转MhGlu基因烟草灰霉病抗性及光合特性研究

以湖北海棠盆栽及组培苗叶片为材料,经NaCl、PEG-6000及4℃下ABA处理后,通过RT-PCR技术克隆了湖北海棠β-1,3-葡聚糖酶基因MhGlu;构建MhGlu基因的植物表达载体,通过农杆菌介导法将MhGlu基因转入烟草中,并通过PCR和RT-PCR检测,成功获得了4个转基因株系T6、T8、T11和T18;以转基因烟草株系T6及T8和非转基因对照植株为材料,对MhGlu基因的功能进行了进一步分析。结果显示:(1)半定量qRT-PCR显示,NaCl、PEG-6000及4℃下ABA处理均可以诱导湖北海棠盆栽及组培苗叶片MhGlu基因的表达;NaCl和PEG-6000处理48h内MhGlu基因的表达随处理时间延长逐渐增强,4℃下ABA处理的MhGlu基因表达量在4h时开始上调,12h时略降低,48h时又达到最大。(2)半定量RT-PCR检测转基因烟草植株几个病程相关基因PRs的表达量,表明过表达的MhGlu基因诱导并增强了烟草病程相关基因NtPR1、NtPR3和NtPR5的表达。(3)用灰霉病侵染烟草叶片,转基因烟草株系T6、T8均表现出较强的抗灰霉病特性。(4)测定烟草植株光合特性参数,转MhGlu基因烟草株系的净光合速率(Pn)、蒸腾速率(Tr)和气孔导度(Gs)较对照组均显著提高,且T8的净光合速率和蒸腾速率均显著高于T6,而T8与T6的气孔导度差异不显著。MhGlu基因在烟草中的过量表达能诱导病程相关基因PRs的表达,激活了烟草的光合特性保护机制,提高了转MhGlu基因烟草植株的灰霉病抗性。     

转柽柳晚期胚胎富集蛋白基因烟草T1代的耐盐性评价

目的:验证转柽柳晚期胚胎富集(LEA)蛋白基因烟草T1代的耐盐性。方法:采用盐胁迫方式,对转柽柳LEA蛋白基因烟草T1代的6个株系及非转基因对照烟草T1代进行不同浓度NaCl胁迫处理,分析了NaCl胁迫下转基因烟草的生长量、根系的发育及盐害程度。结果:各转基因烟草T1代组培苗在150mmol/L的NaCl培养基上根系生长良好,平均增重(鲜重)是非转基因对照的7.72倍,平均高生长是非转基因对照的3.51倍,盐害指数低于或等于50%;而非转基因对照烟草T1代组培苗生长缓慢,根系几乎不能生长发育,盐害指数达65%。结论:柽柳LEA蛋白基因的导入提高了T1代烟草的耐盐性。     

转LEA基因烟草的耐盐性分析

目的:验证柽柳LEA基因的功能,为通过基因工程手段培育耐盐植物提供基础资料。方法:对转LEA基因烟草当代(T0)和子一代(T1)分别进行不同浓度的NaCl胁迫处理,研究转基因烟草的耐盐性。结果:转基因烟草T0代组培苗耐受NaCl的临界浓度为230mmol/L,而对照耐受NaCl的临界浓度为130mmol/L以下;T1代幼苗耐受NaCl的临界浓度为150mmol/L,对照耐受NaCl的临界浓度为100mmol/L以下;在临界浓度转基因烟草的T0代、T1代根系发育良好,生长量明显高于非转基因对照烟草。结论:柽柳LEA基因的转化提高了烟草的耐盐性。     

Spd浸种对盐胁迫下番茄（Solanum lycopersicum)幼苗的保护效应

通过水培试验,研究了100 mmol/L NaCl盐浓度下,0.25 mmol/L Spd浸种处理对两个番茄品种白果强丰(耐盐基因型)和江蔬14号(盐敏感基因型)植株干重、根冠比(R/T)、幼苗叶片和根系抗氧化酶活性及活性氧含量的影响.具体试验处理如下:(a) 对照(蒸馏水浸种+ 0 mmol/L NaCl),(b) NaCl (蒸馏水浸种+ 100 mmol/L NaCl),(c) Spd(0.25 mmol/L Spd浸种 +100 mmol/L NaCl).结果表明,在盐胁迫下,两个番茄品种幼苗叶片和根系内超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性升高,H2O2含量和O·-2产生速率增高,幼苗生长受到抑制,幼苗地上部、地下部干重均明显低于对照,R/T增大,且江蔬14号的变化幅度大于白果强丰.Spd浸种处理降低了盐胁迫下番茄幼苗叶片和根系内O·-2产生速率和H2O2含量,进一步提高了SOD、POD和CAT活性,促进幼苗干重增加,缓解了盐胁迫对植株的伤害.与耐盐基因型番茄品种白果强丰相比,Spd浸种处理对盐敏感基因型番茄品种江蔬14号的作用效果更为明显.总之,Spd浸种处理通过提高盐胁迫下植株体内抗氧化酶活性,降低ROS水平来缓解盐胁迫对番茄幼苗的伤害,提高幼苗耐盐能力.     

NaCl处理对西伯利亚白刺幼苗生长及离子平衡的影响

以1年生西伯利亚白刺水培幼苗为材料,研究了不同浓度NaCl(0、200、400mmol·L~(-1))处理对幼苗生长及不同器官(根、茎、叶)中Na~+、K~+、Ca~(2+)、Mg~(2+)的吸收、运输与分配的影响,探讨西伯利亚白刺的盐适应机制。结果表明:(1)200mmol·L~(-1) NaCl处理促进了西伯利亚白刺幼苗的生长及叶片肉质化程度,400mmol·L-1 NaCl处理显著抑制其生长。(2)随着NaCl处理浓度的升高,西伯利亚白刺幼苗根、茎、叶中Na~+含量显著增加,且叶中Na~+含量显著高于茎和根中;根系中K~+含量显著增加;根、茎、叶中Ca~(2+)、Mg~(2+)含量在200mmol·L~(-1) NaCl处理下保持平稳或上升,而在400mmol·L-1 NaCl处理下显著下降。(3)各器官中K~+/Na~+、Ca~(2+)/Na~+和Mg~(2+)/Na~+比值总体随NaCl处理浓度的升高呈下降趋势,且根部离子比值始终高于叶片和茎。(4)随着NaCl处理浓度的升高,西伯利亚白刺幼苗根-茎SK,Na显著下降,而根-茎SCa,Na、SMg,Na及茎-叶SK,Na、SCa,Na、SMg,Na逐渐提高。研究发现,西伯利亚白刺的盐适应机制主要是通过植株的补偿生长效应及叶片对Na~+的聚积作用实现的,同时也与根系对K~+的扣留及茎叶对K~+、Ca~(2+)、Mg~(2+)选择性运输能力增强有关。     

极端耐盐放线菌白色普氏菌YIM 90005T四氢嘧啶及5-羟基四氢嘧啶合成相关基因的克隆

摘要：【目的】 为了研究耐盐放线菌对高盐环境的适应机理。【方法】 用HPLC定量检测了极端耐盐、丝状产孢放线菌——白色普氏菌（Prauserella alba） YIM 90005T在不同盐浓度下胞内相容性溶质的种类和含量。【结果】 结果发现,四氢嘧啶和5-羟基四氢嘧啶是其主要的相容性溶质。在培养基NaCl浓度为10%时,四氢嘧啶在胞内累积浓度最大,为18.77 μg/mg干菌体重。之后随NaCl浓度的升高,胞内的四氢嘧啶含量逐渐减少,而5-羟基四氢嘧啶的含量逐渐增加,在该菌耐受的最高NaCl浓度下（24% w/v）,胞内5-羟基四氢嘧啶含量达到最大值,为22.98 μg/mg干菌体重。设计兼并引物,利用染色体步移,克隆得到四氢嘧啶及5-羟基四氢嘧啶合成相关基因ectABCD。序列分析表明,ectABCD位于一个操纵子中。进一步对不同NaCl浓度培养条件下ectB,D的表达量进行定量分析,结果表明该基因簇表达量随着培养基中NaCl浓度的增加而增大。【结论】 研究结果证实5-羟基四氢嘧啶是P. alba YIM 90005T在极高盐浓度条件下起渗透调节及保护的相容性溶质。     

NaCl胁迫对高丛越橘幼苗生长和光合生理特性的影响

以2年生北高丛越橘‘蓝丰’幼苗为材料,采用温室盆栽法研究了不同浓度(0、50、100、150和200mmol·L~(-1))NaCl胁迫处理50d对其生长、光合作用和叶绿素荧光特性的影响,以鉴定高丛越橘的耐盐性,为盐碱地区的越橘引种、栽培提供理论依据。结果显示:(1)越橘幼苗地上部鲜质量和地下部鲜质量在50mmol·L~(-1) NaCl处理下与对照无显著差异,当超过100mmol·L~(-1) NaCl时均显著降低;地上部干质量、地下部干质量、总干质量在低于100mmol·L~(-1) NaCl处理下与对照无显著差异,当超过150mmol·L~(-1) NaCl时均显著降低;在不同浓度NaCl处理下,根冠比与对照均无明显差异。(2)越橘幼苗叶片Chl a、Chl b和总Chl含量在低于100mmol·L~(-1) NaCl处理下与对照无显著差异,当超过150mmol·L~(-1) NaCl时均明显降低。(3)越橘叶片净光合速率(Pn)、蒸腾速率(Tr)和气孔导度(Gs)在50mmol·L~(-1) NaCl处理下与对照无显著差异,在超过100mmol·L~(-1) NaCl时均明显下降;在200mmol·L~(-1) NaCl处理下胞间CO2浓度(Ci)、气孔限制值(Ls)明显下降;不同浓度NaCl处理下水分利用效率(WUE)无明显差异;(4)暗适应下最大荧光(Fm)、最大光化学效率(Fv/Fm)、潜在光化学效率(Fv/F0)、PSⅡ光量子产量(ΦPSⅡ)、光合电子传递速率(ETR)、光化学猝灭系数(qP)在低于150mmol·L~(-1) NaCl处理下与对照无显著差异,在200mmol·L~(-1) NaCl时均明显下降。研究表明,越橘幼苗具有一定的耐盐能力,能够忍受100mmol·L~(-1)NaCl胁迫,但在高NaCl浓度胁迫下,气孔限制因素是导致Pn下降的主要原因,此外叶片光合机构受损、叶绿素含量显著减少以及光合电子传递受阻而产生的光抑制,也会导致Pn的下降和生物量的降低。     

外源5-氨基乙酰丙酸对NaCl胁迫下酸枣光合特性的影响

以2年生酸枣幼苗为试验材料,探讨不同浓度NaCl(0、4、8、12g·kg-1)胁迫下喷施5-氨基乙酰丙酸(ALA,75、150mg·L~(-1))对酸枣光合特性的影响。结果显示:(1)不同浓度NaCl胁迫下,外源ALA对酸枣叶片的净光合速率(Pn)、胞间二氧化碳浓度(Ci)、气孔导度(Gs)、蒸腾速率(Tr)及叶绿素含量等具有明显促进作用。(2)在不同浓度NaCl胁迫下,喷施75mg·L~(-1)的ALA仅在NaCl浓度为4和8g·kg-1处理下对酸枣Tr值具有显著提高作用,而喷施150mg·L~(-1) ALA在各NaCl浓度胁迫下对其Pn、Ci和Tr均具有显著促进作用。(3)在重度NaCl胁迫(12g·kg-1)下,喷施150mg·L~(-1) ALA对酸枣叶片叶绿素含量具有显著提高作用,而喷施75mg·L~(-1)浓度ALA却无明显提高。研究表明,在NaCl胁迫条件下,外源ALA能有效改善酸枣叶片光合气体交换参数,提高叶绿素含量,从而缓解NaCl胁迫的伤害,提高其光合能力,并以喷施150mg·L~(-1) ALA的缓解效果更好。     

NaCl胁迫对菜用大豆种子膨大过程中抗氧化系统及渗透调节物质的影响

采用蛭石栽培试验,在100mmol.L-1 NaCl胁迫下,对耐盐性不同的两个菜用大豆品种种子膨大、抗氧化系统及渗透调节物质进行了研究。结果显示:(1)耐盐品种‘绿领特早’种子干质量及大小的增加在NaCl胁迫15d时未受显著影响,而盐敏感品种‘理想高产95-1’种子的膨大受到了显著抑制。(2)NaCl胁迫导致菜用大豆种子超氧阴离子(O2.-)、过氧化氢(H2O2)及丙二醛(MDA)的过量积累,但‘绿领特早’种子的相对积累量均低于‘理想高产95-1’。(3)与‘理想高产95-1’相比,‘绿领特早’在NaCl胁迫期间维持了相对较高的超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)及谷胱甘肽还原酶(GR)活性,以及较高的抗坏血酸(AsA)、可溶性蛋白和可溶性糖含量;胁迫中期维持了相对较高的过氧化物酶(POD)活性,胁迫后期维持了相对较高的过氧化氢酶(CAT)活性;胁迫前中期维持了相对较高的谷胱甘肽(GSH)含量。研究发现,‘绿领特早’种子在NaCl胁迫期间能够保持较高且协调平衡的保护酶SOD、POD、CAT活性和较高的AsA-GSH循环效率,同时维持了较强的渗透调节能力,有效抑制了O2.-和H2O2的过量积累,进而有效降低了细胞膜质过氧化程度,这可能是其耐盐性较强的重要原因之一。     

短期盐胁迫对苦苣菜幼苗叶片抗逆生理指标的影响

采用蛭石中浇灌Hoagland营养液的基质培养方法,以苗期苦苣菜为实验材料,设置5个NaCl处理浓度(0(CK)、66、133、200、250、300mmol·L~(-1)),分别在处理1、2、3d采集苦苣菜叶片,测定其Na~+、K~+、丙二醛(MDA)、可溶性糖,可溶性蛋白质及游离脯氨酸含量,以及超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性,探讨短期盐胁迫下苦苣菜叶片生理指标的变化特征,为进一步研究野生物种的耐盐性及耐盐机制提供理论依据。结果表明:(1)盐胁迫1d和2d、NaCl浓度为66~200mmol·L~(-1)时,苦苣菜叶片Na~+含量无显著变化,K~+含量和K~+/Na~+较对照显著增大,并在200mmol·L~(-1) NaCl处理下达到最大值;盐胁迫3d时,叶片K~+含量与胁迫前2d的变化趋势相似,Na~+含量随盐浓度升高显著增大,K~+/Na~+除66mmol·L~(-1) NaCl处理外均随盐浓度升高而显著减小。(2)盐胁迫1~3d时,苦苣菜叶片可溶性蛋白质、可溶性糖含量均随盐浓度升高而先增后减,可溶性蛋白质含量在胁迫1~2d、可溶性糖在胁迫2~3d时均在200mmol·L~(-1) NaCl下达到最大值;游离脯氨酸含量在盐胁迫前2d时与可溶性蛋白质、可溶性糖的变化趋势相似,在胁迫3d时随盐浓度升高而显著增大且绝大部分与对照差异显著。(3)苦苣菜叶片SOD、POD、CAT活性在盐胁迫1~3d时均随盐浓度升高而先增后减;在胁迫1~2d时,3种酶活性均在250mmol·L~(-1) NaCl下达到最大值且所有盐浓度下均显著大于对照;在盐胁迫3d时,SOD、POD活性在200mmol·L~(-1) NaCl下达到最大值,CAT活性在133mmol·L~(-1) NaCl下达到最大值且除250、300mmol·L~(-1) NaCl外均与对照差异显著。研究发现,苦苣菜具有较强的耐盐性,能够在盐胁迫(66~300mmol·L~(-1))处理1~3d内进行一系列有效的生理调节,增强自身渗透调节能力及抗氧化能力,表现出较强的吸钾拒钠的特性,基本缓解了Na~+的毒害及渗透压力,但是NaCl浓度超过200mmol·L~(-1)后其渗透调节能力和抗氧化能力均显著降低。     

Crystal structures of two solute receptors for L-cystine and L-cysteine, respectively, of the human pathogen Neisseria gonorrhoeae

ATP-binding cassette (ABC) transporters are integral membrane proteins that carry a variety of substrates across biological membranes at the expense of ATP. The here considered prokaryotic canonical importers consist of three entities: an extracellular solute receptor, two membrane-intrinsic proteins forming a translocation pathway, and two cytoplasmic ATP-binding subunits. The ngo0372-74 and ngo2011-14 gene clusters from the human pathogen Neisseria gonorrhoeae were predicted by sequence homology as ABC transporters for the uptake of cystine and cysteine, respectively, and chosen for structural characterization. The structure of the receptor component Ngo0372 was obtained in a ligand-free "open" conformation and in a "closed" conformation when co-crystallized with L-cystine. Our data provide the first structural information of an L-cystine ABC transporter. Dissociation constants of 21 and 33 nM for L-cystine and L-selenocystine, respectively, were determined by isothermal titration calorimetry. In contrast, L-cystathionine and L-djenkolic acid are weak binders, while no binding was detectable for S-methyl-L-cysteine. Mutational analysis of two residues from the binding pocket, Trp97 and Tyr59, revealed that the latter is crucial for L-cystine binding. The structure of the Ngo2014 receptor was obtained in closed conformation in complex with co-purified L-cysteine. The protein binds L-cysteine with a K(d) of 26 nM. Comparison of the structures of both receptors and analysis of the ligand binding sites shed light on the mode of ligand recognition and provides insight into the tight binding of both substrates. Moreover, since L-cystine limitation leads to reduction in virulence of N. gonorrhoeae, Ngo0372 might be suited as target for an antimicrobial vaccine.     

Endopeptidase activity of larval Lacanobia oleracea corpus allatum: metabolism of Manduca sexta allatostatin and allatotropin

The degradation of synthetic Manduca sexta allatostatin (Manse-AS) and allatotropin (Manse-AT) by enzymes associated with the corpus allatum (CA) of larvae of the tomato moth, Lacanobia oleracea, was investigated using reversed-phase high performance liquid chromatography and matrix-assisted laser desorption ionisation-time of flight mass spectrometry. Manduca sexta allatostatin was metabolised by CA extract to Manse-AS5-15, Manse-AS6-15, and Manse-AS7-15, which indicates enzymic cleavage at the C-terminal side of arginine residues R3 and R5 and the N-terminal side of R5, suggesting this is due to a trypsin-like enzyme. In support of this, the same degradation products were identified after Manse-AS was incubated with trypsin, and CA enzymic activity could be inhibited up to 79% by aprotinin. Degradation of Manse-AT by CA extract was also trypsin-like, cleaving at the C-terminal side of the basic residues K3 and R11 to produce Manse-AT4-13 and Manse-AT1-11. Metabolism by trypsin produced the same deletion peptides, but the major product due to this enzyme was Manse-AT4-11. Hydrolysis of Manse-AT by CA could only be partially inhibited by high doses of aprotinin (36%), and the CA extract also cleaved Manse-AT between M8 and T9 to produce Manse-AT1-8. A trypsin-like peptidase appears to be the major enzyme present in the CA of larval L. oleracea that acts to metabolise Manse-AS and Manse-AT. In addition, an unidentified enzyme that cleaves between M and T residues degraded Manse-AT.     

Kinetic and pharmacological analysis of L-[35S]cystine transport into rat brain synaptosomes

The synaptosomal transport of L-[35S]cystine occurs by three mechanisms that are distinguishable on the basis of their ionic dependence, kinetics of transport and the specificity of inhibitors. They are (a) low affinity sodium-dependent transport (Km 463 +/- 86 microM, Vmax 185 +/- 20 nmol mg protein-1 min-1), (b) high affinity sodium-independent transport (Km 6.90 +/- 2.1 microM, Vmax 0.485 +/- 0.060 nmol mg protein(-1) min(-1)) and (c) low affinity sodium-independent transport (Km 327 +/- 29 microM, Vmax 4.18 +/- 0.25 nmol mg protein(-1) min(-1)). The sodium-dependent transport of L-cystine was mediated by the X(AG)- family of glutamate transporters, and accounted for almost 90% of the total quantity of L-[35S]cystine accumulated into synaptosomes. L-glutamate (Ki 11.2 +/- 1.3 microM) was a non-competitive inhibitor of this transporter, and at 100 microM L-glutamate, the Vmax for L-[35S]cystine transport was reduced to 10% of control. L-cystine did not inhibit the high-affinity sodium-dependent transport of D-[3H]aspartate into synaptosomes. L-histidine and glutathione were the most potent inhibitors of the low affinity sodium-independent transport of L-[35S]cystine. L-homocysteate, L-cysteine sulphinate and L-homocysteine sulphinate were also effective inhibitors. 1 mM L-glutamate reduced the sodium-independent transport of L-cystine to 63% of control. These results suggest that the vast majority of the L-cystine transported into synaptosomes occurs by the high-affinity glutamate transporters, but that L-cystine may bind to a site that is distinct from that to which L-glutamate binds. The uptake of L-cystine by this mechanism is sensitive to inhibition by increased extracellular concentrations of L-glutamate. The importance of these results for understanding the mechanism of glutamate-mediated neurotoxicity is discussed.     

Halomonas alkalitolerans sp. nov., a novel moderately halophilic bacteriun isolated from soda meadow saline soil in Daqing, China

A moderately halophilic bacterial strain 15-13(T), which was isolated from soda meadow saline soil in Daqing City, Heilongjiang Province, China, was subjected to a polyphasic taxonomic study. The cells of strain 15-13 were found to be Gram-negative, rod-shaped, and motile. The required growth conditions for strain 15-13(T) were: 1-23% NaCl (optimum, 7%), 10-50°C (optimum, 35°C), and pH 7.0-11.0 (optimum, pH 9.5). The predominant cellular fatty acids were C(18:1) ω7c (60.48%) and C(16:0) (13.96%). The DNA G+C content was 67.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons indicated that strain 15-13(T) clustered within a branch comprising species of the genus Halomonas. The closest phylogenetic neighbor of strain 15-13(T) was Halomonas pantelleriensis DSM 9661(T) (98.9% 16S rRNA gene sequence similarity). The level of DNA-DNA relatedness between the novel isolated strain and H pantelleriensis DSM 9661(T) was 33.8%. On the basis of the phenotypic and phylogenetic data, strain 15-13(T) represents a novel species of the genus Halomonas, for which the name Halomonas alkalitolerans sp. nov. is proposed. The type strain for this novel species is 15-13(T) (=CGMCC 1.9129(T) =NBRC 106539(T)).     

Non-catalytic role of carbonic anhydrase in rat intestinal absorption

Carbonic anhydrase (CA) inhibition reduces NaCl absorption in rat distal ileum, a pH-sensitive, low CA activity tissue, and in distal colon, a CO(2)-sensitive, high CA activity tissue. We hypothesized that CA plays a non-catalytic role in NaCl absorption in these segments. Unidirectional fluxes of Na(+) and Cl(-), and total HCO(3)(-) generation (estimated as the sum of radiolabeled HCO(3)(-) and CO(2) produced from glucose) were measured in Ussing chambers in nominally CO(2), HCO(3)(-)-free HEPES Ringer. Measurements were made in the presence and absence of 0.1 mM methazolamide, a membrane-permeant CA inhibitor. Ringer pH reduction from 7.6 to 7.1 stimulated ileal but not colonic Na(+) and Cl(-) absorption. In the ileum, methazolamide reduced J(ms)(Na) and J(ms)(Cl) and caused net Cl(-) secretion at pH 7.6, and prevented the stimulatory effect of lowering pH. In the colon, methazolamide reduced Na(+) and Cl(-) absorption at pH 7.6. Total HCO(3)(-) generation was minimal in HEPES at pH 7.6 and 7.1 in both segments, was minimally affected by methazolamide, and did not account for the changes in Cl(-) absorption caused by pH or methazolamide. We conclude that CA plays a role in ileal and colonic NaCl absorption independent of its catalytic function.     

Conversion of L-cystine and L-cysteine to taurin by the enzyme systems of liver cells

The kinetics of conversion of sulfur-containing amino acids L-cystine and L-cysteine to taurin by the enzyme system of cattle liver cells was studied, and a mathematical model was developed. It was shown that L-cystine and L-cysteine conversion obeyed the Michaelis-Menten equations of serial-sequential conversions with regard to inhibition by the final product and inactivation. The yield of taurin under the optimized conditions of L-cystine and L-cysteine conversion (temperature, 40 degrees C; pH 1.5 and 3.0, respectively; and addition of enzyme preparations in five equal portions at 2-h intervals) was in the range 80-85% of the substrate weight.     

THE EFFECT OF SODIUM CHLORIDE ON CARBONIC ANHYDRASE ACTIVITY IN MARINE MICROALGAE1

The effects of NaCl on the internal and external carbonic anhydrase (CA) activity of several marine microalgae were studied. Unlike freshwater microalgae in which CA activity is generally inhibited by NaCl, marine microalgae exhibited considerable species-dependent variation when exposed to NaCl. CA activity in Phaeodactylum tricornutum, a diatom, was inhibited, whereas it was activated in the coccolithophorid Pleurochrysis carterae. CA activity in the chlorophyte Dunaliella primolecta was not significantly affected by NaCl. In Dunaliella salina and Dunaliella parva, NaCl inhibited external CA without affecting the internal activity, whereas in Chlorella vulgaris C-133 and Dunaliella peircei only the internal CA was inhibited. Internal CA of Dunaliella tertiolecta was not affected by NaCl, but the external enzyme was significantly enhanced. Salt substitution experiments revealed that chloride (Cl^-) is the ion affecting CA activity; the effect of Cl^- can be replaced by bromide ion. Cl^- affects external CA activity while also affecting the apparent affinity for inorganic carbon during photosynthesis. Microalgae whose internal CA activity was enhanced by Cl^- showed higher intracellular Cl^- concentrations than those species that were inhibited.     

Microchip fluorescence-enhanced immunoaasay for simultaneous quantification of multiple tumor markers

A microchip fluorescence-enhanced immunoassay method was developed for simultaneous detection of carcinoma antigen 125 (CA125) and carbohydrate antigen 15-3 (CA15-3). In this method, CA125 and CA15-3 react with excess amount of fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies (Ab(*)) of CA125 and CA15-3 to form CA125-Ab(125)(*) and CA15-3-Ab(15-3)(*) complexes. Microchip electrophoresis (MCE) separation of free Ab(125)(*), Ab(15-3)(*), and CA125-Ab(125)(*), CA15-3-Ab(15-3)(*) complexes were then performed. The separated species were sensitively detected by laser-induced fluorescence detection (LIF). CA125 and CA15-3 were quantified simultaneously by measuring the fluorescence intensity of CA125-Ab(125)(*) and CA15-3-Ab(15-3)(*) complexes, respectively. Under the optimum conditions, the limits of detection were 0.23 U/mL for CA125 and 0.09 U/mL for CA15-3. The present MCE-LIF method was applied to the determination of CA125 and CA15-3 in serum from healthy subjects and cancer patients. The levels of CA125 and CA15-3 in these sera samples were found to be in the ranges of 15.6-36.1 U/mL and 13.8-28.4 U/mL for healthy subjects, and 192.5-368.3 U/mL and 63.3-198.4 U/mL for cancer patients.     

The prognostic value of the tumor marker CA 15-3 at initial diagnosis of patients with breast cancer

CA 15-3 has been most widely used as a serum tumor marker in follow-up and detection of breast cancer recurrence. In this study we have specifically focused upon the prognostic implications and utility of preoperative CA 15-3 levels. We have identified on our database 414 patients with breast cancer in whom serial levels of the serum tumor marker CA 15-3 had been determined at diagnosis and follow-up. We have analyzed the follow-up and clinical outcomes in these patients and from this data we have assessed the potential of CA 15-3 as a predictor of five-year overall and disease-free survival. Our results show that an initially elevated CA 15-3 level is associated with a very poor prognosis in both early and late stage disease. Elevated pre-biopsy CA 15-3 levels are associated with 14% five-year disease-free survival rates and 17% overall survival rates at five years. In contrast, normal CA 15-3 levels are associated with 47% five-year disease-free survival rates and 54% overall survival rates at five years (p<0.01). Comparison of five-year survival rates between patients with elevated and normal CA 15-3 levels in early breast cancer (stage I and II) also showed significant differences, with survival being 41% and 75%, respectively (p<0.01).     

Conformation of L-cystathionine, a carba analog of cystine, and stereochemistry of hormone-receptor interactions.

The conformation of L-cystathionine, a carba analog of L-cystine, has been studied in the solid state using X-ray diffraction techniques. Crystal of L-cystathionine are tetragonal, space group P41 with cell constants a = 6.691(1) A, c = 21.998(3) A and Z = 4. From diffractometer data to the limit of 2theta = 162 degrees for Cukalpha, the structure was refined using full-matrix least-squares to an R value of 0.061. L-Cystathionine is isostructural chemically to L-cystine and its crystal structure is isomorphous to tetragonal L-cystine (Chaney, M.O. and Steinrauf, L.K. (1974) Acta Crystallogr. 1330, 711--716). The crystal structure of L-cystathionine is disordered, leading to two slightly differing conformers of L-cystathionine (each with half occupancy) with same helical sense but running in opposite directions and occupying the locations of L-cystine molecules in tetragonal L-cystine structure. Their conformational similarity, even when no sterical constraints such as cyclization are present, offers an explanation of the activities of the carba analogs of neuro-hypophysial hormones in terms of the structural integrity of the disulfide-like bridges.