Synthesis and characterization of some metal complexes of a proton transfer salt,and their inhibition studies on carbonic anhydrase isozymes and the evaluation of the results by statistical analysis

Abstract

A novel proton transfer compound (HMeOABT)?⁺?(HDPC)^? (1) and its Fe(III), Co(II), Ni(II) and Cu(II) complexes (2–5) have been prepared and characterized by spectroscopic techniques. Complex 4 has distorted octahedral conformation revealed by single crystal X-ray diffraction method. Structures of the other complexes might be proposed as octahedral according to experimental data. All compounds were also evaluated for their in vitro inhibition effects on hCA I and II for their hydratase and esterase activities. Although there is no inhibition for hydratase activities, all compounds have inhibited the esterase activities of hCA I and II. Data have been analyzed by using a one-way analysis of variance. The comparison of the inhibition studies of 1–5 to parent compounds indicates that 1–5 have superior inhibitory effects. The inhibition effects of 2–5 are also compared to inhibitory properties of the metal complexes of MeOABT and H2DPC, revealing an improved transfection profile.     

Effect of CO2, CO2 and Diamox on photosynthesis and photorespiration in Chlamydomonas reinhardtii (green alga) and Anacystis nidulans (Cyanobacterium, blue-green alga)

Photorespiration by Chlamydomonas reinhardtii and Anacystis nidulans was measured as the oxygen inhibition of CO₂ uptake and the CO₂ compensation points. Net photosynthesis was oxygen dependent in Chlamydomonas grown in 5% CO₂, but CO₂ insensitive in cultures bubbled with air. Anacystis, even when cultured in 5% CO₂, exhibited an CO₂ insensitive net photosynthesis. The CO₂ compensation point of Chlamydomonas grown in cultures bubbled with air and Anacystis grown in 5% CO₂ enriched air, were reached shortly after the measurement was begun and the values were very low, less than 10 μl CO₂ 1^?1; while Chlamydomonas grown in 5% CO₂ enriched air for 4 days showed a high, but temporary CO₂ compensation point (60 μl CO₂ 1^?1). After a two hour adaptation in low CO₂, a stable, low CO₂ compensation point was reached. It seems that photorespiration can only be detected by the methods used in this study when the algae are cultured in high CO₂, but a mechanism exists which blocks photorespiration when the green algae are adapted to low CO₂ concentrations. When Chlamydomonas was treated with Diamox, an inhibitor of carbonic anhydrase, after cultivation in low CO₂ (air), the cells behaved as if they had been grown in high CO₂. They showed an oxygen sensitive net photosynthesis and a high CO₂ compensation point. This indicates that carbonic anhydrase plays an important role in the regulation of a measurable photorespiration in Chlamydomonas. The results are discussed in relation to previous observations of photorespiration measured by enzyme assay, metabolic products and gas exchange properties.     

Carbonic anhydrase inhibitors that directly inhibit anion transport by the human Cl−/HCO3− exchanger,AE1

Carbonic anhydrases (CA, EC 4.2.1.1.) catalyze reversible hydration of CO₂ to HCO₃^?+H⁺. Bicarbonate transport proteins, which catalyze the transmembrane movement of membrane-impermeant bicarbonate, function in cooperation with CA. Since CA and bicarbonate transporters share the substrate, bicarbonate, we examined whether novel competitive inhibitors of CA also have direct inhibitory effects on bicarbonate transporters. We expressed the human erythrocyte membrane Cl^?/HCO₃^? exchanger, AE1, in transfected HEK293 cells as a model bicarbonate transporter. AE1 activity was assessed in both Cl^?/NO₃^? exchange assays, which were independent of CA activity, and in Cl^?/HCO₃^? exchange assays. Transport was measured by following changes of intracellular [Cl^?] and pH, using the intracellular fluorescent reporter dyes 6-methoxy-N-(3-sulfopropyl)quinolinium and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, respectively. We examined the effect of 16 different carbonic anhydrase inhibitors on AE1 transport activity. Among these 12 were newly-reported compounds; two were clinically used non-steroidal anti-inflammatory drugs (celecoxib and valdecoxib) and two were anti-convulsant drugs (topiramate and zonisamide). Celecoxib and four of the novel compounds significantly inhibited AE1 Cl^?/NO₃^? exchange activity with EC₅₀ values in the range 0.22–2.8 μM. It was evident that bulkier compounds had greater AE1 inhibitory potency. Maximum inhibition using 40 μM of each compound was only 22–53% of AE1 transport activity, possibly because assays were performed in the presence of competing substrate. In Cl^?/HCO₃^? exchange assays, which depend on functional CA to produce transport substrate, 40 μM celecoxib inhibited AE1 by 62±4%. We conclude that some carbonic anhydrase inhibitors, including clinically-used celecoxib, will inhibit bicarbonate transport at clinically-significant concentrations.     

贵州山区3种木本植物无机碳利用特性的比较

以生长在喀斯特高原地区玉舍国家森林公园内的成熟银鹊树(Tapiscia sinensis)、白栎(Quercus fabri)和亮叶桦(Betula luminifera)为实验材料,通过对光合作用、叶绿素含量、叶绿素荧光参数、羧化效率(CE)、呼吸速率(Resp)、碳酸酐酶活性(WA)以及稳定碳同位素组成(δ13 C)等指标的测定,分析3种植物不同的无机碳利用特性,为该区生态环境修复选择合适的建群植物种提供依据。结果显示:(1)银鹊树、白栎和亮叶桦分布都较为广泛,银鹊树生长的最佳土壤pH是4.5~5.5,而白栎更倾向于中性到弱酸性土壤,肥沃的酸性土壤则更有益亮叶桦生长;白栎和亮叶桦都能忍受干旱和贫瘠,但是银鹊树不能忍受干旱和高温。(2)银鹊树叶片的Pn、Tr和Gs显著大于白栎和亮叶桦,亮叶桦和白栎的Pn、Tr和Gs分别是银鹊树的69.5%、48.2%、66.7%和28.6%、21.7%、22.2%;亮叶桦叶绿素含量均为银鹊树和白栎的2倍,但3种植物间的WUE则无显著差异。(3)3个树种叶片净光合速率均随着CO2浓度升高呈持续上升的趋势,但它们之间的CO2补偿点和饱和点明显不同。其中,银鹊树和亮叶桦的CO2补偿点均低于50μmol·mol-1,而白栎的则在250~300μmol·mol-1之间;银鹊树的CO2饱和点在1 200μmol·mol-1左右,亮叶桦则在2 300μmol·mol-1左右,而白栎的CO2饱和点明显高于2 300μmol·mol-1。(4)3个树种的CE、Resp和WA均为银鹊树>亮叶桦>白栎;而δ13 C值则以银鹊树最低,亮叶桦和白栎较高。其中,白栎和亮叶桦的CE、Resp、WA分别为银鹊树的5.1%、25.7%、4.0%和45.3%、54.6%、6.8%,且树种间差异显著;白栎和亮叶桦的δ13 C值显著高于银鹊树。研究表明,银鹊树能够吸收大气中的CO2或者在高活性碳酸酐酶作用下转化利用细胞内的HCO3-,它拥有较高的CO2利用能力及无机碳同化效率,因而能够拥有较高的产能;亮叶桦只能获取大气中的CO2作为无机碳源,但它对CO2的利用能力也较高,其产能仅次于银鹊树;白栎同样只能获取大气中的CO2作为无机碳源,同时它对大气中CO2的捕获、利用能力均低于银鹊树和亮叶桦,因而白栎生长非常缓慢,造成其本身对无机碳的需求也最低,所以其产能最低。     

Effects of intraleaf variations in carbonic anhydrase activity and gas exchange on leaf C18OO isoflux in Zea mays

Variation in the C18OO content of atmospheric CO2 (delta18Oa) can be used to distinguish photosynthesis from soil respiration, which is based on carbonic anhydrase (CA)-catalyzed 18O exchange between CO2 and 18O-enriched leaf water (delta18Ow). Here we tested the hypothesis that mean leaf delta18Ow and assimilation rates can be used to estimate whole-leaf C18OO flux (isoflux), ignoring intraleaf variations in CA activity and gas exchange parameters. We observed variations in CA activity along the leaf (> 30% decline from the leaf center toward the leaf ends), which were only partially correlated to those in delta18Ow (7 to 21 per thousand), delta18O and delta13C of leaf organic matter (25 to 30 per thousand and -12.8 to -13.2 per thousand, respectively), and substomatal CO2 concentrations (intercellular CO2 concentrations, c(i), at the leaf center were approximately 40% of those at the leaf tip). The combined effect of these variations produced a leaf-integrated isoflux that was different from that predicted based on bulk leaf values. However, because of canceling effects among the influencing parameters, isoflux overestimations were only approximately 10%. Conversely, use of measured parameters from a leaf segment could produce large errors in predicting leaf-integrated C18OO fluxes.