5''-Aza-CdR 对B细胞淋巴瘤的抑制作用及对瘤细胞CHFR 表达和甲基化的影响

目的:探讨5-氮杂-2'-脱氧胞苷(5'-Aza-Cd R)对人伯基特淋巴瘤Raji裸鼠移植瘤的抑制作用,以及对瘤组织中CHFR表达和甲基化的影响。方法:1皮下接种Raji细胞悬液构建荷瘤裸鼠模型18只,并随机分为实验组和对照组,分别予腹腔内注射等体积的5'-Aza-Cd R(1μg/g)及生理盐水,计算肿瘤体积大小,绘制肿瘤生长曲线,24天后比较两组移植瘤体积大小和重量。2分别从移植瘤组织中提取DNA及RNA,应用实时荧光定量PCR检测CHFR m RNA的表达,甲基化特异性聚合酶链反应(MSP)检测CHFR基因甲基化情况。结果:1实验组移植瘤生长速度较缓慢。实验结束时,实验组移植瘤体积、重量与对照组相比,差异有显著统计学意义(P0.01)。2实验组瘤细胞中CHFR m RNA的表达水平(3.69±1.20)与对照组(1.04±0.30)相比,差异有显著统计学意义(t=3.94,P0.001)。3实验组中的甲基化条带与对照组相比明显变暗,同时出现非甲基化条带,对照组中CHFR基因启动子只有甲基化条带被扩增。结论:5'-Aza-Cd R能抑制人伯基特淋巴瘤的生长,诱导瘤细胞CHFR m RNA表达增加,其机制可能为5'-Aza-Cd R使甲基化的CHFR启动子发生去甲基化。     

顺铂联合鸦胆子油乳对人卵巢癌裸鼠原位移植瘤生长的影响

目的:探讨鸦胆子油乳以及联合顺铂对人卵巢癌裸鼠原位移植瘤生长的抑制作用。方法:构建人卵巢癌裸鼠原位移植瘤模型,随机分为对照组、鸦胆子油乳组、顺铂组和鸦胆子油乳顺铂联合组等4组,每组8只,观察各组裸鼠移植瘤体积、重量、抑瘤率及相对肿瘤增值率(T/C)的变化,检测裸鼠外周血白细胞、血小板数目及血清谷丙转氨酶(ALT)、乳酸脱氢酶(LDH)、血肌酐(CR)的变化。结果:鸦胆子油乳组、顺铂组、联合组均可有效遏制肿瘤细胞生长,各组的抑瘤率分别为23.56%±3.67%、42.57%±3.45%和48.36%±3.08%;各组的肿瘤体积和T/C明显下降,T/C均低于60%,且与对照组比较,差异有统计学意义(P0.05);与对照组比较,鸦胆子油乳组治疗后裸鼠LDH、CR值差异显著(P0.01),顺铂组与联合组ALT、LDH、CR值差异有统计学意义(P0.01);与对照组比较,鸦胆子油乳组白细胞数差异显著(P0.05),而顺铂组、联合组血小板、白细胞数量均明显减少,且差异均具有统计学意义(P0.01)。结论:顺铂联合鸦胆子油乳可以抑制人卵巢癌裸鼠移植瘤的生长,鸦胆子油乳相对于顺铂对人卵巢癌SKOV3细胞裸鼠白细胞及血小板、肝肾功能影响较小,具有低毒作用。     

水飞蓟宾诱导卵巢癌HO-8910细胞凋亡增殖及其机制探讨

目的:探讨水飞蓟宾对人卵巢癌HO-8910细胞增殖的抑制作用及其作用机制。方法:HO-8910细胞分为四组:(1)对照组;(2)水飞蓟宾低浓度组;(3)水飞蓟宾中浓度组;(4)水飞蓟宾高浓度组。通过MTT法测定细胞增值率,流式细胞技术检测细胞凋亡情况,Hoechst染色观查细胞核凋亡,Western-blot检测bax及bcl-2表达。结果:细胞增殖检测结果显示,水飞蓟宾低、中、高浓度组的抑制率(83.00±5.51%、65.33±3.48%、56.67±4.37%)与对照组(97.33±4.25%)比较差异具有统计学意义(P0.05)。流式细胞技术结果显示,水飞蓟宾低、中、高浓度组凋亡细胞比例分别为16.93±2.34%、26.20±2.21%和37.93±1.98%,与对照组(1.43±0.72%)相比差异均有统计学意义(P0.05)。Hoechst染色结果显示,水飞蓟宾低、中、高浓度组凋亡细胞比例分别为12.56±2.55%、25.73±2.05%和39.14±3.69%,与对照组(0.54±0.67%)相比差异均有统计学意义(P0.05)。此外,水飞蓟宾可以升高bax基因表达水平,降低bcl-2基因表达平。结论:水飞蓟宾能明显抑制HO-8910细胞增殖,促进细胞凋亡,通过改变凋亡因子表达诱导卵巢癌HO-8910细胞凋亡。     

肝素结合表皮生长因子抑制剂CRM197 对裸鼠卵巢癌移植瘤 的抑制作用

目的:研究CRM197对裸鼠卵巢癌移植瘤生长的抑制作用探讨其对逆转卵巢癌组织耐药作用及其在卵巢癌治疗中的可行性.方法:用人卵巢癌亲本细胞A2780、耐紫杉醇细胞A2780/Taxol及耐顺铂细胞A2780/DDP分别建立裸鼠卵巢癌移植瘤模型,观察注射CRM197组与阴性对照组裸鼠肿瘤生长情况,免疫组化法测定各组裸鼠肿瘤组织中HB-EGF及P-gp表达情况.结果:HB-EGF及P-gp在各肿瘤组织中不同程度的表达,在注射CRM197的裸鼠肿瘤组织中表达明显降低(P＜0.05),差异具有统计学意义.结论:CRM197通过抑制肝素结合表皮生长因子的信号传导通路激活抑制了裸鼠卵巢癌移植瘤的生长,使裸鼠卵巢癌移植瘤HB-EGF及P-gp表达明显降低.     

胡桃醌不同给药途径对人肝癌细胞BEL-7402裸鼠皮下移植瘤生长的影响

目的采用裸鼠皮下移植瘤模型,通过不同给药途径对胡桃醌抗肿瘤活性和毒性进行评价。方法建立人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,通过腹腔注射和局部注射两个给药途径观察胡桃醌抑制肿瘤生长的效果。结果①以600、300和150μg/kg胡桃醌腹腔注射于人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,发现该剂量胡桃醌对肿瘤生长没有明显的影响;NK细胞活性检测发现,600、300μg/kg胡桃醌对裸鼠免疫功能有影响(P均<0.01),150μg/kg胡桃醌则没有影响(P>0.05);与阳性对照组(5-Fu)相比,600μg/kg胡桃醌组NK细胞活性差异无显著性(P>0.05),300和150μg/kg胡桃醌组NK细胞活性差异有显著性(P<0.05,P<0.01),结果提示胡桃醌对小鼠免疫系统有一定的损伤作用。②以4.5、3和1.5 mg/kg胡桃醌腹腔注射于人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,抑瘤率分别为为78.24%、66.57%、48.94%;4.5、3 mg/kg胡桃醌的抑瘤作用可与阳性对照组比拟(P均>0.05)。但4.5 mg/kg胡桃醌组裸鼠出现明显的皮下脂肪减少、消瘦,并有死亡现象。③以pH 7.4和pH 4.0的600、300和150μg/kg胡桃醌人肝癌BEL-7402细胞裸鼠皮下移植瘤模型局部给药,结果发现不同pH(pH 7.4或4.0)600、300μg/kg的胡桃醌局部注射抑瘤作用与阳性对照组(5-Fu)组差异无显著性(P>0.05),而不同pH的150μg/kg胡桃醌抑瘤作用不明显。同一浓度不同pH药物的抑瘤作用差异无显著性(P均>0.05),但pH 4.0的胡桃醌组肿瘤细胞肝转移较少。结论胡桃醌不同给药途径均可抑制人肝癌BEL-7402细胞裸鼠皮下移植瘤的生长,但有一定的毒副作用,药物安全范围较小。     

DC 荷载alpha-GalCer 对小鼠HepS 肝癌的抑制作用

目的:研究树突状细胞(DC)荷载α-半乳糖神经酰胺(α-Gal Cer)作为自然杀伤T细胞(NKT)刺激物对原发性肝癌Heps荷瘤小鼠的抑制作用。方法:建立小鼠Hep S肝癌移植瘤模型,随机分为3组,对照组(NS对照组)、树突状细胞组(DC组)、DC荷载α-半乳糖神经酰胺组(DC+α-Gal Cer组),每组10只。分别给予尾静脉注射DC、荷载α-Gal Cer的DC4×106/m L,对照组给予同体积的NS,隔天给药1次,共3次。各组于干预后2周取眼球血,用流式细胞术(FCM)检测小鼠外周血CD4+T细胞及CD8+T细胞含量变化。取肿瘤组织,称取各瘤体重量,计算抑瘤率。免疫组化染色比较各组瘤体细胞内Bax凋亡蛋白;Westem blot法检测各组移植瘤组织内Bax的表达。结果:DC荷载α-Gal Cer组CD4+T细胞及CD8+T细胞数较DC组及对照组明显升高(P<0.05),DC组较对照组有上升趋势,但差异无统计学意义(P>0.05)。DC荷载α-Gal Cer组平均瘤体重量较对照组及DC组均显著减轻(P<0.05),DC组与对照组平均瘤重无明显变化(P>0.05)。与NS组及DC组相比,DC荷载α-Gal Cer组移植瘤内Bax阳性细胞数量及Western blot表达量明显升高,差异有统计学意义(P<0.05);DC组与NS组之间无明显差异(P>0.05)。结论:DC荷载α-Gal Cer可活化NKT细胞,激活外周血抗肿瘤免疫细胞杀伤肿瘤,抑制小鼠肝癌移植瘤的生长。     

维生素D_3联合5-氟尿嘧啶对人食管癌Eca-109细胞移植瘤VDR表达的影响

探讨维生素D3、5-氟尿嘧啶单独与联合使用对人食管癌Eca-109细胞移植瘤维生素D受体(vitamin Dreceptor,VDR)的作用.随机分为对照组(C)、预处理组(PT)、维生素D3组(V)、5-氟尿嘧啶组(F)、预处理+5-氟尿嘧啶组(PT+F)、维生素D3+5-氟尿嘧啶组(V+F).体外培养人食管癌Eca-109细胞,BALB/c裸鼠皮下荷瘤,2.5μg/kg1,25-(OH)2维生素D3、25 mg/kg 5-氟尿嘧啶单独与联合腹腔注射,观察瘤体生长情况,逆转录聚合酶链反应(RT-PCR)和蛋白质印迹技术(Western blot)检测裸鼠瘤体组织VDR mRNA与蛋白的表达.研究发现1,25-(OH)2维生素D3、5-氟尿嘧啶均能抑制裸鼠移植瘤的生长,PT、V、F、PT+F、V+F组瘤体体积与C组比较差异有统计学意义(P<0.05);RT-PCR与Western blot结果显示经1,25-(OH)2维生素D3单独与联合5-氟尿嘧啶使用后瘤体组织中VDR mRNA和蛋白表达升高,且联合用药更为显著(P<0.05).结果表明1,25-(OH)2维生素D3、5-氟尿嘧啶均能抑制人食管癌Eca-1...     

RNAi沉默SDF-1基因对人胃癌裸鼠原位移植瘤生物学行为的影响及机制

目的:探讨RNAi技术沉默基质细胞衍生因子-1(stromal cell-derived factor-1,SDF-1)对人胃癌裸鼠原位移植瘤生物学行为的影响及可能机制。方法:利用RNAi-SDF-1细胞及对照组细胞建立人胃癌裸鼠皮下移植瘤,Western Blot检测裸鼠皮下移植瘤SDF-1蛋白表达情况。利用裸鼠皮下移植瘤建立其原位移植瘤模型,8周后处死裸鼠解剖尸体,检测原位移植瘤生长、凋亡及远处脏器转移情况。Western Blot检测SDF-1沉默对通路蛋白Akt、p-Akt、NF-κB、p-NF-κB以及侵袭转移相关基因E-cadherin、MMP-7表达的影响。结果:成功建立RNAi-SDF-1裸鼠原位移植瘤模型。与空白及阴性对照组比较,RNAi-SDF-1组裸鼠原位移植瘤生长缓慢,体积与质量均明显降低,差异有统计学意义(P0.01)。流式细胞术结果显示,RNAi-SDF-1组肿瘤细胞凋亡率明显高于空白及阴性对照组(P0.01)。尸体解剖结果显示,RNAi-SDF-1组肿瘤腹腔淋巴结及肝脏转移率明显低于空白及阴性对照组(P0.05)。Western Blot结果显示,与空白及阴性对照组比较,RNAi-SDF-1组肿瘤组织中Akt、p-Akt、NF-κB、p-NF-κB、MMP-7表达水平明显降低,E-cadherin表达水平明显升高(P0.01)。结论:RNAi-SDF-1能够有效抑制人胃癌SGC7901细胞裸鼠原位移植瘤的生长,诱导肿瘤细胞凋亡,抑制其腹腔淋巴结、肝脏转移,其机制可能与抑制PI3K-Akt、NF-κB信号通路及MMP-7的表达并上调E-cadherin的表达相关。     

EGCG对Tca8113细胞移植瘤小鼠肿瘤生长的抑制作用研究

为了探讨绿茶提取物表没食子儿茶素没食子酸酯(EGCG)对Tca8113鳞状细胞癌细胞移植裸鼠肿瘤的抑制作用及其机制。本研究选取采用Tca8113鳞状细胞癌细胞建立的裸鼠肿瘤模型48只,采用随机数字表法分为空白组(等量生理盐水灌胃)、低剂量组(ECGC 10 mg/kg灌胃)、中剂量组(ECGC 20 mg/kg灌胃)、高剂量组(ECGC 40 mg/kg灌胃)各12只,连续灌胃治疗16 d,测量各组小鼠肿瘤重量及体积、计算抑瘤率,采用流式细胞仪技术测定各组瘤体组织中细胞生长周期的分布,采用免疫组化染色技术测定各组肿瘤组织中Caspase8、Caspase3、Ki-67蛋白的表达。研究显示,低剂量组、中剂量组和高剂量组的瘤体质量和体积均显著的小于空白组(p0.05),且随着EGCG剂量的加大,裸鼠移植瘤体积和质量逐渐减小,而抑瘤率显著增加(p0.05);低剂量组、中剂量组和高剂量组的G0/G1期细胞比例显著高于空白组(p0.05),而S期及G2/M期细胞比例显著降低(p0.05);随着EGCG剂量的增加,各组G0/G1期细胞比例显著增加,而S期及G2/M期细胞比例显著降低(p0.05);低剂量组、中剂量组和高剂量组的肿瘤组织中Caspase8和Caspase3表达显著高于空白组(p0.05),而Ki-67蛋白表达显著低于空白组(p0.05);随着EGCG剂量的增加,各组肿瘤组织中Caspase8和Caspase3蛋白表达显著增加,而Ki-67显著降低(p0.05)。EGCG对Tca8113鳞状细胞癌细胞移植裸鼠肿瘤生长具有明显的抑制作用,其机制可能是通过调节细胞周期及Caspase8、Caspase3和Ki-67蛋白的表达来实现。     

DC荷载α-GalCer对小鼠HepS肝癌的抑制作用

目的:研究树突状细胞(DC)荷载α-半乳糖神经酰胺(α-Gal Cer)作为自然杀伤T细胞(NKT)刺激物对原发性肝癌Heps荷瘤小鼠的抑制作用。方法:建立小鼠Hep S肝癌移植瘤模型,随机分为3组,对照组(NS对照组)、树突状细胞组(DC组)、DC荷载α-半乳糖神经酰胺组(DC+α-Gal Cer组),每组10只。分别给予尾静脉注射DC、荷载α-Gal Cer的DC4×106/m L,对照组给予同体积的NS,隔天给药1次,共3次。各组于干预后2周取眼球血,用流式细胞术(FCM)检测小鼠外周血CD4+T细胞及CD8+T细胞含量变化。取肿瘤组织,称取各瘤体重量,计算抑瘤率。免疫组化染色比较各组瘤体细胞内Bax凋亡蛋白;Westem blot法检测各组移植瘤组织内Bax的表达。结果:DC荷载α-Gal Cer组CD4+T细胞及CD8+T细胞数较DC组及对照组明显升高(P0.05),DC组较对照组有上升趋势,但差异无统计学意义(P0.05)。DC荷载α-Gal Cer组平均瘤体重量较对照组及DC组均显著减轻(P0.05),DC组与对照组平均瘤重无明显变化(P0.05)。与NS组及DC组相比,DC荷载α-Gal Cer组移植瘤内Bax阳性细胞数量及Western blot表达量明显升高,差异有统计学意义(P0.05);DC组与NS组之间无明显差异(P0.05)。结论:DC荷载α-Gal Cer可活化NKT细胞,激活外周血抗肿瘤免疫细胞杀伤肿瘤,抑制小鼠肝癌移植瘤的生长。     

操纵茶树类黄酮3′-羟基化酶生物合成B环-3′,4′-二羟基黄酮类化合物

【目的】操纵茶树类黄酮3′-羟基化酶,生物合成B环-3′,4′-二羟基黄酮类化合物圣草酚、二氢槲皮素和槲皮素。【方法】构建了4个茶树类黄酮3′-羟基化酶基因(CsF3′H)和拟南芥的P450还原酶基因(ATR)融合表达质粒:SUMO-CsF3'H[7-517]::ATR1[49-688]3 AA、SUMO-CsF3'H[28-517]::ATR1[49-688]3 AA、SUMO-CsF3'H[7-517]::ATR2[75-711]3 AA和SUMO-CsF3'H[28-517]::ATR2[75-711]3 AA,分别转化大肠杆菌菌株TOP10、DH5α和BL21,获得12个转化菌株S1–S12;构建了茶树类黄酮3′-羟基化酶基因CsF3′H表达质粒p YES-Dest52-CsF3′H,转化酵母菌株WAT11,得到转化菌株S13;构建了茶树类黄酮3′-羟基化酶基因CsF3′H表达质粒pES-URA-CsF3′H,及茶树黄烷酮3-羟基化酶基因CsF3H与拟南芥黄酮醇合成酶基因At FLS的融合表达质粒pES-HIS-CsF3H::At FLS 9AA,二者共转化酵母菌株WAT11,获得转化菌株S14。【结果】转化SUMO-CsF3'H[28-517]::ATR1[49-688]3 AA质粒的TOP10菌株S6在25°C条件下发酵,转化效率最高,能将1000μmol/L柚皮素、二氢山奈酚和山奈酚,分别转化生成287.93μmol/L圣草酚、131.76μmol/L二氢槲皮素和188.62μmol/L槲皮素。发酵菌株S13能分别将1000μmol/L柚皮素、二氢山奈酚和山奈酚,最多能转化生成734.32μmol/L圣草酚、446.07μmol/L二氢槲皮素和594.64μmol/L槲皮素。喂食S14发酵菌株5 mmol/L的底物柚皮素,在发酵36–48 h中,最多能生成1412.16μmol/L圣草酚、490.25μmol/L山奈酚、445.75μmol/L槲皮素、66.75μmol/L二氢槲皮素和73.50μmol/L二氢山奈酚。【结论】本研究首次将茶树类黄酮3′-羟基化酶基因应用于B环-3′,4′-二羟基黄酮类化合物圣草酚、二氢槲皮素和槲皮素的生物合成。     

Loss of Yme1L perturbates mitochondrial dynamics

Yme1L is an AAA protease that is embedded in the mitochondrial inner membrane with its catalytic domain facing the mitochondrial inner-membrane space. However, how Yme1L regulates mammalian mitochondrial function is still obscure. We find that endogenous Yme1L locates at punctate structures of mitochondria, and that loss of Yme1L in mouse embryonic fibroblast (MEF) cells results in mitochondrial fragmentation and leads to significant increased ‘kiss-and-run'' type of mitochondrial fusion; however, Yme1L knockdown (shYme1L (short hairpin-mediated RNA interference of Yme1L)) cells still remain normal mitochondrial fusion although shYme1L mitochondria have a little bit less fusion and fission rates, and the shYme1L-induced fragmentation is due to a little bit more mitochondrial fission than fusion in cells. Furthermore, shYme1L-induced mitochondrial fragmentation is independent on optic atrophy 1 (OPA1) S1 or S2 processing, and shYme1L results in the stabilization of OPA1 long form (L-OPA1); in addition, the exogenous expression of OPA1 or L-OPA1 facilitates the shYme1L-induced mitochondrial fragmentation, thus this fragmentation induced by shYme1L appears to be associated with L-OPA1''s stability. ShYme1L also causes a slight increase of mitochondrial dynamics proteins of 49 kDa and mitochondrial fission factor (Mff), which recruit mitochondrial key fission factor dynamin-related protein 1 (Drp1) into mitochondria in MEF cells, and loss of Drp1 or Mff inhibits the shYme1L-induced mitochondrial fragmentation. In addition, there is interaction between SLP-2 with Yme1L and shYme1L cells retain stress-induced mitochondrial hyperfusion. Taken together, our results clarify how Yme1L regulates mitochondrial morphology.     

Comparison of full-length genomics sequences between dengue virus serotype 3, parental strain,and its derivatives,and B-cell epitopes prediction from envelope region

Biological markers are normally used to evaluate the candidate of live-attenuated dengue vaccines. D3V 16562 Vero 23 and D3V 16562 Vero 33 which were derivatives of D3V 16562, parental strain, showed the similar biological data. We used molecular techniques and computational tools to evaluate these derivatives. The nucleotide and amino acid sequences of the derivatives were compared to their parent. The secondary structures of untranslated regions and B-cell epitopes were predicted. The results showed that nucleotide substitutions mostly occurred in NS5 and NS5 of V2 was unusual because of amino acid change at 3349 (tryptophan →stop codon). The nucleotide substitutions in 5''UTR, prM, E, NS1, NS2A, NS3, and 3''UTR were 4, 1, 2, 2, 1, 3, and 2, respectively. The secondary structure of 5''UTR of V2 was different from P and V1. The secondary structure of 3''UTR of V2 was similar to P and certainly distinct from V1. Furthermore, B-cell epitopes prediction revealed that there were 21 epitopes of envelope and the interesting epitope was at position 297-309 because it was in domain III in which the neutralizing antibody is induced. For this study, the attenuation of derivatives was caused by the nucleotide substitutions in 5''UTR, 3''UTR, and NS5 regions. The genotypic data and B-cell epitope make the derivatives attractive for the chimeric and peptide DENV vaccine development.     

两湖泊轮虫群落结构的时空动态对水体氮磷含量及“下行效应”的响应

为探讨氮和磷、"上行效应"和"下行效应"在轮虫群落结构时空变动调控中的相对重要性,于2011年7月至2012年6月每月两次采集了芜湖市九莲塘和汀棠湖中的轮虫样品,利用相关分析法和典范对应分析法(CCA)分析了轮虫群落结构的时空变动与水体理化因子及其潜在捕食者密度间的关系。卡尔森营养状态指数表明,两湖均处在富营养化初期。除了总氮和硝态氮的年均浓度、晶囊轮虫(Asplanchna)和剑水蚤(Cyclopoidea)无节幼体的年均密度存在显著差异外(P0.05),两湖泊的其他水环境因子间并未表现出明显的差异(P0.05)。实验室鉴定发现九莲塘中共有轮虫48种,隶属于15科21属;汀棠湖中共有轮虫55种,隶属于15科24属。两湖泊中,疣毛轮虫(Synchaeta)、臂尾轮虫(Brachionus)和异尾轮虫(Trichocerca)均是主要轮虫类群,而密度优势种分别有7种和4种。相关分析结果显示,P含量与九莲塘轮虫群落的均匀度和物种多样性指数间均呈现显著的负相关(P0.05),而N含量却与汀棠湖轮虫群落的物种多样性指数呈现显著的正相关(P0.05);两湖泊中的轮虫物种多样性指数均与水体中的TN∶TP(质量比)间呈现显著的正相关(P0.05)。CCA分析结果显示,九莲塘中桡足类和其无节幼体密度显著高于其他采样批次时的轮虫群落聚成Ⅰ类,汀棠湖中晶囊轮虫密度显著高于其他批次时的轮虫群落聚成Ⅱ类,而其他的轮虫潜在捕食者密度很低或未出现时的轮虫群落聚成Ⅲ类。研究结果表明,湖泊中的TN∶TP(质量比)可能是反映N和P含量对轮虫群落物种多样性影响程度的较为合适的指标。当水体中的轮虫潜在捕食者密度较高时,两湖泊轮虫群落结构的变动均主要取决于"下行效应";而当轮虫潜在捕食者密度很低时,"上行效应"可能是调控两湖泊轮虫群落结构变动的主要因素。     

Crystallographic studies of drug-nucleic acid interactions: Proflavine intercalation between the non-complementary base-pairs of cytidilyl-3′,5′-adenosine

In order to get insights into the binding of dyes and mutagens with denatured and single-stranded nucleic acids and the possible implications in frameshift mutagenesis, a 1:1 complex between the non-self-complementary dinucleoside monophosphate cytidilyl-3′,5′-adenosine (CpA) and proflavine was crystallized. The crystals belong to the tetragonal space group P4₂2₁2 with cell constants $\text{a = b = 19.38(1)}\text{A}\text{?}\text{and}\text{c = 27.10(1)}\text{A}\text{?}$. The asymmetric unit contains one CpA, one proflavine and nine water molecules by weight. The structure was determined using Patterson and direct methods and refined to an R-value of 11% using 2454 diffractometer intensities.The non-self-complementary dinucleoside monophosphate CpA forms a selfpaired parallel chain dimer with a proflavine molecule intercalated between the protonated cytosine-cytosine (C · C) pair and the neutral adenine-adenine (A · A) pair. The dimer complex exhibits a right-handed helical twist and an irregular girth. The neutral A · A pair is doubly hydrogen-bonded through the N(6) and N(7) sites (C(1′)C(1′) distance: 10.97(2) Å) and the protonated C · C pair is triply hydrogen-bonded with a proton shared between the N(3) sites (C(1′)C(1′) distance: 9.59(2) Å). To accommodate the intercalating dye, the sugars of successive nucleotide residues adopt the two fundamental conformations (5′ end: 3′-endo, 3′ end: 2′-endo), the backbone adopts torsion angle values that fluctuate within their preferred conformational domains: the PO bonds (ω, ω′) adopt the characteristic helical (gauche^?-gauche^?) conformation, the CO bonds (φ, φ′) are both in the trans domain and the C(4′)C(5′) bonds (ψ) are in the gauche⁺ region. The bases of both residues are disposed in the preferred anti domain with the glycosyl torsion angles (χ) correlated to the puckering mode of the sugar so that the cytidine residue is C(3′)-endo, low χ (12 dg), and the adenosine residue is C(2′)-endo, high χ (84 °). The intercalated proflavine stacks more extensively with the C · C pair than the A · A pair. Between 4₂-related CpA proflavine units there is a second proflavine which stacks well with both the A · A and the C · C pairs sandwiching it. Both proflavine molecules are positionally disordered. In each of its two disordered sites, the intercalated proflavine forms hydrogen-bonded interactions with only one sugar-phosphate backbone. A total of 26 water sites has been characterized of which only two are fully occupied. These hydration sites are involved in an intricate network of hydrogen bonds with both the dye and CpA and provide insights on the various modes of interactions between water molecules and between water molecules and nucleic acids.The structure of the proflavine-CpA complex shows that intercalation of planar drugs can occur between non-complementary base-pairs. This result can be relevant for understanding the strong binding of acridine dyes to denatured DNA, single-stranded RNA, and single-stranded polynucleotides. Also, the ability of proflayine to promote self-pairs of adenine and cytosine bases could provide a chemical basis for an alternative mechanism of frameshift mutagenesis.     

Effects of increasing the number of players and memory size in the iterated Prisoner's Dilemma: a numerical approach

The Prisoner''s Dilemma has become a paradigm for the evolution of altruistic behaviour. Here we present results of numerical simulations of the infinitely iterated stochastic simultaneous Prisoner''s Dilemma considering players with longer memory, encounters of more than two players as well as different pay-off values. This provides us with a better foundation to compare theoretical results to experimental data. We show that the success of the strategy Pavlov, regardless of its simplicity, is far more general by having an outstanding role in the iterated N-player N-memory Prisoner''s Dilemma. Besides, we study influences of increased memory sizes in the iterated two-player Prisoner''s Dilemma, and present comparisons to results of experiments with first-year students.     

喜盐鸢尾花色形成关键基因的克隆及表达分析

喜盐鸢尾(Iris halophila Pall.)及其变种蓝花喜盐鸢尾(I.halophila Pall.var.sogdiana(Bung)Grubov)因耐盐碱及其多种花色而具有盐碱地园艺开发价值。本文根据喜盐鸢尾内轮花被转录组测序结果,利用基因特异性引物从这2种植物中分别克隆了编码查尔酮合成酶(CHS)、查尔酮异构酶(CHI)、类黄酮-3',5'-羟基化酶类(F3'5'H-like)等基因的部分片段,并对它们在内轮花被中的表达水平进行实时定量PCR分析。序列分析结果确认在喜盐鸢尾中所克隆的CHS(311 bp)、CHI(457 bp)、F3'5'H-like(496 bp)3个基因(部分)未见文献报道与NCBI等数据库记录。其中F3'5'H-like基因与经典的属于细胞色素P450CYP75A亚家族的F3'5'H不同,而与万带兰的F3'5'H-like同属于CYP76AB亚家族,为一类新的蓝花相关基因。实时定量PCR表达分析结果表明,与黄花的喜盐鸢尾相比,蓝花喜盐鸢尾中CHS与F3'5'H-like显著上调表达,可能是其花色不同于喜盐鸢尾的主要原因。     

Sulfolobus acidocaldarius DHH超家族核酸酶Saci0542的酶学特征研究

【目的】以嗜酸嗜热硫化叶菌Sulfolobus acidocaldarius的DHH超家族核酸酶（Saci0542）为例,研究其核酸外切酶活性特点,为阐明其在DNA代谢中的具体功能提供生化基础。【方法】将嗜酸嗜热硫化叶菌DHH超家族核酸酶Saci0542基因在大肠杆菌中重组表达,经亲和层析纯化得到电泳纯的重组蛋白;利用荧光标记的寡核苷酸作为底物,用尿素变性聚丙烯酰胺凝胶电泳技术,鉴定Saci0542的酶学特征。【结果】重组表达的DHH超家族核酸酶Saci0542具有典型的单链核酸特异性的3’-5’外切酶活性。进一步酶学特征表征结果如下:酶活性依赖于二价金属离子Mn²⁺,而Ca²⁺、Mg²⁺、Zn²⁺等二价金属离子对活性没有明显的促进作用;Saci0542在pH5.5–10的广泛范围内均表现出较高酶活性;高于200 mmol/L的NaCl强烈抑制酶活性;最适反应温度为50–55℃;末端磷酸基团抑制3’-5’外切酶活性。【结论】本研究证实,Saci0542是一种Mn²⁺依赖型3’-...