Carbonic anhydrase and C4 photosynthesis: a transgenic analysis

Carbonic anhydrase (CA, EC 4.2.1.1) catalyses the first reaction in the C₄ photosynthetic pathway, the conversion of atmospheric CO₂ to bicarbonate in the mesophyll cytosol. To examine the importance of the enzyme to the functioning of the C₄ photosynthetic pathway, Flaveria bidentis (L.) Kuntze, a C₄ dicot, was genetically transformed with an antisense construct in which the cDNA encoding a putative cytosolic CA (CA3) was placed under the control of a constitutive promoter. Some of the primary transformants had impaired CO₂ assimilation rates and required high CO₂ for growth. The T₁ progeny of four primary transformants were used to examine the quantitative relationship between leaf CA activity and CO₂ assimilation rate. CA activity was determined in leaf extracts with a mass spectrometric technique that measured the rate of ¹⁸O exchange from doubly labelled ¹³C¹⁸O₂. Steady‐state CO₂ assimilation rates were unaffected by a decrease in CA activity until CA activity was less than 20% of wild type when they decreased steeply. Transformants with less than 10% of wild‐type CA activity had very low CO₂ assimilation rates and grew poorly at ambient CO₂ partial pressure. Reduction in CA activity also increased the CO₂ partial pressure required to saturate CO₂ assimilation rates. The present data show that CA activity is essential for the functioning of the C₄ photosynthetic pathway.     

黄顶菊入侵对土壤中主要功能细菌的影响

以我国北方大面积发生的入侵植物黄顶菊为研究对象,对黄顶菊根际土壤中可培养的主要功能细菌进行了分离筛选,通过rep-PCR聚类和多样性分析研究了其群落结构的变化,并利用16S rRNA序列比对,对主要优势菌群进行鉴定.结果表明: 相对于本地植物万寿菊和空白对照,黄顶菊显著增加了根际土壤中固氮菌、有机磷细菌、无机磷细菌和钾细菌的数量.rep-PCR分析显示,黄顶菊根际4种功能细菌的种群结构与本地植物和对照相比有显著差异,3种土壤中相同的聚类群极少.多样性分析表明,黄顶菊根际微生物物种多样性更加丰富,群落结构更加复杂,优势种群比较明显,生态多样性较高.对从3种土壤中分离得到的主要优势菌群的鉴定结果也进一步证明了这一结论.研究结果为阐明黄顶菊入侵的微生态机制提供了理论基础.     

Carbonic anhydrase in the plasma membranes from leaves of C3 and C4 plants

Plasma membranes were isolated from green leaves of maize ( Zea mays ), spinach ( Spinacia oleracea ), Setaria viridis and wheat ( Triticum aestivum cv. Omase) by aqueous two-phase partitioning. Carbonic anhydrase activity was detected in these membranes. The activity was inhibited by specific inhibitors for carbonic anhydrase, acetazolamide and ethoxyzolamide. The carbonic anhydrase activity was markedly enhanced by the addition of Triton X-100 to the plasma membranes. The highest activity was obtained in the presence of 0.015% detergent. The activity was scarcely affected when the plasma membrane vesicles were treated with proteinase K, but largely inactivated by the protease after treating the membranes with Triton X-100. These results indicate that carbonic anhydrase faces the cytoplasmic side of the membrane since plasma membranes purified by aqueous two-phase partitioning are tightly sealed vesicles of right side-out orientation (apoplastic side-out). With leaves of C₄ plants, 20 to 60% of the total carbonic anhydrase activity was found in the microsomal fraction. By contrast, only 1 to 3% of the activity was found in the microsomal fraction from leaves of C₃ plants. Western blot analysis showed that a polypeptide in the spinach plasma membrane cross-reacted with an antiserum raised against spinach chloroplast carbonic anhydrase, and that the molecular mass of the plasma membrane enzyme was higher than that of the chloroplast carbonic anhydrase (28 and 26 kDa, respectively). This indicates the presence of different molecular species of carbonic anhydrase in the chloroplast and the plasma membrane.     

黄顶菊对不同入侵地植物群落及土壤微生物群落的影响

为明确不同入侵地植物群落和土壤生态对黄顶菊入侵的反馈机制,选取天津静海(JH)、河北沧州(CZ)、河北衡水(HS)及河南安阳(AY)4个黄顶菊入侵典型区域,研究黄顶菊对不同入侵地植物群落多样性、土壤理化及土壤微生物群落结构的影响,并进一步揭示植物群落、土壤养分和土壤微生物之间的相关关系。结果表明,黄顶菊入侵显著降低了JH、CZ和HS的植物群落多样性指数(P0.05),改变了四个地区的土壤理化性质,显著升高了不同入侵地真菌PLFA的含量、总PLFA的含量、真菌/细菌和革兰氏阴性菌/革兰氏阳性菌(P0.05),降低了土壤微生物的Margalef丰富度指数(P0.05),但均存在地区间差异;RDA和相关分析的结果表明,硝态氮、全氮的含量对植物群落的影响较大,而铵态氮的含量对土壤微生物群落结构的影响较大,除丰富度指数外,植物群落与土壤微生物群落的多样性指数之间存在显著的负相关关系(P0.05)。总之,黄顶菊改变了入侵地植物群落多样性,并且对入侵地土壤理化性质和土壤微生物群落结构产生了显著影响,且存在地区差异。本研究将为更好的理解外来植物的入侵机制及制定相应的防控策略提供理论依据。     

外来入侵植物黄顶菊在我国的地理分布格局及其时空动态

【目的】黄顶菊是20世纪90年代入侵我国的恶性外来杂草,近年来呈快速扩散蔓延趋势。全面解析黄顶菊在我国的扩散动态对制定早期监测预警等管理措施具有重要意义。【方法】通过查阅文献和标本记录及实地调查等途径重建了黄顶菊在我国扩散的历史过程,分析了其地理分布格局及其主要扩散路线,以及最早入侵地与传入途径。【结果】黄顶菊最早入侵河北省南部的邯郸、衡水,其后主要沿公路扩散蔓延,71%的分布点在距入侵地国道等主要公路2 km的范围内。目前,该杂草已从最初的入侵地向北扩散到天津市,向东扩散到山东省东营市,向西扩散到了山西省黎城市,向南扩散到了河南省开封市。黄顶菊向4个方向扩散的直线距离不等(100～350 km),这种差异与自然屏障和扩散通道相关。太行山脉是黄顶菊向西扩散的地理屏障,但这个屏障已逐渐被突破。至今,黄顶菊已入侵华北地区5个省市(河北、山东、天津、河南和山西)的100余个县。【结论】黄顶菊在我国仍处于快速扩散阶段,未来应该会继续沿公路向南和向西蔓延。因此,亟需在扩散的前沿地带开展监测预警工作,以抑制其在我国的进一步扩散。     

不同植物(组合)对入侵杂草黄顶菊功能性状的影响

【目的】外来植物的入侵能力与其性状之间的关系是入侵生态学中的基本问题之一。外来植物成功入侵具备哪些植物功能性状,一直是研究的热点问题,研究和把握这些性状对外来植物的入侵机理研究具有重要的现实意义。【方法】利用同质园实验,以入侵植物黄顶菊为研究对象,设置不同的植物(或组合)与黄顶菊竞争处理,研究不同植物(组合)对黄顶菊植物功能性状的影响,从而探究不同植物对黄顶菊的抵御能力。【结果】地肤、苘麻、苏丹草和苏丹草与地肤、黄花草木樨组合对黄顶菊有一定抑制作用,混种处理中黄顶菊的生物量、分枝数、株高显著低于黄顶菊单种处理,且对叶片功能性状、根功能性状、植株的全C和全N量等都有一定程度的影响。而反枝苋、反枝苋和紫花苜蓿组合对黄顶菊的生长有一定的促进作用,混种处理中黄顶菊的生物量与全C、全N含量较单种黄顶菊显著增加。【结论】不同植物与黄顶菊竞争生长对黄顶菊的影响不同,地肤和苘麻可以显著地抑制黄顶菊的主要功能性状生长发育,而反枝苋和紫花苜蓿促进黄顶菊生长发育。     

Development of a cheminformatics platform for selectivity analyses of carbonic anhydrase inhibitors

Abstract

The selectivity for a specific human Carbonic Anhydrase (hCA) isoform is an important property a hCA inhibitor (CAI) should be endowed with, in order to constitute a valuable therapeutic tool for the treatment of a desired pathology. In this context, we developed a chemoinformatic platform that allows the analysis of the structure and selectivity profile of known CAIs reported in literature, with the aim of identifying structural motifs connected to ligand selectivity, thus providing useful guidelines for the design of novel ligands selective for the desired hCA isoform. The platform is able to perform ultrafast structure and selectivity analyses through ligand fingerprint similarity, with no need of structural information about the target receptor and ligands’ binding mode. It is easily accessible to the non-expert user through the implementation of a KNIME Analytic Platform workflow and could be extended to analyze the selectivity profile of known ligands of different target proteins.     

Improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). In strain BL21 (DE3) bearing ecaA, the activity of CA was 21.8 U mg⁻¹ protein, whereas non-detectable CA activity was observed in the control strain. Meanwhile, the activity of phosphoenolpyruvate carboxylase (PEPC) increased from 0.2 U mg⁻¹ protein to 1.13 U mg⁻¹ protein. The recombinant bearing ecaA reached a succinate yield of 0.39 mol mol⁻¹ glucose at the end of the fermentation. It was 2.1-fold higher than that of control strain which was just 0.19 mol mol⁻¹ glucose. EcaA gene was also introduced into E. coli DC1515, which was deficient in glucose phosphotransferase, lactate dehydrogenase and pyruvate:formate lyase. Succinate yield can be further increased to 1.26 mol mol⁻¹ glucose. It could be concluded that the enhancement of the supply of HCO₃⁻ in vivo by ecaA overexpression is an effective strategy for the improvement of succinate production in E. coli.     

The structure of a complex between carbonic anhydrase II and a new inhibitor, trifluoromethane sulphonamide

It has recently been shown that aliphatic sulphonamides are good inhibitors of carbonic anhydrase (CA) provided that the pK of the sulphonamide is low. We have determined the structure of the complex between CAII and CF₃SO₂NH₂ by X-ray crystallographic methods. The nitrogen of the sulphonamide is bound to the zinc ion of the enzyme in the usual manner. The other parts of the inhibitor show a different mode of binding from aromatic sulphonamides since the trifluoromethyl group is bound at the hydrophobic part of the active site instead of pointing out from the active site. It should be possible to design new inhibitors specific for the different isoenzymes, starting from the present structure.     

黄顶菊对入侵地群落动态及植物生长生理特征的影响

为明确黄顶菊对入侵地植物群落和土著植物生理生长的影响机制,采用同质园试验对入侵和非入侵土壤的植物群落开展了整个生育期动态监测,并分析了黄顶菊入侵对狗尾草、羽叶鬼针草、灰绿藜、地肤4种土著植物生长和生理特征的影响规律。结果表明:黄顶菊入侵土壤植物群落多样性指数低于非入侵地,且有季节性差异,随生育期的推进差异逐渐减小;黄顶菊对本地植物的生长指标有显著影响(P0.05),随时间变化显著,但存在物种差异;4种植物的净光合速率(Pn)、气孔导度(Cd)、蒸腾速率(Tr)在非入侵土壤生长显著高于入侵地土壤(P0.05);而4种植物在入侵土壤生长的比叶面积(SLA)、比根长(SRL)、比根面积(SRA)显著高于本地土壤(P0.05)。综上,黄顶菊入侵抑制了本地植物的光合效率,减少了生物量的积累,导致本地植物群落的生物多样性水平降低,但表现出季节差异;不同物种对黄顶菊入侵胁迫的响应表现种间特异性,为理解入侵种对群落结构影响和实现入侵生境恢复提供了理论依据。     

Isocoumarins: a new class of selective carbonic anhydrase IX and XII inhibitors

Isocoumarins, isomeric to comarins which act as effective carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, were investigated for the first time as inhibitors of this enzyme. A series of 3-substituted and 3,4-disubstituted isocoumarins incorporating phenylhydrazone, 1-phenyl-pyrazole and pyrazolo-substituted pyrimidine trione/thioxo-pyrimidine dione moieties were investigated for their interaction with four human (h) CA isoforms, hCA I, II, IX and XII, known to be important drug targets. hCA I and II were not inhibited by these compounds, whereas hCA IX and XII were inhibited in the low micromolar range by the less bulky derivatives. The inhibition constants ranged between 2.7–78.9 µM against hCA IX and of 1.2–66.5 µM against hCA XII. As for the coumarins, we hypothesise that the isocoumarins are hydrolysed by the esterase activity of the enzyme with formation of 2-carboxy-phenylacetic aldehydes which act as CA inhibitors. Isocoumarins represent a new class of CA inhibitors.     

A class of carbonic anhydrase IX/XII – selective carboxylate inhibitors

Abstract

A small series of 2,4-dioxothiazolidinyl acetic acids was prepared from thiourea, chloroacetic acid, aromatic aldehydes, and ethyl-2-bromoacetate. They were assayed for the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms of human (h) origin, the cytosolic hCA I and II, and the transmembrane hCA IX and XII, involved among others in tumorigenesis (hCA IX and XII) and glaucoma (hCA II and XII). The two cytosolic isoforms were not inhibited by these carboxylates, which were also rather ineffective as hCA IX inhibitors. On the other hand, they showed submicromolar hCA XII inhibition, with K_Is in the range of 0.30–0.93?µM, making them highly CA XII-selective inhibitors.     

3H-1,2-benzoxathiepine 2,2-dioxides: a new class of isoform-selective carbonic anhydrase inhibitors

A new chemotype with carbonic anhydrase (CA, EC 4.2.1.1) inhibitory action has been discovered, the homo-sulfocoumarins (3H-1,2-benzoxathiepine 2,2-dioxides) which have been designed considering the (sulfo)coumarins as lead molecules. An original synthetic strategy of a panel of such derivatives led to compounds with a unique inhibitory profile and very high selectivity for the inhibition of the tumour associated (CA IX/XII) over the cytosolic (CA I/II) isoforms. Although the CA inhibition mechanism with these new compounds is unknown for the moment, we hypothesize that it may be similar to that of the sulfocoumarins, i.e. hydrolysis to the corresponding sulfonic acids which thereafter anchor to the zinc-coordinated water molecule within the enzyme active site.     

AM真菌影响入侵植物黄顶菊与本土物种狗尾草竞争生长的机理研究

该研究以入侵植物黄顶菊[Flaveria bidentis(L.)Kunt]和本土伴生植物狗尾草为材料,通过筛选出黄顶菊单一优势群落AM真菌,于温室盆栽条件下,采用2物种单播、混播以及接种AM真菌和不接种共6个处理,分析AM真菌对黄顶菊和狗尾草的根系侵染率、相对竞争强度、植株氮磷钾光合利用率、以及丙二醛含量和保护酶活性的影响,探讨AM真菌对黄顶菊与狗尾草竞争生长的机理。结果显示:(1)黄顶菊根际土壤AM真菌共包括4属10种,其中优势种为Glomus constrictum、Glomus perpusillum、Glomus reticulatum;盆栽接种AM真菌后,黄顶菊的根系侵染率显著高于本土伴生植物狗尾草,但接种AM真菌后黄顶菊相对竞争强度显著降低了29.57%,却对狗尾草相对竞争强度无显著影响。(2)接种AM真菌使黄顶菊植株光合氮、磷、钾利用率显著升高,但对伴生植物狗尾草的光合氮、磷、钾利用率均无显著影响。(3)接种AM真菌对黄顶菊植株POD和CAT活性以及MDA含量无显著影响,但显著增加了SOD和APX活性,而伴对生植物狗尾草的POD、CAT和APX活性均显著降低,MDA含量显著提高。研究表明,AM真菌对黄顶菊和狗尾草具有不同的选择性,AM真菌的定植促进了黄顶菊的竞争生长,增加了植株N、P含量、光合养分利用率以及抗氧化酶活性;但显著降低了本土伴生植物狗尾草的N、P吸收以及抗氧化酶活性。因此,AM真菌在竞争生长中对黄顶菊产生了偏利反馈,有助于黄顶菊的入侵。     

The inhibitory effects of phenolic Mannich bases on carbonic anhydrase I and II isoenzymes

Phenolic mono Mannich bases [2-[4-hydroxy-3-(aminomethyl)benzylidene]-2,3-dihydro-1H-inden-1-one (8–15)] and bis Mannich bases [2-[4-hydroxy-3,5-bis(aminomethyl)benzylidene]-2, 3-dihydro-1H-inden-1-one (2–7)] were synthesized starting from 2-(4-hydroxybenzylidene)-2, 3-dihydro-inden-1-one (1). This study was designed in order to investigate the carbonic anhydrase (CA, EC 4.2.1.1) inhibitory properties of a library of compounds incorporating the phenol functional group. All prepared compounds showed a low inhibition percentages on both human (h) isoforms hCA I and hCA II compared to the reference sulfonamide acetazolamide. Mannich bases 2–15 had lower inhibition percentages than the compound 1 on hCA I and hCA II, except compound 14, which is a Mannich base derivative of dipropylamine, which had a similar inhibitory power as compound 1 on hCA II. All compounds synthesized 1–15 were 1.3–1.9 times more effective on hCA II comparing with the effectivenes of the compounds on hCA I.     

Discovery of arjunolic acid as a novel non-zinc binding carbonic anhydrase II inhibitor

Elevated levels of carbonic anhydrase II (CA II) have been shown to be associated with cardiac hypertrophy and heart failure. Although arjunolic acid (AA) has a diverse range of therapeutic applications including cardio-protection, there have been no reports on the effect of AA on CA II. The present study describes for the first time, the novel zinc independent inhibition of CA II by AA. The molecular docking studies of AA indicated that the hydroxyl group at C₂ of the A-ring, which hydrogen bonds with the catalytic site residues (His64, Asn62 and Asn67), along with the gem-dimethyl group at C₂₀ of the E-ring, greatly influences the inhibitory activity, independent of the catalytic zinc, unlike the inhibition observed with most CA II inhibitors. Among the triterpenoids tested viz. arjunolic acid, arjunic acid, asiatic acid, oleanolic acid and ursolic acid, AA was the most potent in inhibiting CA II in vitro with an IC₅₀ of 9 μM. It was interesting to note, that in spite of exhibiting very little differences in their structures, these triterpenoids exhibited vast differences in their inhibitory activities, with IC₅₀ values ranging from 9 μM to as high as 333 μM. Furthermore, AA also inhibited the cytosolic activity of CA in H9c2 cardiomyocytes, as reflected by the decrease in acidification of the intracellular pH (pH_i). The decreased acidification reduced the intracellular calcium levels, which further prevented the mitochondrial membrane depolarization. Thus, these studies provide a better understanding for establishing the novel molecular mechanism involved in CA II inhibition by the non-zinc binding inhibitor AA.     

刈割对外来入侵植物黄顶菊的生长、 气体交换和荧光的影响

为明确不同刈割处理对黄顶菊生长和生理特性的影响,本研究在田间条件下,对黄顶菊在生长季内不同时间进行刈割处理。结果表明,刈割降低了黄顶菊植株各部分的生物量积累,其中以刈割3次效果最为显著,使黄顶菊总生物量、根生物量、茎生物量、叶生物量分别较对照下降82.57%、44.53%、80.04%、91.76%;植株的高度和花序数随刈割次数的增加显著降低,其中刈割3次的花序数为0;刈割1次植株分枝数最大,出现超补偿现象,刈割3次分枝数显著低于其他处理;叶绿素含量除了刈割2次出现增高趋势外,随刈割次数的增加,叶绿素含量逐渐降低;刈割处理使黄顶菊净光合速率(Pn)、气孔导度(Cond)和蒸腾速率(Tr)均显著升高;刈割3次的PSⅡ的最大光化学效率(Fv/Fm)和PSⅡ的潜在活性(Fv/F0)显著低于其它各处理,而初始荧光(F0)则显著增加;生长指标的可塑性指数大于生理指标可塑性指数,表明前者在黄顶菊对刈割处理等物理措施适应方面起到了更为重要的作用。总之,刈割3次处理黄顶菊的各项生长和生理指标所受影响最大,对黄顶菊植株的再生和开花结实抑制效果最为理想。     

黄顶菊与3种本地植物竞争对丛枝菌根真菌种类和多样性的影响

【目的】外来植物黄顶菊对生态环境和农业经济造成了严重危害,了解黄顶菊与3种不同本地植物种植生长对丛生菌根(AM)真菌群落结构和多样性造成的影响,可以从土壤微生物角度进一步解释黄顶菊的入侵机制。【方法】通过同质园小区试验模拟黄顶菊入侵的生态进程,以黄顶菊和3种本地植物狗尾草、藜、黄香草木樨为研究对象,采用AM真菌的形态学鉴定方法,研究黄顶菊与3种本地植物不同种植方式对AM真菌群落结构和多样性的影响。【结果】(1)黄顶菊根际土壤聚集的AM真菌种类与其伴生本地植物种类有关:黄顶菊与狗尾草混种处理中优势种为网状球囊霉和根内球囊霉,而黄顶菊分别与藜、草木樨混种处理中优势种均为网状球囊霉、根内球囊霉和缩球囊霉;(2)黄顶菊分别与狗尾草和黄香草木樨混种处理中AM真菌种类既高于本地单种处理,也高于黄顶菊单种处理,说明随着黄顶菊的入侵和地上植物多样性的改变,AM真菌种类也发生改变;(3)与3种本地植物单种相比,黄顶菊各混种处理和黄顶菊单种处理中黄顶菊根际土壤根内球囊霉的重要值均增加,表明黄顶菊入侵有利于根内球囊霉的生长和发育。【结论】黄顶菊入侵改变了根际土壤AM真菌的群落结构和多样性,AM真菌的改变既与本地植物种类有关,也与入侵程度有关。