Carbonic Anhydrase III S-Glutathionylation Is Necessary for Anti-Oxidant Activity

Carbonic anhydrase isozyme CA3 protects cells against oxidative stress. Ectopic expression of murine Ca3, but not Ca2, protects proto-oncogene Evi1 expressing Rat1 fibroblast cells (ca3low) against hydrogen peroxide (H2O2) induced stress. Ca3 is S-glutathionylated via glutathione adducts with cysteines 181 and 186. Substitution of both Ca3 cysteines with serine fails to protect cells from oxidative stress. Insertion of cysteine at 181 and 186 in Ca2 is insufficient for conferring efficient anti-oxidant activity. This shows for the first time that S-glutathionylation of cys181 and cys186 residues is required for Ca3 anti-oxidant activity but that additional factors are also required.

水泥基材料中Ca^(2+)对矿化微生物的碳酸酐酶产量、活性和矿化的影响

本工作针对一种适用于水泥基材料的耐碱矿化微生物,研究了水泥基材料中Ca^(2+)浓度变化(0~5 mmol/L)对矿化微生物的产酶量、碳酸酐酶活性以及诱导CaCO_(3)矿化能力的影响。对分离纯化不同Ca^(2+)浓度菌液中的碳酸酐酶进行酶产量测定,并通过测定矿化微生物的细菌生长曲线和单菌产酶量分析Ca^(2+)浓度对其的影响机理;基于酯酶法测定不同Ca^(2+)浓度下碳酸酐酶的活性,并利用FTIR测定官能团,分析二级结构变化和氢键变化,进而得到Ca^(2+)浓度对碳酸酐酶活性的影响机理;最后基于热重分析不同Ca^(2+)浓度溶液中矿化微生物的诱导矿化能力,并测定矿化产物晶形。试验结果表明,当Ca^(2+)浓度为2.5 mmol/L时,矿化微生物生长增殖最快,单菌产酶量最高,因此整体酶产量最高;碳酸酐酶活性在Ca^(2+)浓度为5 mmol/L时达到最大值,但Ca^(2+)会破坏碳酸酐酶结构的有序性;当Ca^(2+)浓度为5 mmol/L时,矿化微生物的诱导矿化能力最强,矿化3 d后Ca(OH)_(2)向CaCO_(3)的转化率达到82.67%,且得到的CaCO_(3)晶体形貌主要为球状或椭圆状。

Asymmetric expression of CA2 and CA13 linked to calcification in the bilateral mandibular condyles cause crossed beaks in chickens

Crossed beak is a complex mode of inheritance with prevalence ranging from 0.2 to 7.4% in at least 12 chicken strains worldwide.To reveal the intrinsic factors causing crossed beaks,genes expression patterns in bilateral mandibular condyle between affected and normal birds were characterized by RNA sequencing analysis in the present studies.Crossed beak was induced by short length of unilateral mandibular ramus,and a total of 110differentially expressed genes were up-or down-regulated in the affected(short)mandibular condyle side as compared to the normal side.Carbonic anhydrase 2(CA2)and Carbonic anhydrase 13(CA13)were enriched in the carbonate dehydratase activity,and high-expressed in mandibular condyle and osteoblasts(P<0.05).However,both were low-expressed in short mandibular condyle side of affected birds(P<0.05).The carbonate dehydratase inhibitor experiments confirmed that there is positive association between the calcification and carbonic anhydrase isoenzymes.Quantitative analysis with cetylpyridinium chloride showed a decrease in calcification when the cells were transfected with an anti-CA13 shRNA.Our research suggested that CA2 and CA13 are down-calcified in shortside mandibular condyle,and caused mandibular ramus to grow slowly.CA2 and CA13 have the critical role in crossed beaks by regulating calcification of mandibular condyle.

Cloning and Bioinformation Analysis of Carbonic Anhydrase Gene FsCA1 of Tibet Wild Buckwheat

A carbonic anhydrase( CA) transcript was obtained from the Contig library according to the published sequencing information of the buckwheat transcripts. The full length of the CA gene was amplified by reverse transcription PCR( RT-PCR). The bioinformatics analysis showed that the full length of Fs CA1 gene was 1233 bp and open reading frame was 978 bp,and encoding 325 amino acids. The molecular weight was 35. 11 ku and the isoelectric point was 7. 59; there were 9 α helices,6 β folds,many randon coil and extension chain,containing one signal peptide and one transmembrane region,having a 2 amino acid conserved domains with typical beta-type carbonic anhydrase. Subcellular localization showed that the protein is most likely to appear in the chloroplast. The three-dimensional structure model of Fs CA1 was built by homologous modeling method,indicating that the homo-octamer of buckwheat CA and pea CA could match well,so it can be inferred that buckwheat CA is also homo-octamer. Real-time quantitative PCR was used to detect the expression of Fs CA1 in different organs of buckwheat.The results showed that Fs CA1 had the highest expression level in leaves,then in the stems,and the lowest in roots.

一种靶向CA IX的多肽化合物标记与初步评价

为合成靶向碳酸酐酶IX(CA IX)的多肽探针^(68)Ga-DOTA-CA IX-P1-4-10,建立^(68)Ga-DOTA-CA IX-P1-4-10的标记及质控方法,对pH和前体多肽DOTA-CA IX-P1-4-10浓度对标记率的影响进行研究,确定^(68)Ga-DOTA-CA IX-P1-4-10的标记条件为:反应体系的pH为4.0~5.0(n=6),多肽DOTA-CAIX-P1-4-10浓度为15.0~40.0 mg/L(n=6),反应温度为100℃,反应时间10 min。产品为无色澄明液体、无可见异物,pH为5~7(n=6),放化纯度>99.0%(n=6),比活度为1.0~5.0 GBq/μmol(合成结束后)(n=6)。探针的脂水分配系数为-2.07±0.01(n=6),具有良好的亲水性。^(68)Ga-DOTA-CA IX-P1-4-10在生理盐水和胎牛血清中孵育4 h,放化纯度>95.0%,具有良好的体外稳定性。^(68)Ga-DOTA-CA IX-P1-4-10无异常毒性。初步评价结果表明,^(68)Ga-DOTA-CA IX-P1-4-10标记方法简单、放化纯度高、性质稳定、无异常毒性,具有开展进一步研究的价值。

巴戟天含药血清对成骨-破骨细胞共育体系CAⅡ、NFAT2mRNA表达的影响

目的探讨不同浓度巴戟天含药血清对体外培养成骨-破骨细胞共育体系中碳酸酐酶Ⅱ(CAⅡ)、活化T细胞核因子(NFAT2)mRNA表达的影响。方法取24 h内新生SD乳鼠头盖骨分离培养成骨细胞,取5周龄SD大鼠四肢长骨骨髓基质细胞,加入集落细胞刺激因子(M-CSF)和细胞核因子κB受体活化因子配体(RANKL)诱导培养破骨细胞。采用ALP染色鉴定成骨细胞,TRAP染色、骨吸收陷窝甲苯胺蓝染色、电镜等扫描鉴定破骨细胞,体外建立成骨-破骨细胞共育体系,设置高、中、低3种浓度巴戟天含药血清组和对照组,干预3d后提取各组总RNA,应用Real Time PCR(RT-PCR)方法测定各组CAⅡ、NFAT2mRNA表达并进行统计学分析。结果不同浓度巴戟天含药大鼠血清对成骨-破骨细胞共育体系CAⅡ、NFAT2 mRNA的表达均有抑制作用,且其抑制作用表现出一定的浓度依赖性;各组间差异有统计学意义(P<0.05)。结论巴戟天含药血清可抑制成骨-破骨细胞共育体系CAⅡ、NFAT2mRNA表达,从而达到降低破骨细胞分化成熟及骨吸收活性。

微生物碳酸酐酶诱导CaCO3沉淀的影响因素及生成机理

微生物碳酸酐酶能够加速CO2的水合反应,对于研究全球碳循环具有重要的意义。选用一种产生胞外碳酸酐酶的细菌,研究了温度、pH值和Ca^2+浓度对细菌生长、碳酸酐酶活性的影响。结果表明,25℃碳酸酐酶的活性最高,有利于CaCO3的沉淀;初始pH值为8.5的偏碱性环境中,CaCO3沉淀质量最多;当Ca^2+浓度为50 mmol/L时,细菌的生长繁殖最好,过低的Ca^2+浓度会影响CaCO3的生成,而过高的Ca^2+浓度则会严重影响细菌的生长,降低细菌的活性。最后研究了微生物碳酸酐酶诱导CaCO3沉淀的机理,碳酸酐酶能够加速CO2水化成HCO3-,在碱性环境中、钙源存在的情况下,与OH-和Ca^2+反应生成CaCO3沉淀。

Inorganic Carbon Utilization in Some Marine Phytoplankton Species

In order to learn the ways and possible utilization mechanisms of dissolved inorganic carbon (DIC) in marine phytoplankton species under carbon-replete or -limited conditions, the activity of extracellular carbonic anhydrase (CA) was assayed in different pH, CO 2 and DIC concentrations. Extracellular CA in Amphidinium carterae and Prorocentrum minimum was detected under carbon-replete conditions, while in Melosira sp., Phaeodactylum tricornutum, Skeletonema costatum, Thalassiosira rotula, Emiliania huxleyi and Pleurochrysis carterae, CA activity was assayed under conditions of carbon limitation. No CA activity was found even under carbon-limited conditions in Chaetoceros compressus, Glenodinium foliaceum, Coccolithus pelagicus, Gephrocapsa oceanica and Heterosigma akashiwo. In species without extracellular CA activity, the direct HCO - 3 uptake was investigated using a pH drift technique and the anion exchange inhibitor 4′4′-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) in a closed system. The result showed that direct HCO - 3 transport might occur by an anion exchange mechanism in species Coc. pelagicus and G. oceanica. Of the 13 species investigated, only H. akashiwo did not have the potential for direct uptake or extracellular CA-catalyzed HCO - 3 utilization.

改性文冠果活性炭吸附Ca^2+的研究

采用硝酸对文冠果活性炭进行氧化改性,探讨了Ca^2+溶液初始浓度、吸附温度、时间、pH值对Ca^2+吸附的影响。分析了吸附热力学和动力学,初步探讨了吸附机理。实验表明,当Ca2+的初始浓度为500 mg/L,吸附温度为40℃,吸附时间为120 min,pH值为2时,吸附量最大,可达285.9 mg/g。硝酸改性文冠果活性炭吸附Ca2+符合伪二级动力学模型和Langmuir等温线模型,吉布斯自由能ΔG°<0、焓变ΔH°<0、熵变ΔS°<0,表明该吸附是一个自发的放热过程。

海洋浮游藻类细胞质膜氧化还原活性与细胞外CA活性的关系(简报)

海洋浮游藻类除通过吸收和释放分子与离子来改变其环境的化学成分外,还可通过细胞外表面一些酶的作用引起质膜外化学物质变化。在这方面,海洋浮游藻类一个主要的细胞外表面酶-碳酸酐酶(CA),在经胰蛋白酶处理从细胞质膜上释放出来后,仍保留其催化活性。当细胞外表面CA(简称细胞外CA)具活性时,可催化质膜外HCO_3^-与CO_2的相互转化,为Rubisco(磷酸核酮糖羧化酶)提供一稳定的CO_2流量环境,以维持正常的光合作用。

The Ecological Importance and Influence of Land Use on Carbonic Anhydrase Activity of Waters in the Upstream Section of the Wujiang River Basin

Thirty-four sampling sites for surface water and twenty-seven sampling sites for ground water in areas of different land use were selected in the Wujiang River Basin, upstream section in Guizhou Province, China. Carbonic anhydrase （CA） activity in surface and ground water was analyzed and compared. The results show that CA activity was detected in all surface and ground water samples. In general, average CA activity in surface and ground water was highest from samples in arbor lands, followed by shrub lands, shrub and grass lands, agricultural lands, and residential areas. The average CA activity in ground water from arbor lands, shrub lands, and shrub and grass lands was higher than that in surface water. Water CA activity was positively correlated with HCO3- concentration in surface water and ground water. These results imply that the contribution of CA in water to the karst carbon sink potential of river basins warrants further extensive and in-depth research.

Nitrogen in Relation to Photosynthetic Capacity and Accumulation of Osmoprotectant and Nutrients in Brassica Genotypes Grown Under Salt Stress

Different strategies of the application of nutrients are required to overcome the adverse effects of mustard （Brassica juncea L.） in response to NaCl stress. The objective of the present study was to determine if different added levels of nitrogen （N） in growth medium could alleviate the adverse effects of salt stress on photosynthetic capacity and accumulation of osmoprotectants and nutrients. 14 days mustard seedlings of salt-sensitive （cv. Chuutki） and salt-tolerant （cv. Radha） genotypes were fed with： （i） 0 mmol L^-1 NaCl ＋ 0 mg N kg^-1 sand （control）, （ii） 90 mmol L^-1 NaCl ＋ 30 mg N kg^-1 sand, （iii） 90 mmol L^-1 NaCl ＋60 mg N kg^-1 sand, （iv） 90 mmol L^-1 NaCl ＋90 mg N kg^-1 sand and （v） 90 mmol L^-1NaCl＋ 120 mg N kg^-1 sand. Under the condition of salinity stress, N application caused a significant ameliorative effect on both genotypes with respect to growth attributes [fresh weight （FW） and dry weight （DW）] and physio-biochemical parameters [percent water content （WC）, net photosynthetic rate （PN）, stomatal conductance （gs）, total chlorophyll （Ch1）, carbonic anhydrase （CA） activity and malondialdehyde （MDA）, nitrogen （N）, potassium （K） and sodium （Na） contents, and K/Na ratio] and yield attributes （number of pods/plant, seeds/pod and seed yield/plant）. The salt-tolerant genotype exhibited maximum value for growth, physio-biochemical and yield attributes at 60 mg N kg 1 sand than that of salt-sensitive genotype. These results suggest that application of N may ameliorate most of the attributes and prove to be a physiological remedy to increase the tolerance against the ill effects of salt stress in Brassicas.

Ca(OH)_2改性活性炭及其吸附CO_2的研究

采用Ca(OH)2为化学活化剂,对颗粒活性炭进行改性。结果表明,在Ca(OH)2用量为100mL、改性温度80℃、改性时间2h、干燥时间6h时,改性后的活性炭吸附容量达到最大,为3.91mmol/g。

2个重组海带α-碳酸酐酶(CA)的酶活性比较研究

为了探究海带(Saccharina japonica)α-CA1和α-CA2是否具有催化CO_(2)的可逆水合反应和酯酶活性,作者通过原核表达得到这两种α-CA的可溶性的异源重组蛋白。分别用电极法和酯酶活性检测法来检测重组α-CA1(rα-CA1)和rα-CA2的水合酶活性和酯酶活性,结果发现,rα-CA1的水合酶活性(1.52±0.120U/mg)几乎是rα-CA2(0.54±0.046U/mg)的3倍,表明rα-CA1催化CO_(2)的水合能力明显大于rα-CA2。而两者的酯酶活性并没有显著的差别(rα-CA1比活力:0.697±0.176U/g,rα-CA2比活力:0.743±0.129 U/g),说明催化乙酸对硝基苯酯(p-NPA)生成对硝基苯酚(p-NP)的能力是没有显著差别的。实验结果证实这2个α-CA为功能蛋白,可能参与海带无机碳吸收和储存过程,因此,本研究为海带无机碳吸收和储存机制的解析提供了生物化学依据。

Comparative study of carbonic anhydrase activity in waters among different geological eco-environments of Yangtze River basin and its ecological significance

This study provides the presence of carbonic anhydrase（CA） activity in waters of the Yangtze River basin, China, as well as the correlation of CA activity with HCO-3 concentration and CO2 sink flux. Different degrees of CA activity could be detected in almost all of the water samples from different geological eco-environments in all four seasons. The CA activity of water samples from karst areas was significantly higher than from non-karst areas（PP3-concentration（r = 0.672, P2 sink flux（r = 0.602, P = 0.076） in karst areas. This suggests that CA in waters might have a promoting effect on carbon sinks for atmospheric CO2 in karst river basins. In conditions of similar geological type, higher CA activity was generally detected in water samples taken from areas that exhibited better eco-environments, implying that the CA activity index of waters could be used as an indicator for monitoring ecological environments and protection of river basins. These findings suggest that the role of CA in waters in the karst carbon sink potential of river basins is worthy of further in-depth studies.

Utilizing the γ-Irradiated Sodium Alginate as a Plant Growth Promoter for Enhancing the Growth,Physiological Activities,and Alkaloids Production in Catharanthus roseus L.

Sodium alginate is a polysaccharide that is largely obtained from the brown algae （Sargassum sp.）.It has been used as a wonderful growth promoting substance in its depolymerized form for various plants.The aim of this study was to find out the effects of various concentrations of γ-irradiated sodium alginate （ISA）,viz.,deionized water （control,T0）,20 （T1）,40 （T2）,60 （T3）,80 （T4）,and 100 ppm （T5） on the agricultural performance of Catharanthus roseus L.（Rosea） in terms of growth attributes,photosynthesis,physiological activities,and alkaloid production.The present work revealed that ISA applied as leaf-sprays at concentrations from 20 to 100 ppm might improve growth,photosynthesis,physiological activities,and alkaloid production in C.roseus L.significantly.Of the various ISA concentrations,80 ppm proved to be the best one compared to other concentrations applied.

PHYSIOLOGICAL ASPECTS OF A HIGH-CO_2 REQUIRING MUTANT AND THE HIGH-CO_2 GROWING CELLS OF THE CYANOBACTERIUM SYNECHOCOCCUS PCC7942

A new chemically mutagenic mutant of Synechococcus PCC7942 named high-CO2 re-quiring mutant, which could grow at 4% CO2 but could not grow at air levels of CO2, was isolated. Com-parative studies on some physiological aspects of the mutant and high-cO2 growing cells (growing at 4%CO2 ) were conducted. The result showed that the mutant had lower growing rate, about 1/40th photosyn-thetic affinity to inorganic carbon, 25% lower carbonic anhydrase (CA) activity, lower quenching rate ofchlorophyll fluoresence, and about 1/2 alkalinization rate of the medium. The CA activity responses ofthe two types of cells to different concentrations of CO2 were determined. Upon the addition of inorganiccarbon (Ci), the rate of active Ci uptake described by the rate of chlorophyll fluorescence quenching ofthe mutant was obviously lower compared with that of the high-CO2 growing cells; the size of the internalinorganic carbon pool size determined by the extent of fluorescence quenching Of the mutant was alsosmaller.

改性聚乙烯膜和氧化硅固定化碳酸酐酶

分别以改性聚乙烯(PE)膜和氧化硅(SiO_(2))为载体,考察了不同方法固定化碳酸酐酶(CA)的活性,然后以聚多巴胺/聚乙烯亚胺(PDA/PEI)改性的PE和SiO_(2)固定化CA为研究对象,考察了最适反应条件及稳定性。结果表明:在相同的反应条件下,PDA/PEI-SiO_(2)固定化CA的酶活回收率最高,为58.8%,PDA/PEI-PE固定化CA的酶活回收率为17.1%,使用10次后保留活性分别为84.8%和90.2%;固定化酶的最适反应条件均为35℃、pH 8.5,与游离酶相同;在较高温度(55~65℃)和较高酸浓度(>0.010 mol/L)下的稳定性高于游离酶;Mn2+对游离酶和固定化酶活性均有显著的促进作用,K^(+)、Mg^(2+)无明显影响;PDA/PEI-SiO_(2)和PDA/PEI-PE固定化CA在4℃下贮存10 d,保留活性分别为95.2%和92.4%;当用于CO_(2)水合反应时,CaCO_(3)的生成量分别是游离CA的120%和70%。固定化CA在CO_(2)捕集的工业化应用方面具有很大潜力。

微生物碳酸酐酶对石灰岩的溶蚀驱动作用研究

来源于西南几个不同类型岩溶地区土壤样品分离出来的微生物菌株中,有很多能够产生分泌胞外碳酸酐酶。以一株编号为GLCa102的菌株为代表,模拟岩溶自然环境条件,研究其胞外碳酸酐酶对灰岩的溶蚀驱动作用。结果表明微生物碳酸酐酶能使灰岩溶出的导电离子总量和[Ca2+]提高40%以上,从而对灰岩有显著的酶促溶蚀驱动作用。本研究表明微生物碳酸酐酶在生物岩溶中有重要的作用和地位,同时为深入研究生物对石灰岩的溶蚀作用提供了一定的科学依据。

微藻固定CO_2研究进展

空气中CO2 浓度升高所导致的温室效应已成为重大的环境问题 ,受到人们普遍关注。概述了高效固定CO2 微藻藻种的筛选和培养方法 ,分析了微藻固定CO2 的无机碳利用形式和浓缩机制 ,讨论了高效光生物反应器设计和运行目标 ,简要介绍了微藻 (酶 ) 膜生物反应器集成新技术。并认为今后的研究方向主要是在进一步探索微藻固定CO2 有关机理的基础上 ,构建高效固定CO2 的转基因微藻 ,开发高效膜生物反应集成系统。