Tris对高LET碳重离子辐照下质粒DNA链断裂的保护作用

利用100 keV/μm碳离子束(初始能量为290 MeV/u)照射溶解于纯水、10 mmol/L Tris、1 mmol/L EDTA及TE 缓冲液中的pUC19质粒DNA.通过琼脂糖凝胶电泳技术分析了不同溶液中各种形态DNA分子所占份额,并计算得到不同剂量下平均每个质粒分子中单链断裂(SSB)及双链断裂(DSB)的数目.发现Tris通过抑制SSB和DSB的产生对碳重离子辐照下的质粒DNA有明显的保护作用,而EDTA能够加剧SSB的产生而抑制DSB的形成.     

一氧化氮对小麦叶片老化过程的调节

用不同浓度(0.05、0.10、0.20、0.50 mmol/L)的外源一氧化氮(nitric oxide,NO)供体硝普钠(sodiumnitroprusside,SNP)处理正常生长小麦(Triticum aestivum L.)叶片(二叶一心期时全展第一叶).结果显示低浓度SNP(0.05、0.10、0.20 mmol/L)可以明显降低叶片H2O2和MDA的水平,其中0.10 mmol/LSNP的作用最为明显;而较高浓度SNP(0.50 mmol/L)则作用相反.进一步采用0.10 mmol/L SNP处理不同叶位的小麦叶片(四叶一心期),结果表明低浓度NO对不同老化阶段中叶片的H2O2、O-2和MDA累积都有缓解作用,并明显减缓叶绿素、可溶性叶蛋白尤其是Rubisco的降解,有效延缓了叶片的老化进程.在完整叶绿体体外老化实验中也发现,不同浓度SNP(0.05、0.10、0.20、0.50、1.00、5.00 mmol/L)的作用同样表现双重性,其中0.20 mmol/L SNP对膜结构及Rubisco保护作用最明显.上述结果证实,低浓度外源NO可延缓小麦叶片的老化过程,并可能与其降低叶片活性氧(ROS)水平及缓解氧化损伤有关.     

来源于葡萄球菌的N-乙酰神经氨酸裂合酶的基因克隆及性质

葡萄球菌Staphylococcus hominis来源的N-乙酰神经氨酸裂合酶基因shnal(GenBank Accession No.EFS20452.1)构建至pET-28a质粒并在大肠杆菌中得到表达.通过目的蛋白的纯化和酶学性质研究发现,ShNAL是一个四聚体,裂解方向的最适反应pH为8.0;合成方向的最适反应pH为7.5,最适反应温度为45℃.在45℃下孵育2h对ShNAL的活力基本无影响,高于45℃时,活力迅速下降.该酶在pH 5.0～10.0的环境中比较稳定,4℃下放置24 h酶的残余活力在70％以上.ShNAL对N-乙酰神经氨酸(Neu5Ac)、N-乙酰甘露糖胺(Man)和丙酮酸(Pyr)的Km值分别是(4.0±0.2) mmol/L、(131.7±12.1)mmol/L和(35.14±3.2) mmol/L,kcat/Km值分别为1.9 L/(mmol·s)、0.08 L/(mmol·s)和0.08 L/(mmol·s).     

超滤得到的海蜇酶解产物的降血脂功能评价

目的:探讨超滤对于海蜇酶解产物降血脂功能的提高作用。方法:海蜇通过中性蛋白酶酶解后,将超滤过的酶解产物和未超滤的酶解产物分别按照高、中、低三个剂量组喂食高血脂症大鼠模型42d,测定各组血脂水平并进行对照分析。结论:未超滤组的高、中剂量组和超滤组高中低剂量组喂食42d后均观察到大鼠血清总胆固醇(TC)、甘油三酯(TG)的降低,其中未超滤中剂量组(灌胃剂量5 mg/kg.BW)血清总胆固醇(TC)值为2.45±0.28mmol/L,超滤低剂量组(灌胃剂量0.3mg/kg.BW)血清总胆固醇(TC)值为2.61±0.33mmol/L,均明显低于高脂模型对照组(3.38±0.22 mmol/L),未超滤低剂量组(灌胃剂量3mg/kg.BW)血清总胆固醇(TC)值为2.82±0.38mmol/L,相对于高脂模型对照组(3.38±0.22 mmol/L)无显著差异;未超滤中剂量组(灌胃剂量5 mg/kg.BW)甘油三酯(TG)值为0.90±0.21mmol/L,超滤低剂量组(灌胃剂量0.3mg/kg.BW)甘油三酯(TG)值为0.93±0.14 mmol/L,均明显低于高脂模型对照组(1.21±0.20 mmol/L),未超滤低剂量组(灌胃剂量3mg/kg.BW)甘油三酯(TG)值为1.18±0.12mmol/L,相对于高脂模型对照组(1.21±0.20 mmol/L)无显著差异。结论:海蜇多肽的酶解产物具有降血脂功能,超滤能够有效提高海蜇酶解产物的降血脂活性。     

CTAB选择性沉淀和纯化质粒DNA工艺的建立

通过直接在大肠杆菌碱裂解上清中加入十六烷基三甲基溴化铵(CTAB), 优化CTAB与质粒DNA量的比例、质粒DNA选择性释放溶液的选择和TritonX-114的使用, 建立了简单、易行的大规模质粒DNA纯化工艺。纯化质粒DNA的质量检测结果显示, CTAB纯化的质粒DNA无菌体RNA污染, 菌体基因组DNA、内毒素和蛋白含量分别小于 100 ng/mg、50 EU/mg和10 mg/mg质粒DNA, OD260/OD280比值介于1.75~1.85之间, 超螺旋质粒DNA的比例大于80%, 该工艺纯化的质粒DNA能达到或接近FDA规定的人用质粒DNA的各项指标, 整个过程不使用动物源性酶和苯酚、氯仿、无水乙醇等有毒或易燃、易爆试剂, 成本低廉, 工艺环保。     

火球菌8-氧鸟嘌呤DNA糖苷酶基因克隆、表达纯化和酶学性质研究

【目的】在大肠杆菌中表达火球菌8-氧鸟嘌呤DNA糖苷酶,纯化得到重组火球菌8-氧鸟嘌呤DNA糖苷酶,在此基础上系统研究火球菌8-氧鸟嘌呤DNA糖苷酶的酶学特征。【方法】构建8-氧鸟嘌呤DNA糖苷酶重组表达质粒,将重组质粒转化Escherichia coli Rosetta(DE3),利用IPTG诱导表达重组蛋白,通过Ni2+亲和层析柱纯化重组蛋白;最后利用含8-氧鸟嘌呤损伤的寡核苷酸作为底物,测定8-氧鸟嘌呤DNA糖苷酶的酶学性质。【结果】在大肠杆菌中成功诱导表达了重组火球菌8-氧鸟嘌呤DNA糖苷酶,经Ni2+亲和纯化后蛋白纯度大于95%。在体外鉴定了重组火球菌8-氧鸟嘌呤DNA糖苷酶的酶学性质。结果表明重组火球菌8-氧鸟嘌呤DNA糖苷酶可以切除DNA中的8-氧鸟嘌呤(8-Oxo-G,GO)损伤碱基,并且具有AP裂解酶活性。重组火球菌8-氧鸟嘌呤DNA糖苷酶催化反应的最适pH值和温度分别是pH 8.5和55°C。除Zn2+对火球菌8-氧鸟嘌呤DNA糖苷酶的酶促反应有明显的抑制作用外,实验中测定的其它二价离子(Mn2+,Mg2+,Ca2+,Ni2+,Co2+,Cu2+)对其没有明显的影响。离子强度在50-100 mmol/L范围内对其酶促反应影响不大,超过100 mmol/L时有明显的抑制作用。与8-氧鸟嘌呤互补的碱基差异对火球菌8-氧鸟嘌呤DNA糖苷酶切除8-氧鸟嘌呤损伤的效率影响不大;但与单链DNA相比,双链DNA是优选底物,切割效率如下:GO/C≈GO/G≈GO/T≈GO/AGO/-。【结论】在大肠杆菌中成功表达,并Ni2+亲和纯化了火球菌8-氧鸟嘌呤DNA糖苷酶,生化研究表明制备的重组蛋白具有8-氧鸟嘌呤DNA糖苷酶活性,可能负责切除火球菌基因组DNA中的8-氧鸟嘌呤损伤。     

DCA干预对缺氧复氧肥大心肌细胞能量代谢及细胞凋亡的影响

目的:探讨缺氧复氧损伤诱导体外培养的新生大鼠肥大心肌细胞凋亡及能量代谢途径变化及药物干预的作用.方法:取体外培养的新生大鼠心肌细胞,以血管紧张素Ⅱ诱导其肥大,分4组:一组于3%O2、5%CO2、92%N2三气培养箱中培养12 h,再恢复正常条件培养4h,建立缺氧复氧损伤的肥大心肌细胞模型;另三组加入二氯乙酸盐(DCA)使其终浓度分别为10-3mmol/L、10-4mmol/L和10-5mmol/L,再缺氧复氧相同时间.电镜观察肥大心肌细胞及凋亡细胞的超微结构变化,Hochest33342/PI荧光染色识别凋亡细胞;TUNEL法观察心肌细胞凋亡形态学特征,并记数凋亡心肌细胞数,检测心肌细胞凋亡率;以同位素液闪计数法测定丙酮酸脱氢酶(PDH)内碱脂酰转移酶-1(CPT-1)活性,以及葡萄糖有氧氧化率,葡萄糖酵解率和脂肪酸有氧氧化率.结果:肥大心肌细胞在缺氧12h复氧4h,TUNEL法可检测到阳性的凋亡细胞,凋亡率为(19.99±4.88)%,肥大心肌细胞缺氧培养12h后加入DCA10-3 mmol/L、10-4 mmol/L和10-5 mmol/L再复氧4h检测其凋亡率分别为(16.5±3.24)%、(17.4±3.72)%和(18.4±3.44)%;与正常心肌细胞比较,肥大心肌细胞总的PDH活性没有明显改变.但活化型PDH活性和葡萄糖氧化代谢率(GOR)显著增强,CPT-1活性和脂肪酸有氧氧化代谢率(FOR)显著降低;与对照肥大心肌细胞比较,二氯乙酸(DCA 10-3 mmol/L-DCA 110-3mmol/L)呈剂量依赖性的升高PDH活性和GOR,抑制CPT21活性,FOR和葡萄糖酵解率(glucolysis rate,GLR).结论:DCA对缺氧复氧损伤引起的肥大心肌细胞凋亡有抑制作用.肥大心肌细胞能量代谢向糖代谢转化,DCA可进一步增强糖有氧氧化代谢抑制脂肪酸代谢.     

临床等级质粒DNA的实验室制备

在实验室有效获得100 mg临床试验等级的质粒DNA.摇床发酵3个批次,每批次4 L培养液,产生大约160 g的细菌沉淀物.碱裂解菌体,气浮法结合离心分离浮杂沉淀.裂解液经0.45/0.22 μm囊式过滤器过滤.然后采用阴离子色谱介质富集质粒,异丙醇沉淀.重新溶解沉淀后采用100 kD切向流超滤处理,获得100 mg临床试验等级质粒DNA.体外、体内转染试验对质粒表达效果进行了鉴定.结果表明,纯化的质粒DNA几乎检测不到内毒素、RNA和蛋白质,A260/A280比值在1.82～1.86范围内.纯化的质粒DNA能够满足动物临床试验.     

夏枯草DNA提取及RAPD反应条件的优化

用改进后的SDS法从夏枯草植物的新鲜叶片中提取基因组DNA,通过正交设计和单因素结合的方法对RAPD-PCR反应条件进行优化,确定了适合夏枯草DNA的最佳扩增体系:20μL的PCR反应体系中,模板DNA浓度1.0-2.0 mg/L,引物浓度1.00-1.50 mmol/L,Mg2 浓度2.0-2.5 mmol/L,dNTP浓度0.20-0.25 mmol/L,Taq酶用量0.5 U,10×BufferMg2 free 2μL,BSA浓度0.25-0.50μg/μL。     

高压氧对肾脏缺血再灌注损伤的保护作用及其机制研究

目的:观察高压氧(hyperbaric oxygen,HBO)对肾脏缺血再灌注损伤的保护作用并探讨其作用机制.方法:56只SD大鼠被随机分为三组,假手术组(n=8);I/R组(n=24),夹闭双肾动脉45分钟后恢复血流灌注;I/R+HBO组(n=24),夹闭双肾动脉45分钟并在恢复血流后1h、24 h、48 h行HBO治疗,每次HBO后采血并取双肾,比色法测定血浆尿素氮(BUN)、肌酐(Cr)值,原位末端标记(TUNEL)法检测肾小管上皮细胞凋亡情况,实时定量PCR法检测促凋亡基因Bax的mRNA含量.结果:与sham组(BUN值为9.563± 1.384 mmol/L;Cr值为45.912±2.685 mmo1/L,TUNEL值为2.088％)比较,I/R组大鼠再灌注1小时尿素氮(12.5±1.487 mmol/L)和血肌酐水平(51.388±3.092 mmol/L)升高,但差异无统计学意义,而TUNEL阳性细胞数(9.775％)和Bax的mR-NA(3.219± 0.427)表达水平均显著升高(P＜0.05),再灌注24小时及48小时后尿素氮(28.087± 2.012 mmol/L、41.225± 1.397mmol/L)和血肌酐(241.75± 11.853 mmol/L、278.75± 12.578 mmol/L)水平、TUNEL阳性细胞数(12.512％、14.413％)和Bax的mRNA(5.541±0.227、6.407± 0.291)表达水平均显著升高(P＜0.05);而HBO治疗可显著降低再灌注24小时及48小时的大鼠尿素氮(14.15±1.397 mmol/L、25.962± 2.497 mmol/L)和血肌酐(146.375± 8.782 mmolL、210.125± 11.519 mmol/L)水平(P＜0.05),但仍显著高于假手术组(P＜0.05).结论:HBO治疗可以改善I/R后肾功能,其作用机制可能与在早期明显降低Bax的mRNA表达,减轻肾小管上皮细胞凋亡有关.     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.