重组TFPI缺失突变体TFPI1-161在毕赤酵母中的高效表达及功能鉴定

人组织因子途径抑制物(TFPI)是一种体内天然存在的外源性凝血途径特异性抑制物。缺失突变体TFPI1-161包括TFPI的N末端、K1和K2结构域,是一种研究TFPi结构与功能及其相互关系的理想对照分子。以克隆质粒pGEM-3Zf(-)-TFPi为模板,用PCR方法获得TFPI1-161基因,构建表达质粒pPIC9K-TFPi1-161并转化毕赤酵母GS115。通过筛选多拷贝转化子及优化发酵培养条件,首次在毕赤酵母中高效表达了TFPI1-161经纯化后最终产量高于酿酒酵母20倍以上。由于糖基化程度不同,TFPI1-161表达为TFPI1-161(24kD)和TFPI1-161(27kD)两种分子形式,其等电点分别为4、8和4．9。根据等电点差异,二可通过阴离子交换层析得到分离,其活性无显性差异。经分子筛和阴离子交换层析分离纯化后,从4L发酵培养液中可分别获得1.4g TFPI1-161(24kD)和1.8gTFPI1-161(27kD),其比活性分别达12880u／mg和12400u／mg,回收率达55％。经稀释的凝血酶原时间及发色底物法检测,重组TFPI1-161具有良好的抗凝及抑制FXa活性的作用。为获得大量TFPI1-161提供了一种廉价高效的蛋白表达纯化方式,为进一步的基础及临床前研究奠定了基础。     

CH50真核表达载体pCH503的构建及小鼠体内表达的趋化与抗肿瘤活行

构建重组FN多肽CH50真核表达载体并在小鼠体内表达,研究其趋化与抗肿瘤作用.采用重组DNA技术构建表达质粒;体内进行基因转染,采用RT-PCR鉴定导入基因的表达;通过肝素亲和层析、SDS-PAGE和Western blot鉴定表达产物;腹腔细胞计数、Giemsa染色分析以及肌肉组织切片与染色观察体内基因转染后的趋化作用;小鼠黑色素瘤模型研究基因转染抑制肿瘤的作用.从CH50原核表达载体获得重组多肽的cDNA,5＇端加上小鼠IFN-γ5＇端非编码区和信号肽编码区的cDNA,3＇端加上人FN cDNA的3＇端非编码区;将重组cDNA插入pREP8质粒,即构建出pCH503质粒.巨噬细胞在体内经pCH503转染,然后在体外培养,能够产生CH50多肽.以pCH503分别进行腹腔基因转染和肌肉内基因转染,均可对免疫细胞产生趋化作用;pCH503体内转染可以使小鼠腹腔内黑色素肿瘤结节数降低50%～601.CH50真核表达载体pCH503可在小鼠体内表达,体内基因转染可趋化免疫细胞和抑制肿瘤结节形成,在肿瘤综合治疗中有重要意义.     

p53相关蛋白激酶结合蛋白CGI-121

利用酵母双杂交技术从人的睾丸cDNA文库中鉴定得到p53相关蛋白激酶(PRPK)结合蛋白CGI-121,体外实验表明,重组CGI-121能抑制PRPK磷酸化p53第15位的Ser,未磷酸化p53进入泛素蛋白酶体途径,导致细胞增殖或肿瘤发生;然而,体内过表达CGI-121并没有显著的抑制PRPK磷酸化p53.Michael Downey等在研究cdcl3基因缺陷型酿酒酵母中筛选得到cdcl3-1突变体的抑制基因CGI-121,CGI-121是真核生物一个新的保守复合物--KEOPS复合物组成之一.KEOPS复合物具有促进端粒延伸和使端粒拆开的功能.CGI-121突变体在热敏感cdcl3-1酵母突变株中可以减少ssDNA的积累和缩短端粒;同时,在端粒功能异常芽殖酵母中CGI-121和piD261/Bud32促进端粒的拆开.然而,基因调控自身表达的机制以及在PRPK信号途径和KEOPS复合物中的扮演的角色有待于进一步研究.     

抑癌基因OVCA1 A34D突变对其生物半衰期的影响

目的为探索抑癌基因OVCA1结构与功能的关系,阐明其在肿瘤发生发展中的作用机制,本研究构建OVCA1A34D突变体并探讨该位点突变对其生物半衰期的影响。方法以本实验室制备并保存的含有人OVCA1基因全长序列的质粒为模板,采用分子克隆方法构建了GFP-tagged-OVCA1A34D突变体重组质粒,并经测序证实。应用脂质体法将突变体质粒转染入体外培养细胞,观察OVCA1A34D突变体蛋白在细胞中的表达。采用蛋白合成抑制剂放线菌酮抑制蛋白合成后检测OVCA1A34D基因突变对其蛋白生物半衰期的影响。结果成功构建了GFP-tagged-OVCA1A34D突变体重组质粒,并在细胞中成功表达。OVCA1A34D突变体蛋白的半衰期与野生体相比明显延长。结论 OVCA1A34D位点突变可导致其生物半衰期延长。     

扩展青霉脂肪酶K56R叠加突变对热稳定性的影响

目的:扩展青霉脂肪酶随机突变体ep8是一株热稳定性比野生型有所提高的突变体.获得热稳定性提高的优良菌株.方法:在ep8的基础上利用重叠延伸PCR构建叠加突变重组质粒pPIC3.5K-ep8一K56R,将该质粒电转毕赤酵母(Pichia paaoris)GS115进行异源表达.结果:该叠加突变脂肪酶在毕赤酵母中获得了活性表达.15%SDS-PACE结果分析表明突变脂肪酶PEL-ep8-K56R-GS分子量与野生型PEL-GS一致,约为28kDa.叠加突变脂肪酶在37℃时酶活为852U/mL、野生型为760u/mL、随机突变体为824u/mL,叠加突变体酶活相比野生型提高了21.1%,相比随机突变体提高了3.4%.热稳定性分析数据表明叠加突变脂肪酶Tm值为40.1℃、野生型为38.7℃、随机突变体为39.9℃,Tm值相比野生型提高了1.4℃,相比随机突变体提高了0.2℃.     

rhsBLyS及其突变体对小鼠血清免疫球蛋白分泌及B细胞增殖分化的影响

为了进一步研究所构建的人可溶性B淋巴细胞刺激因子 (hsBLyS)及其突变体hsBY A(Cys14 6→Ala14 6 )和hsBY V(Cys14 6→Val14 6 )在体内的生物活性 ,将 3种重组蛋白注射入BALb C小鼠体内 ,进而分析重组蛋白对小鼠血清免疫球蛋白分泌和脾脏B细胞增殖分化的影响 ,并对 3种蛋白的体内生物活性进行了比较 .结果表明 ,3种蛋白均能促进小鼠血清总IgG和IgM的分泌明显增加 ,统计学检验显示突变体rhsBY V诱导这 2种免疫球蛋白分泌的能力最强 ;3种蛋白还诱导小鼠脾脏增大、脾脏成熟B细胞的相对数量增加 .该高活性突变体hsBY V的获得提示 ,Cys14 6并非BLyS发挥正常功能所必需 ,将Cys14 6突变成Val14 6有可能提高BLyS的生物活性 .     

重组TFPI缺失突变体TFPI1-161在毕赤酵母中的高效表达及功能鉴定

人组织因子途径抑制物 (TFPI)是一种体内天然存在的外源性凝血途径特异性抑制物。缺失突变体TFPI_1-161仅包括TFPI的N末端、K1和K2结构域 ,是一种研究TFPI结构与功能及其相互关系的理想对照分子。以克隆质粒Pgem 3Zf(-) TFPI为模板 ,用PCR方法获得TFPI_1-161 基因 ,构建表达质粒Ppic9k TFPI_1-161并转化毕赤酵母GS115。通过筛选多拷贝转化子及优化发酵培养条件 ,首次在毕赤酵母中高效表达了TFPI_1-161,经纯化后最终产量高于酿酒酵母 20倍以上。由于糖基化程度不同 ,TFPI_1-161 表达为TFPI1_1-161 (2 4kD)和TFPI_1-161 (27kD)两种分子形式 ,其等电点分别为 4.8和 4.9。根据等电点差异 ,二者可通过阴离子交换层析得到分离 ,其活性无显著性差异。经分子筛和阴离子交换层析分离纯化后 ,从 4L发酵培养液中可分别获得 1.4gTFPI_1-161(2.4kD)和1.8gTFPI_1-161 (2.7kD) ,其比活性分别达12880u/mg和12400u/mg.回收率达55%.经稀释的凝血酶原时间及发色底物法检测,重组FTPI_1-161具有良好的抗凝及抑制Fxa活性的作用。为获得大量TFPI_1-161提供了一种廉价高效的的蛋白表达纯化方式。为进一步的基础及临床前研究奠定了基础。     

CH50真核表达载体pCH503的构建及小鼠体内表达的趋化与抗肿瘤活性

构建重组 FN多肽 CH50真核表达载体并在小鼠体内表达 ,研究其趋化与抗肿瘤作用 .采用重组 DNA技术构建表达质粒 ;体内进行基因转染 ,采用 RT- PCR鉴定导入基因的表达 ;通过肝素亲和层析、SDS- PAGE和 Western blot鉴定表达产物 ;腹腔细胞计数、Giemsa染色分析以及肌肉组织切片与染色观察体内基因转染后的趋化作用 ;小鼠黑色素瘤模型研究基因转染抑制肿瘤的作用 .从 CH50原核表达载体获得重组多肽的 c DNA,5′端加上小鼠 IFN- 5′端非编码区和信号肽编码区的 c DNA,3′端加上人 FN c DNA的 3′端非编码区 ;将重组 c DNA插入 p REP8质粒 ,即构建出p CH50 3质粒 .巨噬细胞在体内经 p CH50 3转染 ,然后在体外培养 ,能够产生 CH50多肽 .以p CH50 3分别进行腹腔基因转染和肌肉内基因转染 ,均可对免疫细胞产生趋化作用 ;p CH50 3体内转染可以使小鼠腹腔内黑色素肿瘤结节数降低 50 %～ 60 % . CH50真核表达载体 p CH50 3可在小鼠体内表达 ,体内基因转染可趋化免疫细胞和抑制肿瘤结节形成 ,在肿瘤综合治疗中有重要意义 .     

鸡Mx蛋白基因诱变修饰及抗病活性

[目的]进一步研究鸡Mx蛋白第631位氨基酸的变异与鸡群抗病性的相关性.[方法]本实验利用PCR突变技术将鸡Mx蛋白基因的全长cDNA第2032位的碱基由G突变为A(既631位氨基酸的改变),并将突变的Mx基因插入真核表达载体pcDNA3.0,重组表达载体转染COS-Ⅰ细胞后,进行RT-PCR与间接免疫荧光(IFA)鉴定.[结果]对鸡Mx蛋白基因的cDNA进行PCR诱变修饰正确,构建了能够正确表达鸡Mx蛋白的重组真核表达载体;诱变修饰重组Mx蛋白对抗新城疫病毒(NDV)感染分析结果显示,重组Mx蛋白具有较强的抗新城疫病毒生物活性.[结论]为下一步研究鸡Mx蛋白的抗病机理与制备抗病转基因鸡奠定了坚实的基础.     

人可溶性gp190在酵母Pichia pastoris中的表达

人可溶性gPl90(sgp190)是白血病抑制因子(LIF)的可溶性受体,可能在LIF行使众多生物学功能中扮演着重要的角色。为获得这一蛋白以研究它在人体内的生物学活性,在酵母Pichia pastoris中实现了重组sgp190分泌表达。利用基因重组技术,将编码人可溶性gP190(LlF受体α亚基gP190胞外区)cDNA克隆到毕赤酵母Pichia pastoris分泌表达载体pPIC9K,构建了重组表达质粒pPIC9K-sgp190。原生质体法转染Pichia pastoris GS115菌株,经过G418筛选,得到了高效分泌表达sgp190蛋白的毕赤酵母菌株GS115／pPIC9K-sgp190,sgp190蛋白占摇瓶培养表达上清中总蛋白质的26％。经初步纯化,对表达产物的性质鉴定表明,其分子量约125kD,具有免疫活性。对sgp190体外活性分析表明它能够抑制LIF诱导滋养层细胞分泌hCG。     

Inhibition of tissue factor-factor VIIa-catalyzed factor X activation by factor Xa-tissue factor pathway inhibitor. A rotating disc study on the effect of phospholipid membrane composition.

The physiological inhibitor of tissue factor (TF).factor VIIa (FVIIa), full-length tissue factor pathway inhibitor (TFPI(FL)) in complex with factor Xa (FXa), has a high affinity for anionic phospholipid membranes. The role of anionic phospholipids in the inhibition of TF.FVIIa-catalyzed FX activation was investigated. FXa generation at a rotating disc coated with TF embedded in a membrane composed of pure phosphatidylcholine (TF.PC) or 25% phosphatidylserine and 75% phosphatidylcholine (TF.PSPC) was measured in the presence of preformed complexes of FXa.TFPI(FL) or FXa.TFPI(1-161) (TFPI lacking the third Kunitz domain and C terminus). At TF.PC, FXa.TFPI(FL) and FXa.TFPI(1-161) showed similar rate constants of inhibition (0.07 x 10(8) M(-1) s(-1) and 0.1 x 10(8) M(-1) s(-1), respectively). With phosphatidylserine present, the rate constant of inhibition for FXa.TFPI(FL) increased 3-fold compared with a 9-fold increase in the rate constant for FXa. TFPI(1-161). Incubation of TF.PSPC with FXa.TFPI(FL) in the absence of FVIIa followed by depletion of solution FXa.TFPI(FL) showed that FXa.TFPI(FL) remained bound at the membrane and pursued its inhibitory activity. This was not observed with FXa.TFPI(1-161) or at TF.PC membranes. These data suggest that the membrane-bound pool of FXa.TFPI(FL) may be of physiological importance in an on-site regulation of TF.FVIIa activity.     

Semisynthesis of ent-norstrobane diterpenoids as potential inhibitor for factor Xa

A semisynthesis of two ent-strobane diterpenoids strobols C (7) and D (14) was accomplished via a Wagnar-Meerwein rearrangement. Compounds 7, 14, and the intermediate products were evaluated for their inhibition on factor Xa (FXa). Among all the compounds screened for FXa inhibitory activity, three compounds 6, 7, and 9 showed significant inhibitory activities with IC₅₀ values of 1067?±?164, 81?±?11, 1023?±?89?nM, respectively. The inhibitory activity on FXa described in this study highlight the importance of structural modification based on natural products in the development of FXa inhibitors.     

Elimination kinetics and protein binding of theophylline in the rabbit

The detailed elimination kinetics of theophylline were studied in 27 rabbits. Each received a 10 mg/kg intravenous bolus of aminophylline. The theophylline half-life ($\text{T}\text{1}\text{2}$) was 3.8 ± 0.63 hr. The apparent volume of distribution (V_D) and total body clearance (TBC) for theophylline were 439 ± 60 ml/kg and 81.0 ± 17.3 ml/kg·hr respectively. Theophylline protein binding was determined in 10 animals. The mean bound fraction was 74.3 ± 3.9% (range, 68.3–78.0%); the fraction bound was concentration indifferent over a serum concentration range of 5–20 μgm/ml.     

Oxidation products of phospholipid-containing delta-9 fatty acids specifically impair the activity of tissue factor pathway inhibitor

In the present study, we explored the active components in oxidized low-density lipoprotein (ox-LDL) that reduce the catalytic activity of tissue factor pathway inhibitor (TFPI), a Kunitz-type protease inhibitor of the extrinsic blood coagulation pathway. The active fraction was extracted from the phospholipid fraction of ox-LDL and separated. The oxidation products of 1- and/or 2-oleoyl phosphatidylcholine (PC) or phosphatidylethanolamine were the most potent compounds, while those of arachidonyl PC possessed only a weak inhibitory effect on the TFPI activity. These oxidized phospholipids associated strongly with rTFPI containing the carboxyl-terminal domain. When rTFPI was incubated with purified oxononanoyl PC (9CHO-PC) and its carboxylic form (9COOH-PC), the catalytic activity was specifically impaired, though neither oxovaleroyl PC (5CHO-PC) nor lyso-phospholipids reduced the TFPI activity. We conclude that the oxidation products of delta-9 unsaturated phospholipid in the lipoproteins are the active components that impair the anti-coagulation activity of TFPI.     

UV-induced mutagenesis at the hypoxanthine—guanine phosphoribosyl transferase locus in two L5178Y mouse lymphoma cell strains with different UV sensitivities

Two strains of L5178Y mouse lymphoma cells, L5178Y-R (LY-R) and L5178Y-S (LY-S), differ markedly in their sensitivity to 254 nm UV radiation (D₀ = 0.7 and 5.5 J/m²; n = 6.0 and 2.0 for LY-R and LY-S cells, respctively). In this study, the frequency o hypoxanthine-guanine-phosporibosyl-transferase-deficient mutants was determined, using 6-thioguanine (TG) as a selective agent, in populations of LY-R and LY-S cells exposed to various fluences of UV radiation. The spontaneous mutation frequency for LY-R cells was (3.7 ± 0.6) × 10^?5 TG^r mutants per viable cell, and the UV induction rate was (2.2 ± 0.8) × 10^?4 TG^r mutants per viable cell, per J/m². Both spontaneous and induced mutantion frequencies were much lower for LY-S cells. The sopntaneous mutation frequency for these cells were too low to make its measurement practicable ( < 0.0013 × 10^?5 TG^r mutants per viable cell). Mutation induction rate was (4.2 ± 2.2) × 10^?7 TG^r mutants per viable cell, per J/m². These differences in mutability do not appear to be due to gene duplication in LY-S cells, or to selective growth disadvantage of LY-S-derived TG-resistant mutants. Possible mechanisms underlying the differences in mutability of LY-R and LY-S cells are considered.     

The onset of photosynthetic acclimation to elevated CO2 partial pressure in field-grown Pinus radiata D. Don. after 4 years

The effects of CO₂ enrichment on photosynthesis and ribulose‐1,5‐bisphosphate carboxylase/oxygenase (rubisco) were studied in current year and 1‐year‐old needles of the same branch of field‐grown Pinus radiata D. Don trees. All measurements were made in the fourth year of growth in large, open‐top chambers continuously maintained at ambient (36 Pa) or elevated (65 Pa) CO₂ partial pressures. Photosynthetic rates of the 1‐year‐old needles made at the growth CO₂ partial pressure averaged 10·5 ± 0·5 μmol m^?2 s^?1 in the 36 Pa grown trees and 11·8 ± 0·4 μmol m^?2 s^?1 in the 65 Pa grown trees, and were not significantly different from each other. The photosynthetic capacity of 1‐year‐old needles was reduced by 25% from 23·0 ± 1·8 μmol m^?2 s^?1 in the 36 Pa CO₂ grown trees to 17·3 ± 0·7 μmol m^?2 s^?1 in the 65 Pa grown trees. Growth in elevated CO₂ also resulted in a 25% reduction in V_cmax (maximum carboxylation rate), a 23% reduction in J_max (RuBP regeneration capacity mediated by maximum electron transport rate) and a 30% reduction in Rubisco activity and content. Total non‐structural carbohydrates (TNC) as a fraction of total dry mass increased from 12·8 ± 0·4% in 1‐year‐old needles from the 36 Pa grown trees to 14·2 ± 0·7% in 1‐year‐old needles from the 65 Pa grown trees and leaf nitrogen content decreased from 1·30 ± 0·02 to 1·09 ± 0·10 g m^?2. The current‐year needles were not of sufficient size for gas exchange measurements, but none of the biochemical parameters measured (Rubisco, leaf chlorophyll, TNC and N), were effected by growth in elevated CO₂. These results demonstrate that photosynthetic acclimation, which was not found in the first 2 years of this experiment, can develop over time in field‐grown trees and may be regulated by source‐sink balance, sugar feedback mechanisms and nitrogen allocation.     

Seasonal variability of the parameters of the Ball–Berry model of stomatal conductance in maize (Zea mays L.) and sunflower (Helianthus annuus L.) under well‐watered and water‐stressed conditions

The Ball–Berry (BB) model of stomatal conductance (g_s) is frequently coupled with a model of assimilation to estimate water and carbon exchanges in plant canopies. The empirical slope (m) and ‘residual’ g_s (g₀) parameters of the BB model influence transpiration estimates, but the time‐intensive nature of measurement limits species‐specific data on seasonal and stress responses. We measured m and g₀ seasonally and under different water availability for maize and sunflower. The statistical method used to estimate parameters impacted values nominally when inter‐plant variability was low, but had substantial impact with larger inter‐plant variability. Values for maize (m = 4.53 ± 0.65; g₀ = 0.017 ± 0.016 mol m^?2 s^?1) were 40% higher than other published values. In maize, we found no seasonal changes in m or g₀, supporting the use of constant seasonal values, but water stress reduced both parameters. In sunflower, inter‐plant variability of m and g₀ was large (m = 8.84 ± 3.77; g₀ = 0.354 ± 0.226 mol m^?2 s^?1), presenting a challenge to clear interpretation of seasonal and water stress responses – m values were stable seasonally, even as g₀ values trended downward, and m values trended downward with water stress while g₀ values declined substantially.     

The metabolism of synthetic estrogens in non-users and users of oral contraceptives

Using both pulse injections and constant infusions of ³H-mestranol (³H-ME) (1) and ³H-ethinyl estradiol (³H-EE) we have studied the metabolism of these compounds in non-users and users of oral contraceptives. Following pulse injection of ³H-ME the disappearance of radioactivity could be described as a function which was the sum of two exponentials. Studied by both types of administration there was no difference in the metabolism of ³H-ME in the two groups; the overall mean ± SE metabolic clearance rate (MCR) was 690 ± 45 1/day/m², the mean ratio of the concentrations of radioactivity as EE following administration of ME (CR_BB^{M, E}) was 0. 23 ± 0. 02 and the mean [ρ]_BB^{M, E} (fraction of administered ME measured in blood as EE) was 0. 19 (95% confidence limits = 0.15 – 0. 23).Following pulse injection of ³H-EE the disappearance of radioactivity was best described as a function which is the sum of three exponentials. Results from both types of administration revealed no difference in the metabolism of ³H-EE between non-users. The overall mean ± SE MCR^EE was 630 ± 30 I/day/m². The MCR^EE is significantly (0. 02 > P > 0. 01) less than the mean MCR for estradiol reported previously, in both non-users and users of oral contraceptives. The use of oral contraceptives containing estrogens and progestins does not appear to influence the metabolism of the estrogen used. Approximately 20% of mestranol is converted to and appears in the blood as ethinyl estradiol.     

Serum cortisol levels affect the metabolic clearance rate of progesterone in female baboons.

Since interactions between progesterone (P₄), Cortisol (F), cortisone (E) and corticosteroid binding globulin (CBG) may influence the metabolic clearance rates (MCR) of these steroids, the effect of altering circulating F concentrations on clearance of the steroids was determined. MCR of P₄, F and E were determined by the iv constant infusion method in 6 pregnant and 6 nonpregnant baboons (Papio papio). Serum F concentrations were altered by iv infusion of 5 mg F/90 min or im injection of betamethasone (3 mg bi-daily for 2 days). Mean MCR-P₄ (1/d/kg ± SE) was greater (P < 0.01) in pregnant (92.8 ± 8.5) than in nonpregnant (53.9 ± 4.4) animals while mean MCR-F was similar in both groups ($\text{10.8 ± 1.2}\text{vs}\text{13.0 ± 1.5}$, respectively). Mean MCR-E was also similar in pregnant (30.8 ± 4.9) and nonpregnant (34.1 ± 4.5) baboons. Mean serum F concentrations (/gmg/100 ml ± SE) in 4 nonpregnant (42.0 ± 8.6) and 4 pregnant (52.2 ± 10.0) baboons were increased (P < 0.05) 60% by F administration but MCR-P₄, -F and -E were unaltered. Betamethasone treatment reduced (P < 0.05) serum F 75% in both groups. In nonpregnant baboons, betamethasone treatment reduced (P < 0.01) MCR-P₄ (37.3 ± 3.9), MCR-F (7.4 ± 1.6) and MCR-E (18.5 ± 3.7). Betamethasone treatment of pregnant animals reduced (P < 0.01) MCR-P₄ (56.5 ± 7.4), MCR-F (6.3 ± 0.8) and MCR-E (14.6 ± 2.6). Infusion of F into betamethasone-treated animals increased serum F levels and increased MCR-P4, -F and -E. It is concluded that variations in serum F levels affect the clearance of F, E and P₄ presumably because of the mutual interactions of these steroids with CBG.     

The cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942 has a sodium-dependent chloride transporter

Synechococcus R-2 (PCC 7942) actively accumulated Cl^? in the light and dark, under control conditions (BG-11 media: pH_o, 7·5; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 molm^?3). In BG-11 medium [Cl^?], was 17·2±0·848 mol m^?3 (light), electrochemical potential of Cl^? (ΔμCl^?_i,o) =+211±2mV; [Cl^?]_i= 1·24±0·11 mol m^?3(dark), ΔμCl^?_i,o=+133±4mV. Cl^? fluxes, but not permeabilities, were much higher in the light: ?Cl^?_i,o= 4·01±5·4 nmol m^?2 s^?1, ^PCl^?_i,o= 47±5pm s^?1 (light); ?Cl^?_i,o= 0·395±0·071 nmol m^?2 s^?1, _PCl^?_i,o= 69±14 pm s^?1 (dark). Chloride fluxes are inhibited by acid pH_o (pH_o 5; ?Cl^?_i,o= 0·14±0·04 nmol m^?2 s^?1); optimal at pH_o 7·5 and not strongly inhibited by alkaline pH_o (pH_o 10; ?Cl^?1_i,o= 1·7±0·14 nmol m^?2 s^?1). A Cl^?_in/2H⁺_in coporter could not account for the accumulation of Cl^? alkaline pH_o. Permeability of Cl^? is very low, below 100pm s^?1 under all conditions used, and appears to be maximal at pH_o 7·5 (50–70 pm s^?1) and minimal in acid pH_o (20pm s^?1). DCCD (dicyclohexyl-carbodiimide) inhibited ?Cl^?_i,o in the light about 75% and [Cl^?]_i fell to 2·2±0·26 (4) mol m^?3. Valinomycin had no effect but monensin severely inhibited Cl^? uptake ([Cl^?]_i= 1·02±0·32 mol m^?3; ?Cl^?_i,o= 0·20±0·1 nmol m^?2 s^?1). Vanadate (200 mmol m^?3) accelerated the Cl^? flux (?Cl^?_i,o= 5·28±0·64 nmol m^?2 s^?1) but slightly decreased accumulation of Cl^? ([Cl^?], = 13·9±1·3 mol m^?3) in BG-11 medium but had no significant effect in Na⁺-free media. DCMU (dichlorophenyldimethylurea) did not reduce [Cl^?], or ?Cl^?_i,o to that found in the dark ([Cl^?]_i= 8·41±0·76 mol m^?3; ?Cl^?_i,o= 2·06±0·36 nmol m^?2 s^?1). Synechococcus also actively accumulated Cl^? in Na⁺-free media, [Cl^?]_i was lower but ΔΨ_i,o hyperpolarized in Na⁺-free media and so the ΔμCl^?_i,o was little changed ([Cl^?]_i= 7·98±0·698 mol m^?3; ΔμCl^?_i,o=+203±3 mV). Net Cl^? uptake was stimulated by Na⁺; Li⁺ acted as a partial analogue for Na⁺. Synechococcus has a Na⁺ activated Cl^? transporter which is probably a primary 2Cl^?/ATP pump. The Cl^? pump is voltage sensitive. ΔμCl^?_i,o is directly proportional to ΔΨ_i,o(P»0·01%): ΔμCl^?_i,o= -1·487 (±0·102) ×ΔΨ_i,o, r= -0·983, n= 31. The ΔμCl^?_i,o increased (more positive) as the Δμ_i,o became more negative. The ΔμCl^?_i,o has no known function, but might provide a driving force for the uptake of micronutrients.