe, North America, India and China. It affects wheat, barley, oat and triticale and causes yield loss of up to 80%. At present, Transferring resistance genes against CCN into wheat cultivars and breeding varieties are considered one of the most effective methods for controlling the CCN. However, there are very limited reports concerning the cloning studies of resistance genes against the cereal cyst nematode. According to the sequence of Rccn4 which had high similarity to the nucleotide binding site (NBS) coding region of cereal cyst nematode resistance gene, Cre3， We designed three 3’ nested primers. Using single oligonucleotide nested PCR (SON-PCR) we successfully amplified one band, Rccn-L, of 1264bp from E-10 which is the wheat-Ae.variabilis translocation line containing the cereal cyst nematode resistance gene of Ae.variabilis. We found that this band of interesting is the 3’ flanking sequence of 1209bp in size of Rccn4. The coding region was 1026bp, which contained an incomplete open reading frame and a terminator codon, without initiation codon and intron, encoding a peptide of 342 amino acid residues, and shared 86﹪nucleotide sequence identity with Cre3. This peptide had a conserved LRR domain, containing the imperfect repeats,XXLXXLXXL, which contains 17﹪ leucine residues and shares, respectively, 89﹪ nucleotide sequence and 78﹪ amino acid sequence identity with the LRR sequence of Cre3 locus. This research firstly used SON-PCR in the research of plant genome successfully, which indicated that SON-PCR is another method of cloning plant gene. At the same time, According to the conversed motif of NBS and LRR region of cereal cyst nematode resistance gene Cre3 from wild wheat (Triticum tauschlii L.) and the known NBS-LRR group resistance genes, we designed two pairs of specific primers for NBS and LRR region respectively. One band of approximately 530bp was amplified using the specific primers for conversed NBS region and one band of approximately 1200bp was amplified with the specific primers for conversed LRR region. After sequencing, we found that these two sequences included 32bp common nucleotide sequence and have 1675 bp in total, which was registered as RCCN in the Genbank. RCCN contained a NBS-LRR domain and an incomplete open reading frame without initiation codon, terminator codon and inxon. Its exon encodes a peptide of 557 amino acid residues. The molecular weight of the protein from the amino acid was 63.537 KDa. The amino acid sequence of RCCN contained conserved motif: ILDD, ESKILVTTRSK, KGSPLAARTVGG, RRCFAYCS, EGF，LRR. RCCN shares 87.8﹪ nucleotide sequence and 77﹪ amino acid sequence identity with cereal cyst nematode gene Cre3. It might be a novel cereal cyst nematode resistance gene. These research results of cloning the resistance genes against cereal cyst nematode bring a great promise for transferring resistance genes into wheat cultivars and breeding new wheat varieties against cereal cyst nematode by gene engineering. And these results also lay the hard foundation for the expressing researches of these genes.

小麦抗禾谷孢囊线虫（ Heterodera avenae）相关基因的研究

禾谷孢囊线虫严重影响禾谷类作物的产量，在小麦中由禾谷孢囊线虫引起的产量损失可达30-100%。尤其在澳大利亚、欧洲、印度和中东危害严重，目前禾谷孢囊线虫已成为危害我国作物的主要病源。控制禾谷孢囊线虫的方法主要有：作物轮作、杀线虫剂、寄主抗性等等，其中基因工程方法培育抗线虫小麦品种被认为是最经济有效的方法。分离抗禾谷类孢囊线虫基因对揭示抗性基因结构与功能及其表达调控具有重要意义。 尽管小麦是重要的粮食作物，在小麦中已发现的抗禾谷孢囊线虫的基因很少，而比其近缘属如节节麦、易变山羊草、偏凸山羊草中含有丰富的抗源。目前已鉴定出禾谷孢囊线虫抗性位点Cre，并发现了9个禾谷孢囊线虫抗性基因(Cre1,2, 3, 4, 5, 6, 7, 8, and R) ，其中只有Cre1和Cre8直接从普通小麦中获得。从节节麦中获得的Cre3基因能最有效的控制线虫数量，其次是Cre1和Cre8。这些基因的克隆对于了解禾谷孢囊线虫抗性机制及进一步的育种应用都是非常关键的。然而，目前为止仅有Cre3基因通过图位克隆的方法从节节麦中被分离得到。该基因已被克隆得到的多数线虫抗性基因一样均属于核苷酸结合位点区（NBS）－亮氨酸重复序列区（LRR）基因家族。目前，已有很多抗性基因被分离，这些已知的NBS-LRR类抗性基因的保守序列为应用PCR的方法克隆新的抗性基因提供了可能。 因此本课题的目的是采用保守区同源克隆、3′RACE 和5′RACE 等方法从抗禾谷孢囊线虫小麦-易变山羊草小片段易位系E10 中克隆小麦抗禾谷孢囊线虫基因全序列，进而通过半定量PCR 和荧光定量PCR 研究该基因的表达模式。同时通过mRNA 差别显示技术和任意引物PCR（RAP-PCR）技术分离克隆植物禾谷孢囊线虫抗性基因及其相关基因，为阐明植物抗病性分子机制以及改良作物抗病性和作物育种提供基础，为通过分子标记辅助育种和基因工程方法实现高效、定向转移抗病基因到优良小麦品种奠定了重要的理论和物质基础。主要研究结果： 1． 本实验根据此前从抗禾谷孢囊线虫材料E-10 扩增得到的与来自节节麦的抗禾谷孢囊线虫Cre3 基因及其他的NBS-LRR 类抗性基因的NBS 和LRR 保守区序列设计了两对特异性引物，从E10 中扩增到532bp 和1175bp 的两个目标条带，它们有一个32bp 的共同序列，连接构成总长为1675bp 的NBS-LRR 编码区（命名为RCCN）。根据RCCN设计引物，利用NBS-LRR区序列设计引物，通过5′RACE 和3′RACE 技术采用3′-Full RACE Core Set(TaKaRa)和5'-Full RACE Kit (TaKaRa)试剂盒，反转录后通过嵌套引物GSP1 和GSP2 分别进行两轮基因特异性扩增，分别将NBS_LRR 区向5′端和3′端延伸了1173bp 和449bp，并包含了起始密码子和终止密码子。根据拼接的得到的序列重新设计引物扩增进行全基因扩增的结果与上面获得的一致。拼接后得到全长2775 bp 的基因序列（记作CreZ, GenBank 号：EU327996）。CreZ 基因包括完整的开放阅读框，全长2775 bp，编码924个氨基酸。序列分析表明它与已知的禾谷孢囊线虫抗性基因Cre3的一致性很高，并且它与已经报到的NBS-LRR 类疾病抗性基因有着相同的保守结构域。推测CreZ基因可能是一个新的NBS-LRR 类禾谷孢囊线虫抗性基因，该基因的获得为通过基因工程途径培育抗禾谷孢囊线虫小麦新品种奠定了基础，并为抗禾谷孢囊线虫基因的调控表达研究提供了参考。 2． 通过半定量PCR和SYBR Green荧光定量PCR技术对CreZ基因的相对表达模式进行了研究。以α-tubulin 2作为参照，采用半定量PCR 分析CreZ 基因在不同接种时期1d, 5d, 10, 15d 的E-10的根和叶的的表达情况。在内参扩增一致的条件下，CreZ 在E-10的根部随着侵染时间的增加表达量有明显的增加，在没有侵染的E-10的根部其表达量没有明显变化，而在叶中没有检测表达，说明该基因只在抗性材料的根部表达。SYBR Green定量PCR分析接种前后E10根部基因CreZ基因的表达水平为检测CreZ基因的表达建立了一套灵敏、可靠的SYBRGreen I 荧光定量PCR 检测方法。接种禾谷孢囊线虫后E10根内CreZ基因的相对表达水平显著高于接种前。随接种时间的延长持续增加，最终CreZ基因的相对表达量达到未接种的对照植株的10.95倍。小麦禾谷孢囊线虫抗性基因CreZ的表达量与胁迫呈正相关，表明其与小麦的的禾谷孢囊线虫抗性密切相关，推测CreZ基因可能是一个新的禾谷孢囊线虫候选抗性基因。 3． 针对小麦基因组庞大、重复序列较多，禾谷孢囊线虫抗性基因及其相关基因的片断难以有效克隆的问题，通过mRNA 差别显示技术及RAP-PCR 技术分离克隆植物禾谷孢囊线虫抗性及其相关基因。试验最终得到154 条差异表达条带，将回收得到的差异条带的二次PCR 扩增产物经纯化后点到带正电的尼龙膜上，进行反向Northern 杂交筛选，最终筛选得到102 个阳性差异点。将其中81 个进行测序，并将序列提交到Genbank 中的dbEST 数据库，分别获得登录号(FE192210 -FE192265，FE193048- FE193074 )。序列比对分析发现，其中26 个序列与已知功能的基因序列同源；有28 条EST 序列在已有核酸数据库中未找到同源已知基因和EST，属新的ESTs 序列；另外27 个EST 序列与已知核酸数据库中的ESTs 具有一定相似性，但功能未知。其所得ESTs 序列补充了Genbank ESTs 数据库，为今后进一步开展抗禾谷类孢囊线虫基因研究工作打下了基础。结合本试验功能基因的相关信息，对小麦接种禾谷孢囊线虫后产生的抗性机制进行了探讨。接种禾谷孢囊线虫后植物在mRNA 水平上的应答是相当复杂的，同时植物的抗病机制是一个复杂的过程，涉及到多个代谢途径的相互作用。 The cereal cyst nematode (CCN), Heterodera avenae Woll, causes severe yieldreductions in cereal crops. The losses caused by CCN can be up to 30-100% in somewheat fields. At present, cereal cyst nematode has become the major disease sourcein China and it also damaged heavily in Australia, Europe, India and Middle East.The damage caused by CCN can be mitigated through several methods, includingcrop rotation, nematicide application, cultural practice, host resistance, and others.Of these methods, incorporating resistance genes into wheat cultivars and breedingresistant lines is considered to be the most cost-effective control measure forreducing nematode populations. Although wheat is an economically important crop around the world, far fewergenes resistant to CCN were found in wheat than were detected in its relatives, suchas Aegilops taucchi, Aegilops variabilis and Aegilops ventricosa. Cloning these genesis essential for understanding the mechanism of this resistance and for furtherapplication in breeding. Because of the huge genome and high repeat sequencescontent, the efficient methods to clone genes from cereal crops, are still lacking. A resistance locus, Cre, has been identified and 9 genes resistant to CCN (designatedCre1, 2, 3, 4, 5, 6, 7, 8, and R) have been described, in which Cre1 and Cre8 werederived directly from common wheat. The Cre3 locus, which was derived from Ae.tauschii, has the greatest impact on reducing the number of female cysts, followed byCre1 and Cre8. Cloning these genes is essential for understanding the mechanism ofthis resistance and for further application in breeding. However, to this point, only Cre3, a NBS-LRR disease resistance gene, has been obtained through mappingcloning in Ae. tauschii. The majority of nematode resistance genes cloned so far belong to a super familywhich contains highly conserved nucleotide-binding sites (NBS) and leucine-richrepeat (LRR) domains. To date, many NBS-LRR resistance genes have been isolated.The conserved sequences of these recognized NBS-LRR resistance genes provide thepossibility to isolate novel resistance genes using a PCR-based strategy. The aim of the present study was to clone the resistance gene of CCN fromWheat/Aegilops variabilis small fragment chromosome translocation line E10 whichis resistant to CCN and investigate the espression profiles of this gene withsemi-quantitative PCR and real-time PCR. Another purpose of this study is cloningthe relational resistance gene for CCN by mRNA differential display PCR andRAP-PCR. These works will offer a foundation for disease defence of crop andbreeding and directional transferring resistance gene into wheat with geneengineering. Primary results as following: 1．According to the conversed motif of NBS and LRR region of cereal cystnematode resistance gene Cre3 from wild wheat (Triticum tauschlii) and the knownNBS-LRR group resistance genes, we designed two pairs of specific primers for NBSand LRR region respectively. One band of approximately 530bp was amplified usingthe specific primers for conversed NBS region and one band of approximately 1175bpwas amplified with the specific primers for conversed LRR region. After sequencing,we found that these two sequences included 32bp common nucleotide having 1675bpin total, which was registered as RCCN in the Genbank. Based on the conservedregions of known resistance genes, a NBS-LRR type CCN resistance gene analog wasisolated from the CCN resistant line E-10 of the wheat near isogenic lines (NILs), by5′RACE and 3′ RACE.designated as CreZ (GenBank accession number: EU327996) .It contained a comlete ORF of 2775 bp and encoded 924 amino acids. Sequencecomparison indicated that it shared 92% nucleotide and 87% amino acid identitieswith those of the known CCN-resistance gene Cre3 and it had the same characteristic of the conserved motifs as other established NBS-LRR disease resistance genes. 2． Usingα-tubulin 2 as exoteric reference, semi-quantitative PCR and real-timePCR analysis were conducted. The expression profiling of CreZ indicated that it wasspecifically expressed in the roots of resistant plants and its relative expression levelincreased sharply when the plants were inoculated with cereal cyst nematodes. therelative expression level of the 15days-infected E10 is the 10.95 times as that ofuninfected E10,ultimately. It was inferred that the CreZ gene be a novel potentialresistance gene to CCN. 3．We cloned the relational resistance gene for CCN by mRNA differentialdisplay PCR and arbitrarily primed PCR fingerprinting of RNA from wheat whichpossess huge and high repeat sequence content genomes. Total 154 differentialexpression bands were separated and second amplified by PCR. The products werenylon membrane. The 102 positive clones were filtrated by reverse northern dot blotand 81 of those were sent to sequence. The EST sequences were submitted toGenbank (Genbank accession: FE192210 - FE192265, FE193048 - FE193074). Thesequences alignment analysis indicated 26 of them were identical with known genes;28 were not found identical sequence in nucleic acid database; another 27 ests wereidentical with some known ests, but their functions were not clear. These ESTsenriched Genbank ESTs database and offered foundation for further research ofresistance gene of CCN.

链霉菌S227抗耐药菌活性成分的研究

本论文以从四川峨嵋山森林土壤中分离筛选获得的一株产抗耐药性活性化合物的链霉菌S227为材料，对发酵液中活性物质的分离纯化及抗耐药性活性进行了研究。 建立了抗耐药性活性的定性、定量检测方法。建立的管蝶法活性定量检测的标准回归方程为：D=4.8229Ln(C)+3.6326 R=0.9972 ；纸片法活性定量检测的标准回归方程为：D=5.5Ln(C)-12.794 R=0.999。 根据建立的样品活性的检测方法，测定了发酵液的初始活性。实验证明活性物质的温度、pH稳定性好。 通过活性的定性、定量追踪方法，分别利用等体积的石油醚、乙酸乙酯、正丁醇在不同的pH梯度下萃取，确定了pH3条件下正丁醇能最大程度的萃取活性物质，说明活性物质极性很大。对正丁醇萃取相经过两次硅胶柱层析及薄层层析分离得到具有抗耐药菌活性的纯化样品S227-4。 经过核磁共振氢谱、碳谱数据分析初步确定S227-4为四聚糖，通过糖的水解实验初步确定S227-4由葡萄糖和半乳糖组成。 纸片法活性检测表明S227-4具有抗耐药菌活性。采用MIC测定法对该样品抗耐药活性进行研究。在证明该样品本身不具有抗菌活性的基础上，以临床分离的耐药性金黄色葡萄球菌为指示菌，考察了该样品与抗生素联合使用时对耐药菌抗生素MIC（最小抑菌浓度）值的影响，结果表明在不影响菌体生长的浓度条件下，该样品能明显降低多株耐药菌对多种抗生素的MIC值，不同程度地恢复所测试耐药菌对相应抗生素的敏感性。如S227-4与青霉素钠联用可以使S. aureus 12352的MIC降低8倍，而与红霉素联用可以使S. aureus 12334的MIC降低128倍。 The purification process and the activity of the anti bacterial drug resistance compounds produced by Streptomyces S227 isolated from the forest soil sample of the Mountain E’MEI in Sichuan Province were studied in this thesis. Quantitative and qualitative activity assay methods of the active compounds were established. The regression equation of the tube method was D=4.8229Ln(C)+3.6326, R=0.9972. The regression equation of the paper method was D=5.5Ln(C)-12.794, R=0.999. According to the established activity assay method, the incipient activity of the broth was evaluated. And it was proved that the stability of the active compounds was good. By quantitative and qualitative activity tracing method, petroleum ether, ethyl acetate and butanol were used to extract the active compounds at different pH. The result showed that butanol was the most effective agent for active component recovery at pH3. From the butanol extraction a purified sample, S227-4, was isolated by silica gel column chromatography and thin-layer chromatography . S227-4 was proved to be a tetra- saccharide by 1H-NMR and 13C-NMR. And its monosaccharides include glucose and galactose by hydrolysate analysis. The anti-drug resistant activity of S227-4 was tested in vitro by MICs assay using different drug resistant Staphylococcus aureus strains isolated clinically. The sample itself showed no anti-microbial activity in growth inhibitory experiment, but when it was used together with different antibiotics, it could remarkably decrease the MICs of different clinically isolated drug-resistant bacterial strains to these antibiotics. For example, when S227-4 was used with penicillin, the MIC of S. aureus 12352 decreased 8 times compared with that when penicillin was used alone. Meanwhile when it was used with erythromycin the MIC of S. aureus 12334 deceased 128 times compared with erythromycin alone.

Direct formation of giant vesicles from synthetic polypeptides

This report describes direct formation of giant vesicles from a series of poly(L-lysine)-block-poly(L-phenylalanine) (PLL-b-PPA) block copolymers from their water solution. These polymers are prepared by successive ring-opening polymerization (ROP) of the two alpha-amino acid N-carboxyanhydrides and then removing the side chain protecting groups by acidolysis. The structures of the copolymers are confirmed by nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and size exclusion chromatography ( SEC). The vesicles are studied by atomic force microscopy (AFM), field emission scanning electron microscopy (ESEM), and confocal laser scanning microscopy (CLSM). Rhodamine B is used as a fluorescent probe to confirm the existence of the vesicle with an aqueous interior. The vesicle size is in the range 0.55-6 mu m, depending on the absolute and relative lengths of the two blocks, on initial polymer concentration, and on solution pH. The vesicles are still stable in water for 2 months after preparation. Addition of the copolymer to DNA solution results in complex formation with it. The complex assumes the morphology of irregular particles of less than 2 mu m. It is expected to be used in drug and gene delivery.

A biodegradable triblock copolymer poly(ethylene glycol)-b-poly(L-lactide)-b-poly(L-lysine): Synthesis, self-assembly and RGD peptide modification

A novel biodegradable triblock copolymer poly(ethylene glycol)-b-poly(L-lactide)-b-poly(L-lysine) (PEG-PLA-PLL) was synthesized by acidolysis of poly(ethylene glycol)-b-poly(L-lactide)-b-poly(F-benzyloxycarbonyl-L-lysine) (PEG-PLA-PZLL) obtained by the ring-opening polymerization (ROP) of epsilon-benzyloxycarbonyl-L-lysine N-carboxyanhydride (ZLys NCA) with amino-terminated PEG-PLA-NH2 as a macro-initiator, and the pendant amino groups of the lysine residues were modified with a peptide known to modulate cellular functions, Gly-Arg-Gly-Asp-Ser-Tyr (GRGDSY, abbreviated as RGD) in the presence of 1,1'-carbonyldiimidazole (CDI). The structures of PEG-PLA-PLL/RGD and its precursors were confirmed by H-1 NMR, FT-IR, amino acid analysis and XPS analysis. The cell adhesion and cell spread on the PEG-PLA-PLL/RGD film were enhanced compared to those on pure PLA film. Therefore, the novel RGD-grafted triblock copolymer is promising for cell or tissue engineering applications. Both copolymers PEG-PLA-PZLL and PEG-PLA-PLL showed an amphiphilic nature and could self-assemble into micelles of homogeneous spherical morphology. The micelles were determined by fluorescence technique, dynamic light scattering (DLS), and field emission scanning electron microscopy (ESEM) and could be expected to find application in drug and gene delivery systems.

Micellization and reversible pH-sensitive phase transfer of the hyperbranched multiarm PEI-PBLG copolymer

A novel, hyperbranched, amphiphilic multiarm biodegradable polyethylenimine-poly(gamma-benZyl-L-gluta- mate) (PEI-PBLG) copolymer was prepared by the ring-opening polymerization of gamma-benzyl-L-glutamate-N-car-boxyanhydride (BLG-NCA) with hyperbranched PEI as a macroinitiator. The copolymer could self-assemble into core-shell micelles in aqueous solution with highly hydrophobic micelle cores. As the PBLG content was increased, the size of the micelles increased and the critical micelle concentration (CMC) decreased. The surface of the micelles had a positive potential. The cationic micelles were capable of complexing with plasmid DNA (pDNA), which could be released subsequently by treatment with polyanions. The PEI-PBLG copolymer formed unimolecular micelles in chloroform solution. ne pH-sensitive phase-transfer behavior exhibited two critical pH points for triggering the encapsulation and release of guest molecules. Both the encapsulation and release processes were rapid and reversible. Under strong acidic or alkaline conditions, the release process became partially or completely irreversible.

Targeting aberrant kinase activity in myeloid leukaemias

Acute myeloid leukaemia (AML) is the most common form of acute leukaemia in adults. Its treatment has remained largely unchanged for the past 30 years. Chronic myeloid leukaemia (CML) represents a tremendous success story in the era of targeted therapy but significant challenges remain including the development of drug resistance and disease persistence due to presence of CML stem cells. The Aurora family of kinases is essential for cell cycle regulation and their aberrant expression in cancer prompted the development of small molecules that selectively inhibit their activity. Chapter 2 of this thesis outlines the efficacy and mechanism of action of alisertib, a novel inhibitor of Aurora A kinase, in preclinical models of CML. Alisertib possessed equipotent activity against CML cells expressing unmutated and mutated forms of BCR-ABL. Notably, this agent retained high activity against the T315I and E255K BCR-ABL mutations, which confer the greatest degree of resistance to standard CML therapy. Chapter 3 explores the activity of alisertib in preclinical models of AML. Alisertib disrupted cell viability, diminished clonogenic survival, induced expression of the forkhead box O3 (FOXO3a) targets p27 and BCL-2 interacting mediator (BIM), and triggered apoptosis. A link between Aurora A expression and sensitivity to ara-C was established. Chapter 4 outlines the role of the proto-oncogene serine/threonine-protein (PIM) kinases in resistance to ara-C in AML. We report that the novel small molecule PIM kinase inhibitor SGI-1776 disrupted cell viability and induced apoptosis in AML. We establish a link between ara-C resistance and PIM over-expression. Finally, chapter 5 explores how the preclinical work outlined in this thesis may be translated into clinical studies that may lead to novel therapeutic approaches for patients with refractory myeloid leukaemia.

Paclitaxel versus doxorubicin as first-line single-agent chemotherapy for metastatic breast cancer: a European Organization for Research and Treatment of Cancer Randomized Study with cross-over.

PURPOSE: To compare the efficacy of paclitaxel versus doxorubicin given as single agents in first-line therapy of advanced breast cancer (primary end point, progression-free survival ¿PFS) and to explore the degree of cross-resistance between the two agents. PATIENTS AND METHODS: Three hundred thirty-one patients were randomized to receive either paclitaxel 200 mg/m(2), 3-hour infusion every 3 weeks, or doxorubicin 75 mg/m(2), intravenous bolus every 3 weeks. Seven courses were planned unless progression or unacceptable toxicity occurred before the seven courses were finished. Patients who progressed within the seven courses underwent early cross-over to the alternative drug, while a delayed cross-over was optional for the remainder of patients at the time of disease progression. RESULTS: Objective response in first-line therapy was significantly better (P =.003) for doxorubicin (response rate ¿RR, 41%) than for paclitaxel (RR, 25%), with doxorubicin achieving a longer median PFS (7.5 months for doxorubicin v 3.9 months for paclitaxel, P <.001). In second-line therapy, cross-over to doxorubicin (91 patients) and to paclitaxel (77 patients) gave response rates of 30% and 16%, respectively. The median survival durations of 18.3 months for doxorubicin and 15.6 months for paclitaxel were not significantly different (P =.38). The doxorubicin arm had greater toxicity, but this was counterbalanced by better symptom control. CONCLUSION: At the dosages and schedules used in the present study, doxorubicin achieves better disease and symptom control than paclitaxel in first-line treatment. Doxorubicin and paclitaxel are not totally cross-resistant, which supports further investigation of these drugs in combination or in sequence, both in advanced disease and in the adjuvant setting.

An EORTC-IDBBC phase I study of gemcitabine and continuous infusion 5-fluorouracil in patients with metastatic breast cancer resistant to anthracyclines or pre-treated with both anthracyclines and taxanes.

The aim of this study was to determine the maximum tolerated dose (MTD), dose-limiting toxicities (DLT), and potential activity of combined gemcitabine and continuous infusion 5-fluorouracil (5-FU) in metastatic breast cancer (MBC) patients that are resistant to anthracyclines or have been pretreated with both anthracyclines and taxanes. 15 patients with MBC were studied at three European Organization for Research and Treatment of Cancer centres. 13 patients had received both anthracylines and taxanes. Gemcitabine was given intravenously (i.v.) on days 1 and 8, and 5-FU as a continuous i.v. infusion on days 1 through to 14, both drugs given in a 21-day schedule at four different dose levels. Both were given at doses commonly used for the single agents for the last dose level (dose level 4). One of 6 patients at level 4 (gemcitabine 1200 mg/m2 and 5-FU 250 mg/m2/day) had a DLT, a grade 3 stomatitis and skin toxicity. One DLT, a grade 3 transaminase rise and thrombosis, occurred in a patient at level 2 (gemcitabine 1000 mg/m2 and 5-FU 200 mg/m2/day). Thus, the MTD was not reached. One partial response and four disease stabilisations were observed. Only 1 patient withdrew from the treatment due to toxicity. The MTD was not reached in the phase I study. The combination of gemcitabine and 5-FU is well tolerated at doses up to 1200 mg/m2 given on days 1 and 8 and 250 mg/m2/day given on days 1 through to 14, respectively, every 21 days. The clinical benefit rate (responses plus no change of at least 6 months) was 33% with one partial response, suggesting that MBC patients with prior anthracycline and taxane therapy may derive significant benefit from this combination with minimal toxicity.

HER2: a 'predictive factor' ready to use in the daily management of breast cancer patients?

The past few years have witnessed an exponential increase in studies trying to identify molecular markers in patients with breast tumours that might predict for the success or failure of hormonal therapy or chemotherapy. HER2, a tyrosine kinase membrane receptor of the epidermal growth factor receptor family, has been the most widely studied marker in this respect. This paper attempts to critically review to what extent HER2 may improve 'treatment individualisation' for the breast cancer patient. Copyright (C) 2000.

Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of america.

Cryptococcosis is a global invasive mycosis associated with significant morbidity and mortality. These guidelines for its management have been built on the previous Infectious Diseases Society of America guidelines from 2000 and include new sections. There is a discussion of the management of cryptococcal meningoencephalitis in 3 risk groups: (1) human immunodeficiency virus (HIV)-infected individuals, (2) organ transplant recipients, and (3) non-HIV-infected and nontransplant hosts. There are specific recommendations for other unique risk populations, such as children, pregnant women, persons in resource-limited environments, and those with Cryptococcus gattii infection. Recommendations for management also include other sites of infection, including strategies for pulmonary cryptococcosis. Emphasis has been placed on potential complications in management of cryptococcal infection, including increased intracranial pressure, immune reconstitution inflammatory syndrome (IRIS), drug resistance, and cryptococcomas. Three key management principles have been articulated: (1) induction therapy for meningoencephalitis using fungicidal regimens, such as a polyene and flucytosine, followed by suppressive regimens using fluconazole; (2) importance of early recognition and treatment of increased intracranial pressure and/or IRIS; and (3) the use of lipid formulations of amphotericin B regimens in patients with renal impairment. Cryptococcosis remains a challenging management issue, with little new drug development or recent definitive studies. However, if the diagnosis is made early, if clinicians adhere to the basic principles of these guidelines, and if the underlying disease is controlled, then cryptococcosis can be managed successfully in the vast majority of patients.

Changing epidemiology of serious bacterial infections in febrile infants without localizing signs.

OBJECTIVE: Historically, management of infants with fever without localizing signs (FWLS) has generated much controversy, with attempts to risk stratify based on several criteria. Advances in medical practice may have altered the epidemiology of serious bacterial infections (SBIs) in this population. We conducted this study to test the hypothesis that the rate of SBIs in this patient population has changed over time. PATIENTS AND METHODS: We performed a retrospective review of all infants meeting FWLS criteria at our institution from 1997-2006. We examined all clinical and outcome data and performed statistical analysis of SBI rates and ampicillin resistance rates. RESULTS: 668 infants met criteria for FWLS. The overall rate of SBIs was 10.8%, with a significant increase from 2002-2006 (52/361, 14.4%) compared to 1997-2001 (20/307, 6.5%) (p = 0.001). This increase was driven by an increase in E. coli urinary tract infections (UTI), particularly in older infants (31-90 days). CONCLUSIONS: We observed a significant increase in E. coli UTI among FWLS infants with high rates of ampicillin resistance. The reasons are likely to be multifactorial, but the results themselves emphasize the need to examine urine in all febrile infants <90 days and consider local resistance patterns when choosing empiric antibiotics.

The missense of smell: functional variability in the human odorant receptor repertoire.

Humans have ~400 intact odorant receptors, but each individual has a unique set of genetic variations that lead to variation in olfactory perception. We used a heterologous assay to determine how often genetic polymorphisms in odorant receptors alter receptor function. We identified agonists for 18 odorant receptors and found that 63% of the odorant receptors we examined had polymorphisms that altered in vitro function. On average, two individuals have functional differences at over 30% of their odorant receptor alleles. To show that these in vitro results are relevant to olfactory perception, we verified that variations in OR10G4 genotype explain over 15% of the observed variation in perceived intensity and over 10% of the observed variation in perceived valence for the high-affinity in vitro agonist guaiacol but do not explain phenotype variation for the lower-affinity agonists vanillin and ethyl vanillin.