Clonal adaptation of cancer cells in flatfish liver to environmental contamination by changes in expression of P-gp related MXR, CYP450, GST-A and G6PDH activity

Progression from eosinophilic foci to persistent basophilic foci and carcinomas was observed in pollution-induced hepatocellular carcinogenesis in European flounder (Platichthys flesus L.) in a similar sequence as in chemically induced liver cancer in mammals. Image analysis was used to quantify enzyme activity and protein expression as visualised by enzyme and immunohistochemistry in various stages of toxipathic lesions and carcinogenesis. In eosinophilic foci, growth advantage was achieved by increased production of NADPH and pentoses for biosynthesis and cell proliferation due to increased G6PDH capacity during the first steps of clonal adaptation. Simultaneously, oxyradical production by CYP450, phase I was reduced. Overexpression of P-gp mediating multi-xenobiotic resistance was noted in basophilic cell types which persisted during progression towards carcinomas. This was accompanied by increased protein levels of oxyradical scavenging GST-A. These changes are consistent with adaptation and phenotypic expression of the multidrug or xenobiotic resistance (MDR/MXR) type in hepatocarcinomas of European flounder.     

Cell responses to xenobiotics: comparison of MCF7 multi-drug- and mussel blood cell multi-xenobiotic-defense mechanisms

Multi-drug resistance (MDR) in MCF7 breast cancer cells and multi-xenobiotic resistance (MXR) in mussel (Mytilus edulis) blood cells (MBC) are well known mechanisms that contribute to the decrease in intracellular concentrations of many unrelated but cytotoxic compounds. In the present work, we have carried out comparative investigations of the MDR/MXR protective mechanisms using a rapid colorimetric assay for cell viability and calcein accumulation for MDR/MXR activities. These studies were performed using cultured MCF7 and MBC before and after in vitro exposure to xenobiotics. Our results indicate that a 5-day exposure to doxorubicin or vincristine decreased calcein accumulation in MBC which is consistent with an induction of multi-xenobiotic resistance. The increase in calcein accumulation provoked by 1-h treatment with 50 microM verapamil was much lower in MBC when compared to the P-glycoprotein overexpressing MCF7 cell line. We conclude that such microplate assays could be used in primary cultures of MBC to estimate the effects of various chemicals on MXR activity.     

饲料蛋白质水平对拟目乌贼生长前期的生长性能、肌肉成分和酶活的影响

以鱼粉、豆粕和明胶为主要蛋白源配制成蛋白质水平分别为31.5%、35.0%、38.5%、42.0%、45.5%、49.0%的试验饲料。选取初始均重(3.99±0.08)g的拟目乌贼(Sepia lycidas)540只,随机分成6组,每组3个重复,每个重复30只。结果表明:拟目乌贼的生长性能指标(增重率、特定生长率、成活率)均在饲料蛋白质水平为45.91%时达到最佳,除与蛋白质水平为49.72%时差异不显著(P0.05)外,与其他各组均有显著差异(P0.05)。饲料系数随蛋白质水平的升高呈先降后升的趋势,在饲料蛋白质水平为45.91%时达到最低,显著低于除49.72%组外的其他各组;蛋白质效率随着饲料中蛋白质水平的升高而显著下降(P0.05)。饲料蛋白质水平对肥满度与肝体比影响不显著(P0.05)。胃蛋白酶、胰蛋白酶、肠道脂肪酶和肝脏超氧化物歧化酶活性,随饲料蛋白质水平的增加呈先升后降的趋势,均在饲料蛋白质水平为45.91%时达到最高,胃蛋白酶和胰蛋白酶活性除与蛋白质水平为42.22%时差异不显著(P0.05)外,与其他各组均有显著差异(P0.05);脂肪酶和肝脏超氧化物歧化酶活性与其他各组均有显著差异(P0.05),而肠道淀粉酶活性则相反。肝脏谷丙转氨酶和谷草转氨酶活性随饲料蛋白质水平的增加呈先升后稳定的趋势,均在饲料蛋白质水平为49.72%时达到最大,显著高于除45.91%组外的其他各组(P0.05);而饲料蛋白质水平对碱性磷酸酶活性影响不显著(P0.05)。乌贼肌肉粗蛋白质在饲料蛋白质水平45.91%时含量最高,显著高于除49.72%组外的其他各组(P0.05);粗脂肪含量最低,显著低于其他各组(P0.05);而肌肉水分和粗灰分含量不受饲料蛋白质水平的影响(P0.05)。拟目乌贼生长前期(4~10 g)配合饲料适宜的蛋白质水平为45.91%。     

脱水对条斑紫菜叶状体psbA和psbD基因表达量的影响

从处于不同脱水复水阶段的条斑紫菜(Porphyra yezoensis Ueda)叶状体中提取总RNA,用荧光定量PCR技术检测条斑紫菜不同脱水和复水过程中psbA和psbD基因的转录水平上的相对表达量的变化.实验结果表明,psbA和psbD基因转录水平上的相对表达量的变化趋势相似但变化幅度不一致.在相对含水量约为71...     

大菱鲆CD55负调控补体激活及其广谱细菌结合能力

CD55蛋白在哺乳动物中是一种补体调控因子,但其在鱼类中的功能未知。本研究探索了大菱鲆(Scophthalmus maximus) CD55(SmCD55)蛋白的功能。结果表明,SmCD55蛋白由344个氨基酸残基构成,含有一个N端信号肽和3个补体调控蛋白(CCP)功能域。SmCD55基因在大菱鲆多种组织中均有表达,其中,在肌肉中表达量最低,在脑组织的表达量最高。通过原核表达获得了重组的SmCD55(rSmCD55)蛋白,发现rSmCD55蛋白能够抑制大菱鲆血清的溶血活性和杀菌活性,表明其负调控补体系统的激活。此外,本研究还发现rSmCD55蛋白可以结合包括重要水产病原菌迟缓爱德华氏菌(Edwardsiella tarda)、荧光假单胞杆菌(Pseudomonas fluorescens)、鳗弧菌(Vibrio anguilla­rum)、哈维氏弧菌(Vibrio harveyi)和海豚链球菌(Strep­tococcus iniae)在内的多种细菌,其中与哈维氏弧菌的结合能力最强。以上这些研究结果丰富了鱼类补体系统的理论知识,加深了对鱼类补体激活调控的了解。     

饲料蛋白水平对奥尼罗非鱼(Oreochromis niloticus × O. aureus)生长、免疫功能以及抗病力的影响

采用单因素实验设计方法,进行了饲料蛋白水平对奥尼罗非鱼(Oreochromis niloticus×O.aureus)生长、免疫功能和抗无乳链球菌能力影响的研究。结果表明,饲料蛋白水平对奥尼罗非鱼生长和饲料利用率以及成活率不产生显著影响,蛋白质效率和蛋白质沉积率伴随着蛋白水平的升高显著下降。35%(D35)饲料组奥尼罗非鱼肝脏过氧化氢酶含量显著高于其它饲料组,然而奥尼罗非鱼肝脏溶菌酶、超氧化物歧化酶活性以及丙二醛含量并不受饲料蛋白水平的影响。无乳链球菌攻毒实验结果表明,蛋白水平对奥尼罗非鱼在攻毒后24h和48h后的累积死亡率是不存在显著影响。本实验的结果表明,24.8%(D25)蛋白水平饲料能够满足奥尼罗非鱼正常生长、以及维持机体正常的非特异性免疫功能和抗病力的营养需求。     

Effect of Protein Level and Varying Protein – Lipid Concentrations on Growth Characteristics of Juvenile Spotted Grunter Pomadasys Commersonnii (Haemulidae)

A 60-day feeding trial was conducted to evaluate the effects of dietary crude protein level on growth, feed conversion and protein efficiency ratio of juvenile (3–6 g) spotted grunter Pomadasys commersonnii. In the experiment, six semi-purified diets containing casein and fishmeal as protein sources, and with crude protein levels ranging from 35 to 60%, were fed to three replicate groups of fish per treatment in a recirculating system at the optimum temperature for growth. The relationships between dietary crude protein level and growth parameters were analysed by broken-line regression models. Results suggest that 48 – 50% dietary protein is needed for optimum growth and feed conversion for P. commersonnii. In a second experiment, three protein levels (35, 45 and 55%) at three different lipid concentrations (6, 8 and 12%) were used to formulate nine semi-purified diets that were fed for 60 days to triplicate groups of fish per treatment. Results suggest that a diet of at least 45% protein with a 12% lipid inclusion level is required for the best specific growth rate (5.96% per day) and feed conversion ratio (1.72) at this specific stage in the growth phase of P. commersonnii.     

新型蛋白源替代饲料中鱼粉对星斑川鲽(Platichthys stellatus)幼鱼氨基酸组成的影响

以新型蛋白源(大豆浓缩蛋白、脱酚棉籽蛋白、喷雾血球蛋白粉)分别替代基础饲料(D0)中17% (D17)、35% (D35)、52% (D52)和 69% (D69)的鱼粉, 配制 5 种等氮等能的饲料, 饲喂星斑川鲽幼鱼[初始体重(75.6±0.18)g] 60d, 以考察鱼粉替代对星斑川鲽幼鱼氨基酸组成的影响。结果表明, 试验组肌肉 Arg 和 His 含量显著增大(P<0.05), Thr 含量差异不显著(P>0.05), 其它必需氨基酸(EAA)含量均显著降低(P<0.05); 试验组全鱼 EAA 总量无显著性差异(P>0.05)。 D69 组肝脏天冬氨酸氨基转移酶(AST)和天冬氨酸氨基转移酶/丙氨酸氨基转移酶(AST/ALT)显著低于其它各组(P<0.05); 血清 AST及AST/ALT随鱼粉替代量的增加显著增大(P<0.05)。 本试验条件下, 当替代饲料中35%的鱼粉时, 饲料 EAA 消化率及试验鱼肌肉和全鱼氨基酸组成均无显著性差异, 替代比例进一步加大, 会对试验鱼肝脏生理功能产生影响。     

动物蛋白酶解物与无机氮、磷源的组合对小球藻(Chlorella vulgaris)生长和生化指标的影响

采用三因素不完全正交设计方法,通过对细胞密度、干重、叶绿素和可溶性蛋白质含量的测定.确定了N、P源与蛋白酶解物之间的最佳组合,研究碳酸氢铵作为N源、磷酸二氢钠作为P源与动物蛋白酶解物之间的组合对小球藻生长和生化指标的影响.结果表明:(1)动物蛋白酶解物与N、P源间存在互作效应--影响小球藻细胞密度、叶绿素的主要因素分别是P浓度、蛋白酶解物浓度;而N浓度及蛋白酶解物浓度同时对小球藻的蛋白质含量起主要作用;(2)同时添加N 50mg/L、P 4mg/L、蛋白酶解物12ml/L能显著提高小球藻生物量,促进叶绿素和藻体蛋白质的合成(P<0.05).     

刺激隐核虫ADP/ATP 载体蛋白基因的克隆和分子特征分析

刺激隐核虫(Cryptocaryon irritans)是严重危害海水鱼类的寄生纤毛虫,为了对其进行分子水平的研究,采用SMART技术构建了刺激隐核虫滋养体期的cDNA文库,并随机挑取克隆做EST测序,获得大量克隆的基因。从中挑选ATP/ADP载体蛋白基因同源物(CiAAC)的克隆进一步测序获得全长的cDNA序列。该序列全长为1029bp,包含编码287个氨基酸的开放阅读框。用RT-PCR分析了其在刺激隐核虫各时期的表达情况,并应用生物信息学分析方法,对CiAAC基因进行不同物种的系统进化树分析、功能区分析、物理性质分析、疏水性分析、跨膜结构域预测、亚细胞定位、二级结构预测等等。结果表明:CiAAC蛋白在刺激隐核虫各时期均有表达;据预测,它为六次跨膜蛋白,与卵形肠虫(Nyctotherus ovalis)的ADP/ATP载体蛋白的同源性最高,相似性达65%;蛋白为疏水性,并定位于刺激隐核虫的细胞质中。随后实验中通过对基因的定点诱变,将纤毛虫的非通用密码进行改造后,构建了pGEX-4T-1/CiAAC表达载体。以上实验为病原生物刺激隐核虫CiAAC载体蛋白的研究及应用提供了基础资料。     

几种鲑鳟鱼血清蛋白非变性聚丙烯酰胺凝胶电泳的研究

采用非变性聚丙烯凝胶电泳的方法分析了硬头鳟、金鳟、虹鳟、北极红点鲑和溪红点鲑血清蛋白的组成成分及其含量。结果显示, 硬头鳟、金鳟和虹鳟三者之间的遗传距离最小, 硬头鳟、金鳟和虹鳟三者与溪红点鲑的遗传距离最大; 硬头鳟、金鳟和虹鳟三者与北极红点鲑处于同一亚组, 而溪红点鲑独处于另一亚组。研究结果表明硬头鳟、金鳟和虹鳟三者与溪红点鲑的亲缘关系远于与北极红点鲑的关系; 同时还表明, 以聚丙烯酰胺凝胶电泳分析鲑鳟鱼血清蛋白的方法简单易行、重复性好。本研究结果对鲑鳟鱼的种属鉴定、良种选育、品种改良和遗传结构分析提供了试验依据; 虹鳟的繁养殖需进一步规范化和标准化, 减少蛋白遗传基因的流失, 从根本上预防大规模疾病的暴发。     

金鼓鱼幼鱼对蛋白质的适宜需要量研究

为确定金鼓鱼(Scatophagus argus)日粮中蛋白质的适宜添加量,作者以酪蛋白为蛋白源、鱼油为脂肪源,配制脂肪水平为8%,蛋白水平分别为24%、28%、32%、36%、40%的5种配合饲料(D1~D5),在海上网箱中以初始体质量为4.3 g左右的幼鱼开展养殖实验。8周后,测定实验鱼增质量率、特定生长率、蛋白质效率、饲料系数、消化酶和一些免疫及抗氧化指标。结果显示,随着饲料蛋白水平的升高,金鼓鱼的增质量率、特定生长率、蛋白质效率等均呈现先上升后下降的趋势,以D3组最高,3项指标均显著高于D1、D2组;饵料系数呈现先下降再上升趋势,以D3组最低,且显著低于D1、D2组。饲料蛋白水平也显著影响金鼓鱼的相关生理生化指标,其中D3和D4组的全鱼蛋白含量、消化道蛋白酶活性和一些免疫抗氧化指标最高,全鱼脂肪含量最低。根据饲料蛋白含量与增质量率的线性回归分析,确定金鼓鱼幼鱼饲料中的适宜蛋白水平为35%。     

CYP1A- and CYP3A-immunopositive protein levels in digestive gland of the mussel Mytilus galloprovincialis from the Mediterranean Sea

CYP1A-immunopositive protein can be elevated in response to planar PAHs and PCBs in Mytilus sp. digestive gland whilst CYP3A-immunopositive protein has been associated with testosterone 6β-hydroxylation in fish. Levels of CYP1A- and CYP3A-immunopositive protein were determined in Mytilus galloprovincialis digestive gland microsomes collected from 12 sites in the Mediterranean Sea during May and September 2001. CYP1A-immunopositive protein was significantly highest at contaminated sites whilst CYP3A-immunopositive protein was significantly lowest. A weak negative correlation (r²=0.21) was seen between CYP1A- and CYP3A-immunopositive protein. Little evidence of differences at the different sampling times was observed. These results confirm previous work indicating elevation of CYP1A-immunopositive protein in Mytilus sp. digestive gland at contaminated sites. Further study is required to characterise CYP3A-like expression in Mytilus and to elucidate the consequences of possible CYP3A-like down-regulation at contaminated sites.     

半滑舌鳎(Cynoglossus semilaevis Günther)生长激素体外重组表达及活性分析

根据半滑舌鳎(Cynoglossus semilaevis Günther)生长激素(GH)基因的c DNA全长序列设计引物克隆得到其全长552个碱基成熟肽序列。利用RT-PCR方法将扩增片段克隆到原核表达载体p ET-28a上,实现了GH成熟肽在大肠杆菌BL21(DE3)中的融合表达。融合蛋白分子量为26 k Da,在IPTG诱导4h时目的蛋白表达量最高,占细菌总蛋白的41.5%,主要以包涵体形式存在。Western-blotting分析表明GH融合蛋白可特异性地被6×His抗体识别。诱导表达后的菌液沉淀经纯化和复性后,获得大小为26 k Da的纯化GH融合蛋白。以ELISA方法检测纯化后的GH融合蛋白显示其具有免疫学活性。本研究为认识半滑舌鳎生长轴的调控机制提供了基础资料。     

不同饵料组成对花鲈幼鱼生长和生理活性影响研究

为探究不同饵料组成对北方人工繁育花鲈(Lateolabrax maculatus)幼鱼生长和生理活性的影响,优化花鲈幼鱼饵料组成,作者采用投喂不同组成的饵料研究其对花鲈幼鱼的生长和消化酶活性的影响,实验采用63日龄幼鱼,设4个处理组,分别组1投喂卤虫(Artemia)、组2高蛋白配合饲料、组3低蛋白配合饲料和组4冰鲜桡足类,共40 d。结果表明,组2的生长速度、最终体长、体质量、特定生长率、绝对生长率、增质量率均大于其余3组,组3的生长状况次之,但与其他2组并无显著性差异(P0.05);除组1的蛋白酶最大活性出现在实验中期,其他3组的蛋白酶变化与其生长状况相符,最大值均出现在103日龄,各组间差异显著;4组的淀粉酶活性呈上升趋势,在103日龄时,组1的淀粉酶活性大于组2,并显著大于组3和组4;组1的脂肪酶活性在实验后期迅速上升,并在103日龄达到最大值,而其余3组的脂肪酶活性呈下降趋势,103日龄时组3和组4脂肪酶活性显著小于组2和组1(P0.05)。研究表明,花鲈幼鱼摄食高蛋白配合饲料生长状况最好,摄食低蛋白配合饲料生长状况次之,摄食卤虫状况最差,出于经济的考虑,建议投喂低蛋白配合饲料。     

Multidrug resistance in the embryos and larvae of the mussel Mytilus edulis.

Cells exhibiting the multidrug resistance (MDR) phenotype demonstrate a decreased intracellular drug accumulation due to an active outward transport and decreased intracellular flux. This study demonstrates the inhibition of MDR in mussel (Mytilus edulis) embryos and larvae based on a simple bioassay. The development of embryos was assessed and abnormalities identified at key stages of development, including gastrulation, trochophore and prodissoconch stages. The incidence of developmental abnormalities was significantly increased in the presence of vinblastine, MMS, chloroquine, mitomycin-C, cadmium chloride and colchicine, compared to clean seawater. Consistently, there was a further increase in the number and severity of deformities observed when each toxin was added in the presence of verapamil. Larval growth was also significantly impaired in the presence of verapamil. Increased accumulation of fluorescent MDR dyes, such as rhodamine B, has been measured and shown to be verapamil sensitive. This bioassay encompasses a period of intense cellular activity during which the impairment of a number of critical processes results in abnormal growth and development.     

海洋球石藻病毒(Coccolithovirus)硫氧还蛋白(Trx)在毕赤酵母(Pichia pastoris)中的表达及其活性分析

从实验室保存的pBS-Trx重组质粒中克隆球石藻病毒EhV-Trx基因,构建毕赤酵母重组表达载体pPIC9K-EhV-Trx,将重组质粒电转化毕赤酵母GS115,诱导分泌表达并对重组蛋白进行二硫键还原酶活性分析。结果表明,EhV-Trx基因开放阅读框为591bp,编码197个氨基酸;在毕赤酵母GS115中成功诱导表达重组EhV-Trx,经SDS-PAGE分析目的蛋白分子量约为27.8kDa;重组EhV-Trx具有二硫键还原酶的活性,能有效打开胰岛素A、B两条链的二硫键,有望开发成一种新型的硫氧还蛋白脱敏制剂应用于食品安全领域。     

刺激隐核虫甘油醛-3-磷酸脱氢酶的分子特征

从刺激隐核虫(Cryptocaryon irritans)滋养体/包囊前体c DNA文库中筛选出了甘油醛-3-磷酸脱氢酶基因(Ci GAPDH),定点诱变Ci GAPDH基因开放阅读框内的非通用密码子后,构建其原核表达载体p GEX-4T-3/Ci GAPDH,转化到大肠杆菌BL21(DE3)中,用异丙基硫式-B-D-半乳糖苷诱导表达,结果大肠杆菌成功表达了r Ci GAPDH蛋白。用抗r Ci GAPDH蛋白的鼠血清进行免疫印迹分析,结果抗血清能够识别刺激隐核虫各期虫体的天然Ci GAPDH蛋白,其表观分子质量为37.3 ku,与根据氨基酸序列推算的理论值相符;实验表明r Ci GAPDH蛋白具有很好的免疫原性,而且Ci GAPDH在生活史的各阶段均有表达,符合持家基因的特征。利用间接免疫荧光抗体实验(IFAT)检测天然Ci GAPDH蛋白在刺激隐核虫幼虫上的定位,结果表明天然Ci GAPDH蛋白主要分布在幼虫的细胞质内,且在胞口位置分布最多。对Ci GAPDH的进一步研究可能为刺激隐核虫感染的药物靶点的寻找多条线索。     

基于鲎试剂生产的中国鲎血液质量监测方法研究

为了研究中国鲎血液质量的监测方法,实验选择9只中国鲎检测其血液中铜含量、血细胞数量、血浆蛋白含量、血淋巴蛋白含量等指标,并进行相关性分析。结果表明,中国鲎血液中铜含量与血细胞数相关性r=0.917,与血浆蛋白含量相关性r=0.995,与血淋巴蛋白含量相关性r=0.983,均呈现极显著相关性。中国鲎血液中铜含量与血细胞数的相关关系为Y₁=6.14+0.86X(R²=0.840),与血浆蛋白含量的相关关系为Y₂=2.08+0.78X(R²=0.989),与血淋巴蛋白含量的相关关系为Y₃=1.87+0.62X(R²=0.966)。中国鲎血液中铜含量不但能够直接反映血蓝蛋白的含量,而且可以反映血细胞数、血浆蛋白含量、血淋巴蛋白含量。因此,以中国鲎血液中铜含量作为监测血液质量的指标,可以推动中国鲎可持续采血技术的发展与完善,对中国鲎的利用与保护具有重要意义。