聚磷激酶基因在假单胞菌中的整合和表达

为了构建高效除磷的微生物,将来源于大肠杆菌的聚磷激酶基因(ppk)插入广宿主载体pBBR1MCS-2多克隆位点区,得到质粒pBBR1MCS-2-ppk.以该质粒为模板,通过PCR扩增出携带有载体启动子和终止子序列的ppk基因,插入自杀型质粒pUTmini-Tn5中得到重组质粒pUTmini-Tn5-ppk.pUTmini-Tn5-ppk经三亲接合作用进入Pseudomonas putidaKT2440,同时mini-Tn5通过转座作用将ppk整合到宿主菌株的染色体DNA中,获得基因工程菌Pseudomonas putidaKT2440-PPK,用于表达ppk.RT-PCR结果显示,ppk基因在KT2440-PPK中得到较高量的表达,而在原始菌株KT2440中表达微弱.人工模拟污水实验结果表明,接种1 h时KT2440-PPK中聚磷含量达到最大,为3.05 mg/g,约是对照菌株KT2440的15倍.测定模拟污水中磷酸盐的含量表明,KT2440-PPK可以去除该模拟污水中90%以上的磷酸盐.     

False elevation of amniotic fluid enzymes of relevance for prenatal diagnosis: Creatine kinase measurement in relation to duchenne muscular dystrophy

The creatine kinase activity of amniotic fluid was measured in samples collected at fetoscopy. In our first study, the control sample range was 0-25 IU/1, although four samples had activities of 35–85 IU/1. Elevated values did not correlate with the activities in the fetal or maternal circulations. Electrophoresis revealed the presence of the BB isozyme of creatine kinase rather than just the MM form as expected. This suggested that the source of the elevated enzyme activity was from the myometrium, damaged by insertion of the trocar and cannula. In a further series the first 2 ml of amniotic fluid withdrawn yielded a much higher creatine kinase activity than a second aliquot. A control series of such second samples (first 2 ml discarded) gave an activity range of 0–7 IU/1 with no spuriously high values. This compares favourably with a series from single samplings taken by amniocentesis. Normal creatine kinase activities were found in the amniotic fluids from 20 pregnancies at risk for Duchenne muscular dystrophy. We conclude that for accurate measurement of amniotic fluid enzyme activity the first portion withdrawn should be discarded. Amniotic fluid creatine kinase activity is of no value for the prenatal diagnosis of Duchenne muscular dystrophy.     

城市污水处理厂Candidatus Accumulibacter的菌群结构及定量分析

Candidatus Accumulibacter是污水生物除磷系统中的优势聚磷菌.本研究采用编码聚合磷酸盐激酶的功能基因(polyphosphate kinase 1gene,ppk1)作为遗传标记,对9个污水处理厂共12个活性污泥样品中Accumulibacter的进化枝水平的丰度及其菌群结构进行了研究.实时定量PCR(Quantitative PCR,QPCR)的结果显示,Accumulibacter的6个进化枝(IA、IIA、IIB、IIC、IID、IIF)均存在于12个污泥样品中.A2O及其改良工艺中总Accumulibacter丰度最高,达到总菌的22.77%.所有样品中Accumulibacter均以Type-II为主,其中,IIC、IID的丰度均达到了108cells·g-1(以MLSS计),占Accumulibacter的平均比例分别为75.34%和14.87%,是污水处理厂中的优势聚磷菌.分支IA丰度最低,占总Accumulibacter比例平均只有0.32%.系统发育分析结果显示,每个样品中Accumulibacter分支多样化,均含有IIA、IIC、IID分支.QPCR和系统发育分析都证实了生物除磷效果与Accumulibacter的群落结构密切相关,除磷效果不好可能和IID占总Accumulibacter比例较高有关.     

几种化合物对温石棉诱导入胚肺细胞蛋白激酶C活性的影响

为了探讨几种化合物对温石棉诱导人胚肺（HEL）细胞蛋白激酶C（PKC）活性的影响，该研究用不同浓度的柠檬酸铝、混合稀土或亚硒酸钠溶液浸泡温石棉1小时后，再将其与HEL细胞共同孵育24小时，测定了HEL细胞胞浆和胞膜中PKC的活性。其结果显示：随着石棉剂量的增加，HEL细胞胞浆和胞膜中的PKC活性逐渐升高，均呈明显的剂量依赖性；经3种化合物预处理的石棉，其所诱导的HEL细胞胞浆和胞膜中的PKC活性均明显低于未处理温石棉组。笔者提示：温石棉可能通过改变细胞内PKC的活性进一步促进肿瘤的发生；用3种化合物预处理温石棉，均可抑制温石棉对HEL细胞PKC活性的诱导作用。     

多聚磷酸盐激酶基因在污水生物除磷中的功能

为验证多聚磷酸盐激酶基因(ppk)在污水生物除磷中的功能.采用Red敲除系统,以p KD4质粒为模板,设计同源短臂,扩增外源线性DNA片段,将外源线性DNA片段电转化整合入已导入p KD46的大肠杆菌ATCC25922野生型菌株.获得重组菌E.coli/ppk~-Kan~+.将p CP20导入大肠杆菌E.coli/ppk~-Kan~+以消除卡那霉素抗性基因,通过负抗性筛选及正反向引物验证,构建无抗生素抗性的ppk基因缺失工程菌株E.coli/ppk~-Kan~-.比较工程菌株和野生型菌株的生长特性,并比较两者在缺磷诱导/富磷及多次厌氧/好氧诱导条件下的除磷性能.结果表明采用Red重组系统,通过无痕敲除,成功构建了大肠杆菌ppk基因缺失菌株E.coli/ppk~-Kan~-.敲除后的工程菌株和野生型菌株生长整体没有差异,但是4 h前对数期工程菌株生长快于野生型菌株,8 h后稳定期工程菌株生长慢于野生型菌株,表明ppk影响菌体的生长;缺磷诱导/富磷条件下,工程菌株并未表现出因ppk缺失而影响其除磷能力;经过5次厌氧/好氧诱导,两菌菌体含磷量保持在1%~2%,没有因诱导次数的增加而表现出菌体含磷量增加的趋势,也未发现厌氧有PHB好氧有聚磷颗粒生成,表明ppk基因的缺失并没有引起菌体除磷能力的下降.ppk并未表现出明显的与污水生物除磷相关的功能.     

Effects of a phycotoxin,okadaic acid,on oyster heart cell survival

Okadaic acid (OA) is a dinoflagellate toxin which accumulates in shellfish producing diarrhetic shellfish poisoning (DSP) in humans. It was found that OA is a highly selective inhibitor of protein phosphatase types 1 (PP1) and 2A (PP2A) which produces a marked increase in phosphorylation of several proteins, including p38 mitogen-activated protein (MAP) kinase. The cytotoxicity attributed to OA and the effects on p38 MAP kinase and calcium current were examined in the oyster Crassostrea gigas in this study. Data showed that p38 MAP kinase is strongly expressed in oyster heart and that OA bioaccumulated in cultured heart cells. Hence the effects of OA was tested in vitro and in vivo on oysters. OA was found to (i) exert a positive chronotropic effect on cultured atrial cardiomyocytes which is related to an increase in calcium current via PKC as shown by patch clamp measurements, (ii) produce an activation/phosphorylation of MAP kinase as shown by Westernblot while the non-phosphorylated p38 remained constant during treatment, (iii) did not induce a pro-apoptotic effect. Data suggest that OA may also stimulate the anti-apoptotic pathway by phosphatase inhibition.     

Effects of aluminum on NAD kinase activity in chloroplast fraction from leaves of rice seedlings

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MUCT短程硝化和反硝化除磷系统中Candidatus Accu- mulibacter的代谢活性和菌群结构

采用实现亚硝酸型硝化的MUCT工艺处理低C/N实际生活污水,在短程硝化的基础上实现反硝化除磷.研究短程硝化建立与破坏过程中,亚硝酸盐积累率的变化对系统除磷性能及Candidatus Accumulibacter菌群结构的影响.结果表明:MUCT除磷以反硝化除磷为主,平均反硝化除磷率高达88%.磷去除率与亚硝酸盐积累率具有很好的正相关性.短程硝化阶段磷的平均去除率比全程硝化阶段高30%以上,证明了亚硝酸盐更适合作为低C/N比污水反硝化除磷的电子受体.以多聚磷酸盐激酶基因(ppk1)作为遗传标记,采用实时荧光定量PCR方法考察不同亚硝酸盐积累率下Accumulibacter的丰度、各主要进化分支的菌群结构和相对丰度.当系统处于全程硝化状态时,存在少量以硝酸盐为电子受体的Acc-I型反硝化聚磷菌,低于总Accumulibacter的5%;当系统进入短程硝化状态后,Acc-I逐渐消失.运行期间以亚硝酸盐作为电子受体进行反硝化除磷的Acc-IID始终是优势聚磷菌,达到总Accumulibacter的92%以上,甚至接近100%,保证了亚硝酸型反硝化除磷的稳定运行,亚硝酸盐浓度是影响其丰度变化的重要因素.