Human multidrug-resistant cell lines: increased mdr1 expression can precede gene amplification

The development of simultaneous resistance to multiple structurally unrelated drugs is a major impediment to cancer chemotherapy. Multidrug resistance in human KB carcinoma cells selected in colchicine, vinblastine, or Adriamycin is associated with amplification of specific DNA sequences (the multidrug resistance locus, mdr1). During colchicine selection resistance is initially accompanied by elevated expression of a 4.5-kilobase mdr1 messenger RNA (mRNA) without amplification of the corresponding genomic sequences. During selection for increased levels of resistance, expression of this mRNA is increased simultaneously with amplification of mdr1 DNA. Increased expression and amplification of mdr1 sequences were also found in multidrug-resistant sublines of human leukemia and ovarian carcinoma cells. These results suggest that increased expression of mdr1 mRNA is a common mechanism for multidrug resistance in human cells. Activation of the mdr1 gene by mutations or epigenetic changes may precede its amplification during the development of resistance.     

Cell surface P-glycoprotein associated with multidrug resistance in mammalian cell lines

The plasma membranes of hamster, mouse, and human tumor cell lines that display multiple resistance to drugs were examined by gel electrophoresis and immunoblotting. In every case, increased expression of a 170,000-dalton surface antigen was found to be correlated with multidrug resistance. This membrane component is of identical molecular size and shares some immunogenic homology with the previously characterized P-glycoprotein of colchicine-resistant Chinese hamster ovary cells. This finding may have application to cancer therapy.     

Role of the glutathione redox cycle in acquired and de novo multidrug resistance

Drug resistance represents a major obstacle to successful cancer chemotherapy. However, the specific biochemical mechanisms responsible for clinical drug resistance are unknown. In these studies resistance to the antitumor agent adriamycin was found to involve two mechanisms, one that decreased drug accumulation by the P170 mechanism and another that altered the glutathione redox cycle, an important pathway in the detoxification of reactive oxygen. This dual mechanism of drug resistance was demonstrated in cell lines that had acquired the multidrug-resistant phenotype and in human colorectal cancer cells with de novo resistance. These studies support a model of acquired and de novo multidrug resistance that includes alterations in both drug accumulation and the glutathione redox cycle.     

中国野生华东葡萄抗霜霉病抑制消减文库构建及初步分析

 【目的】分离和获得中国野生华东葡萄抗霜霉病相关基因片段的EST序列。【方法】以高抗霜霉病的中国野生华东葡萄（V.pseudoreticulata）白河-35-1 株系为材料,以田间接种葡萄霜霉菌的幼嫩叶片为处理,以田间自然生长的未接种幼嫩叶片为对照,分别提取RNA,采用抑制消减杂交技术构建抗霜霉病基因的消减正交和反交两个文库,在文库中,选择3个EST序列,用半定量PT-PCR检验构建文库中EST片段的表达情况。【结果】构建的文库中EST片段的大小在150～900 bp,经筛选文库,得到正交库有效的ESTs 85条,反交库ESTs 29条,所获序列经过BLASTn和BLASTx网上序列比对,其中有78条ESTs与GenBank中其它植物相关基因序列有较高的同源性,31条为未知功能序列,已登录GenBank 114条ESTs,登录号:FG106789-FG106902。进一步利用半定量RT-PCR 对文库中3个基因的表达情况进行了分析,证明这些EST序列是可以表达的。【结论】经初步分析这些序列的功能,涉及抗病信号传导、能量代谢、蛋白质代谢、核酸代谢、光合作用及膜运输和代谢等方面,其中获得与抗病直接相关的ESTs 23条,下一步是通过对获得的EST序列进行末端快速分析（RACE）技术,获得抗霜霉病基因全长序列,并进行原核和真核表达研究验证基因全长序列功能,为研究葡萄抗霜霉病的基因提供依据。     

The shared antibiotic resistome of soil bacteria and human pathogens

Soil microbiota represent one of the ancient evolutionary origins of antibiotic resistance and have been proposed as a reservoir of resistance genes available for exchange with clinical pathogens. Using a high-throughput functional metagenomic approach in conjunction with a pipeline for the de novo assembly of short-read sequence data from functional selections (termed PARFuMS), we provide evidence for recent exchange of antibiotic resistance genes between environmental bacteria and clinical pathogens. We describe multidrug-resistant soil bacteria containing resistance cassettes against five classes of antibiotics (β-lactams, aminoglycosides, amphenicols, sulfonamides, and tetracyclines) that have perfect nucleotide identity to genes from diverse human pathogens. This identity encompasses noncoding regions as well as multiple mobilization sequences, offering not only evidence of lateral exchange but also a mechanism by which antibiotic resistance disseminates.     

Senescence of nickel-transformed cells by an X chromosome: possible epigenetic control

Transfer of a normal Chinese hamster X chromosome (carried in a mouse A9 donor cell line) to a nickel-transformed Chinese hamster cell line with an Xq chromosome deletion resulted in senescense of these previously immortal cells. At early passages of the A9/CX donor cells, the hamster X chromosome was highly active, inducing senescence in 100% of the colonies obtained after its transfer into the nickel-transformed cells. However, senescence was reduced to 50% when Chinese hamster X chromosomes were transferred from later passage A9 cells. Full senescing activity of the intact hamster X chromosome was restored by treatment of the donor mouse cells with 5-azacytidine, which induced demethylation of DNA. These results suggest that a senescence gene or genes, which may be located on the Chinese hamster X chromosome, can be regulated by DNA methylation, and that escape from senescence and possibly loss of tumor suppressor gene activity can occur by epigenetic mechanisms.     

烟草cDNA-AFLP体系中选择性引物筛选

为确定cDNA-AFLP技术在烟草中的最适合的选择性碱基数目,本实验对低钾胁迫下的烟草根系进行了cDNA-AFLP分析,通过240对引物组合的筛选,共得到2100个转录衍生片段,并根据不同选择性碱基的组合对2100个片段进行了分析。结果表明,在扩增产物过多时,应将引物中选择性碱基的数量调整到3个较为合适。     

新疆焉耆地区4种动物源的沙门菌耐药性及耐药基因检测

为探明新疆焉耆地区4种动物(牛、鸡、猪、羊)源沙门菌对临床常用抗菌药物的耐药性及其携带耐药基因的情况,通过选择性培养基和管家基因invA的PCR检测,分离、鉴定沙门菌;采用琼脂稀释法检测其对11种抗菌药物的最小抑菌浓度;在分离株中用PCR的方法检测14种耐药基因。结果表明：共分离出不同动物源沙门菌191株,分离率从高到低依次为牛源(62.0%)、鸡源(28.3%)、猪源(9.0%)和羊源(8.5%);不同动物源沙门菌对被检抗菌药物显示出不同程度的耐药性,鸡源、羊源、猪源、牛源的耐药严重程度依次降低,鸡源沙门菌耐药谱宽,多药耐药在0~3、5~7耐有分布,对四环素和氟苯尼考耐药率在70.0%以上,羊源沙门菌对氨苄西林的耐药率(88.2%)高于其他动物源的,多药耐药以7耐(23.5%)为主,牛源沙门菌对环丙沙星的耐药率(66.1%)高于其他动物源的,多药耐药以4耐(38.7%)为主,不同动物源沙门菌均对磷霉素、亚胺培南和多粘菌素敏感,耐药谱型多样化;不同动物源沙门菌中除qnrS和mcr–1基因未检出外,其余被检耐药基因均有检出,blaCMY–2和tetA基因仅在猪源沙门菌中被检出,不同动物源沙门菌均以同时携带blaTEM、blaOXA、tetB、oqxA、oqxB、aadA2、ant(3")–Ia、aac(6'')–Ib–cr、floR耐药基因为主,牛源沙门菌对以上耐药基因检出率最高,在35%以上,羊源与猪源沙门菌对以上耐药基因检出率在20%左右,鸡源沙门菌对以上耐药基因检出率不到10%。     

Serum concentration: effects on cycle and x-ray sensitivity of mammalian cells

If the content of serum in the culture medium of exponentially growing Chinese hamster cells is below optimum (15 percent), the doubling time and the resistance to x-irradiation of the cells are increased. In synchronously dividing populations the increase in doubling time is primarily caused by increase in duration of the postmitotic (G(1)) phase of the cells; this phase is relatively radiation resistant. The response of the cells growing synchronously is related quantitatively to the response of the cells dividing randomly.     

烟草青枯菌FQY_4的抗生素类抗/耐药性相关基因分析

为探明烟草青枯菌基因组中抗/耐药性基因作用机理。在烟草青枯菌FQY_4基因组测序的基础上,结合培养青枯菌的半选择性培养基中抗生素的使用,采用基因注释及同源基因比较法,分析青枯菌FQY_4基因组携带的抗/耐药性相关基因。结果显示:青枯菌FQY_4染色体上和巨大质粒上分别有6个和12个与多抗/耐药性相关的基因,包括多粘菌素耐药蛋白基因、膜融合蛋白CmeA基因、二甲基腺苷转移酶基因、膦胺霉素耐药性蛋白基因、外膜多耐药性系统相关基因和抗四环素基因序列。     

茉莉酸甲酯诱导的小麦白粉病抗性与9个抗病相关基因表达间的关系

[目的]为了明确茉莉酸对小麦白粉病抗性的诱导作用和对抗病相关基因的激活作用,以及抗病性变化与基因表达变化之间的相关性,探索小麦抗白粉病分子机理。[方法]以田间表现不同的代表性感白粉病小麦品种＂中国春＂、＂濮麦9号＂和＂周麦18＂为材料,用茉莉酸甲酯（methyljasmonate,MeJA）处理小麦幼苗叶片进行诱导,通过离体叶段培养法接种白粉菌（Blumeria graminis f.sp.tritici,Bgt）进行抗性鉴定;用实时定量PCR技术检测叶片中PR1（PR1.1）、PR2（β,1-3葡聚糖苷酶）、PR3（几丁质酶）、PR4、PR5（类甜蛋白）、PR9（TaPERO,过氧化物酶）、PR10、TaGLP2a（类胚素蛋白）和Ta-JA2（茉莉酸甲酯诱导蛋白）基因的表达变化。[结果]MeJA处理可以显著提高＂中国春＂、＂濮麦9号＂和＂周麦18＂对白粉菌的抗性水平。诱导抗性可以从MeJA处理后12～96h检测到,24h达到峰值。虽然存在一定差异,但MeJA对3个品种中除TaGLP2a外的8个抗病相关基因的表达有显著激活作用,在处理12、24或48h后达到峰值。MeJA对PR9和PR1的诱导作用最强,可达100倍,对PR2、PR4、PR5、PR3、PR10和Ta-JA2的诱导作用强,可达10-70倍;对TaGLP2a没有明显作用。茉莉酸诱导的抗病性提高与8个抗病相关基因的表达增强呈正相关。[结论]茉莉酸诱导的抗病性增强与抗病相关基因的表达增强呈正相关,茉莉酸信号传导途径在小麦抗白粉病反应中起作用,对这一途径的调控可提高小麦的白粉病抗性。     

Sequence and expression of human estrogen receptor complementary DNA

The mechanism by which the estrogen receptor and other steroid hormone receptors regulate gene expression in eukaryotic cells is not well understood. In this study, a complementary DNA clone containing the entire translated portion of the messenger RNA for the estrogen receptor from MCF-7 human breast cancer cells was sequenced and then expressed in Chinese hamster ovary (CHO-K1) cells to give a functional protein. An open reading frame of 1785 nucleotides in the complementary DNA corresponded to a polypeptide of 595 amino acids and a molecular weight of 66,200, which is in good agreement with published molecular weight values of 65,000 to 70,000 for the estrogen receptor. Homogenates of transformed Chinese hamster ovary cells containing a protein that bound [3H]estradiol and sedimented as a 4S complex in salt-containing sucrose gradients and as an 8 to 9S complex in the absence of salt. Interaction of this receptor-[3H]estradiol complex with a monoclonal antibody that is specific for primate ER confirms the identity of the expressed complementary DNA as human estrogen receptor. Amino acid sequence comparisons revealed significant regional homology among the human estrogen receptor, the human glucocorticoid receptor, and the putative v-erbA oncogene product. This suggests that steroid receptor genes and the avian erythroblastosis viral oncogene are derived from a common primordial gene. The homologous region, which is rich in cysteine, lysine, and arginine, may represent the DNA-binding domain of these proteins.     

中药复方制剂对大肠埃希菌多重耐药基因AcrA的影响

试验分别用精提和粗提的中药复方制剂"连黄"作用多重耐药的大肠埃希菌,提取其基因组DNA,并以大肠埃希菌AcrA的编码序列设计引物,成功扩增出AcrA基因中1 005bp大小的片段,与中药作用前的多重耐药基因AcrA的碱基序列进行对比分析.从分子生物学水平上探讨中药复方制剂时大肠埃希菌多重耐药基因AcrA的影响.结果表明.精提和粗提的中药复方制剂均能改变AcrA基因的编码序列,但精提制剂较粗提制剂对AcrA基因的影响更大.     

Transmission of antibiotic resistance genes in agroecosystems: an overview

The use of antibiotics in human medicine and animal husbandry has resulted in the continuous release of antibiotics into the environment, which imposes high selection pressure on bacteria to develop antibiotic resistance. The spread and aggregation of antibiotic resistance genes (ARGs) in multidrug-resistant pathogens is one of the most intractable clinical challenges. Numerous studies have been conducted to profile the patterns of ARGs in agricultural ecosystems, as this is closely related to human health and wellbeing. This paper provides an overview of the transmission of ARGs in agricultural ecosystems resulting from the application of animal manures and other organic amendments. The future need to control and mitigate the spread of antibiotic resistance in agricultural ecosystems is also discussed, particularly from a holistic perspective, and requires multiple sector efforts to translate fundamental knowledge into effective strategies.     

规模化畜禽养殖场粪便中多重耐药菌分离鉴定及其耐药特征

为了解禽畜粪便中多重耐药菌的污染特征,本研究对鸡粪、牛粪、猪粪和有机肥这4种不同样品中多重耐药菌进行了计数分析,并对不同来源的多重耐药菌株进行了分离和纯化,进一步开展了基于16S rDNA序列比对的分子生物学鉴定以确定其种属地位,以及基于药敏试验的耐药性特征分析。结果表明:不同养殖动物粪便中四环素、恩诺沙星、磺胺甲恶唑和泰乐菌素4种抗生素多重耐药菌的绝对数量和相对数量排序均为鸡粪>猪粪>牛粪,粪源有机肥中可培养的多重耐药菌的绝对数量和相对数量在堆肥后均有所下降。通过对耐药菌株鉴定和分类学分析,发现禽畜粪便中多耐药菌的菌门集中分布在Proteobacteria、Firmicutes和Actinobacteria,多重耐药菌的优势菌属为Escherichia、Corynebacterium、Kurthia;而有机肥样品中多重耐药菌的优势菌属为Staphylococcus、Glutamicibacter。菌株的药敏试验表明,3类动物粪污中的多重耐药菌都对红霉素、四环素有着较高的耐药率,均高于80%,而对阿米卡星这种抗生素表现出较好的敏感性,其耐药率低于30%。通过对Ⅰ、Ⅱ类整合子基因盒的扩增,发现PCR产物的大小从0.8 kb到1.8 kb不等,基因盒ad A2、dfr A17、dfr A1及sat2在养殖场粪污中检出率均较高。     

Mechanisms involved in biocontrol and plant induced resistance by richoderma. asperellum (T. harzianum T-203)

Growing awareness of the environmental damage caused by the use of chemical substances for plant disease control in agriculture has raised the need to study biological alternatives, such as activating the defense response of plant crops by inducers not toxic to the environment. Trichoderma spp. are effective biocontrol agents for a number of soilborne pathogens, and are also known for their ability to enhance plant growth and to induce systemic resistance (ISR) in plants. In our laborator…     

镜鲤抗疱疹病毒(CyHV-3)F4抗病品系病毒表达量评估

镜鲤抗疱疹病毒新品种选育到F4,抗病性状已达到稳定。本研究以F4为实验材料,利用实时荧光定量PCR技术以病毒TK和ORF72基因为病毒标志基因,分析镜鲤抗疱疹病毒（CyHV-3）选育抗病品系F4在病毒感染不同阶段中的表达量,从而从病毒表达角度探讨F4抗病选育效果及其病毒表达模式。结果显示：（1）两个基因不同感染阶段脾、肾组织表达量趋势一致,24h-144h呈上升趋势,144h-288h呈下降趋势。选育组脾在144h相比其他时期差异显著（P<0.05）;未选育组脾、肾及选育组肾在144h相比其他时期差异极显著（P<0.01）（2）脾组织两个基因表达量均低于肾组织。选育组和未选育组两个基因均在96h-144h差异显著,其中选育组两个基因在96h、ORF72基因144h差异显著(P<0.05),其他时期差异极显著(P<0.01)。（3）选育组两个基因表达量低于未选育组。脾组织两个基因均在96h-168h差异极显著(P<0.01),其中ORF72基因在192h、216h也差异极显著(P<0.01);肾组织两个基因在96h-168h差异极显著(P<0.01);其中ORF72基因在192h也差异极显著(P<0.01)。以上结果说明选育组抗病性能强于未选育组,其中肾组织抗病性能强于脾组织,且经选育肾组织抗病免疫性能得到很大的提高。目前,未见其他鱼类的选育种在抗病性状上达到一个稳定阶段,本研究在对于病毒表达量的研究当中可对其他鱼类提供重要的指导作用。