神经节苷脂GM3对重建Ca 2+ -ATP酶构象的调节

应用稳态荧光和纳秒时间分辨瞬态荧光技术,以不同性质猝灭剂探测了神经节苷脂GM3诱发的Ca ²⁺-ATP酶构象的变化.结果显示,GM3可使重建的Ca ²⁺-ATP酶蛋白内源荧光寿命明显延长;并且能不同程度地减弱离子性猝灭剂碘化钾(I^-)和脂溶性猝灭剂竹红菌乙素(HB)对Ca ²⁺-ATP酶色氨酸(Trp)内源荧光的猝灭程度.进一步用时间分辨荧光猝灭动力学分析,当体系中有GM3存在时,HB对该蛋白不同荧光寿命组分的Trp内源荧光猝灭的幅度减小.猝灭常数(K_sv)明显降低.说明GM3依靠其亲水糖链和疏水的神经酰胺链作用,不仅可以改变重建Ca ²⁺-ATP酶蛋白嵌于膜脂疏水区内部的构象,使位于膜脂疏水区不同部位的Trp残基更加趋向排列于亲水-疏水域界面;而且还使Ca ²⁺-ATP酶亲水-疏水结构域之间更趋接近,致使整个酶蛋白分子呈现较“紧凑”的构象,表达较高的酶活力.     

GM1对肌质网Ca~(2+)-ATPase活性及膜流动性的影响

外源性GM1对肌质网Ca2+-ATPase的水解及转运活性都有明显的抑制作用.在GM1浓度为0～8nmol/mg蛋白质范围内抑制作用具有浓度依赖性.当GM1浓度达到8nmol/mg蛋白质时,酶活性受到最大抑制,此时水解活性降低51%,转运活性降低49%.荧光偏振测定结果表明:GM1参入后,肌质网膜流动性降低.     

GM3与Ca2+-ATP酶的重建及其冷冻断裂电镜观察

应用生物膜的分离与重建技术, 将GM3、大豆磷脂与肌质网Ca²⁺-ATP酶共同重建在脂质体上, 酶活力明显增加. 经负染、冷冻断裂复型后电镜等形态学方法证实形成的脂酶体囊泡封闭性好,脂酶体上Ca²⁺-ATP酶蛋白颗粒均匀、直径增大.     

神经革苷脂GM3对肌质网Ca^2＋—ATP酶活力的调节

研究了神经节苷脂ＧＭ３参入肌质网膜后Ｃａ＾２＋－ＡＴＰ酶活力的变化。结果表明：ＧＭ３参入肌质网膜后,对肌质网Ｃａ＾２＋－ＡＴＰ酶活性（ＡＴＰ水解活力与转运活力）有明显的激活作用。当参入的ＧＭ３浓度为８μｍｏｌ／Ｌ、参入时间为１２０ｍｉｎ、温度为３０℃时,对Ｃａ＾２＋－ＡＴＰ酶的激活作用最大。     

γ-干扰素时间分辨免疫荧光分析方法的建立

采用生物素-Eu³⁺标记链亲和素双抗体夹心时间分辨免疫荧光分析技术(TRIFMA),建立新的γ-干扰素检测技术,提高γ-干扰素检测方法的灵敏度.用固相包被的兔多抗捕获样品中IFN-γ,以具有中和IFN-γ抗病毒活性的生物素化单抗作为二抗体,再加Eu³⁺标记链亲和素并荧光检测.已知不同浓度标准IFN-γ CPS值的标准曲线,判断待检样品中IFN-γ量.本方法最低检测值为0.02 μg/L,检测范围为0.02～400 μg/L,而TNF-α,IL-2和IFN-α等细胞因子无交叉反应.对基因工程IFN-γ的生产,纯化过程中定性, 定量监控以及对培养细胞上清中IFN-γ量的检测等都有实用价值.     

神经节苷脂GM_3对肌质网Ca~（2＋）－ATP酶活力的调节

研究了神经节苷脂ＧＭ_３参入肌质网膜后Ｃａ~（２＋）－ＡＴＰ酶活力的变化,结果表明：ＧＭ_３参入肌质网膜后,对肌质网Ｃａ~（２＋）ＡＴＰ酶活性（ＡＴＰ水解活力与转运活力）有明显的激活作用．当参入的ＧＭ_３浓度为８μｍｏｌ／Ｌ、参入时间为１２０ｍｉｎ、温度为３０℃时,对Ｃａ~（２＋）－ＡＴＰ酶的激活作用最大．     

钆对兔肌质网膜脂与膜蛋白的影响

利用荧光偏振、顺磁共振波谱及圆二色谱研究了轧对肌质网膜脂和膜蛋白的影响。结果表明,Ｇｄ３＋降低肌质网膜脂不同层次的流动性,并使Ｃａ２＋－ＡＴＰ酶的旋转运动加快,低度的浓Ｇｄ３＋使肌质网膜Ｃａ２＋－ＡＴＰ酶的α－螺旋含量减少,随着其浓度的增加,则使其α－螺旋含量增加。     

吸附金(Au3+)的真菌筛选

通过筛选获得3株吸附Au³⁺能力较强的真菌。对其中一株芽枝状枝孢(Cladosporimmcladosporioides AS 3.3995)进行了吸附Au³⁺的条件研究。结果表明,溶液的pH值、温度、时间和Au³⁺的浓度对菌的吸附作用有影响。最适pH为5以下,温度在30—50℃之间。该菌的最大吸附量为140 mgAu³⁺/g(干细胞)。电镜观察表明,Au³⁺在细胞壁的表面上慢慢地还     

稀土La3+跨PC12细胞膜行为研究

使用AR-CM-M1C阳离子测定系统,发展Fura-2荧光测定技术,将其应用于测定细胞内游离稀土离子La³⁺,并以此研究了La³⁺跨PC12细胞（大鼠嗜铬细胞瘤细胞）膜的行为.结果表明：在模拟细胞内离子组分,pH=7.05的溶液中,测得La³⁺-Fura-2的表观解离常数为3.27×10^-11 mol·L^-1.对于PC12细胞,静息条件下La³⁺不能跨越细胞膜进入胞内.与钙离子通道相关的KCl和去甲肾上腺素均不能刺激稀土La³⁺过膜.用哇巴因（ouabain）使胞内Na⁺超载后,La³⁺可过膜进入细胞内,且过膜量与胞外La³⁺浓度和胞内Na⁺超载程度有一定的浓度依赖关系,提示La³⁺可以经由Na⁺／La³⁺交换机制过膜而进入细胞内.     

胡杨质膜的纯化及其H＋-ATPase活性的研究

用Dextran T-500, PEG 3350两相分配法分离并纯化了悬浮培养的胡杨细胞质膜.不同聚合物浓度(5.5%、5.7%、5.9%、6.1%、6.3%、6.5%)和KCl浓度(0、5、10、15 mmol/L)对分离效果影响的研究结果表明, 采用聚合物浓度为5.9%和无盐存在的两相分配体系可获得纯度较高的胡杨细胞质膜.纯化的质膜H^＋-ATPase的活力提高8倍,且酶定向程度较高,这为进一步研究胡杨细胞质膜特性及获得高纯度H^＋-ATPase提供了良好基础.     

Influence of exogenous application of glycinebetaine on antioxidative system and growth of salt-stressed soybean seedlings (Glycine max L.)

Glycinebetaine is one of the most competitive compounds which play an important role in salt stress in plants. In this study, the enhanced salt tolerance in soybean (Glycine max L.) by exogenous application of glycinebetaine was evaluated. To improve salt tolerance at the seedling stage, GB was applied in four different concentrations (0, 5, 25 and 50 mM) as a pre-sowing seed treatment. Salinity stress in the form of a final concentration of 150 mM sodium chloride (NaCl) over a 15 day period drastically affected the plants as indicated by increased proline, MDA and Na⁺ content of soybean plants. In contrast, supplementation with 50 mM GB improved growth of soybean plants under NaCl as evidenced by a decrease in proline, MDA and Na⁺ content of soybean plants. Further analysis showed that treatments with GB, resulted in increasing of CAT and SOD activity of soybean seedlings in salt stress. We propose that the role of GB in increasing tolerance to salinity stress in soybean may result from either its antioxidant capacity by direct scavenging of H₂O₂ or its role in activating CAT activity which is mandatory in scavenging H₂O₂.     

Na(+) interaction with the pore of Shaker B K(+) channels: zero and low K(+) conditions.

The Shaker B K(+) conductance (G(K)) collapses (in a reversible manner) if the membrane is depolarized and then repolarized in, 0 K(+), Na(+)-containing solutions (Gómez-Lagunas, F. 1997. J. Physiol. 499:3-15; Gómez-Lagunas, F. 1999. Biophys. J. 77:2988-2998). In this work, the role of Na(+) ions in the collapse of G(K) in 0-K(+) solutions, and in the behavior of the channels in low K(+) was studied. The main findings are as follows. First, in 0-K(+) solutions, the presence of Na(+) ions is an important factor that speeds the collapse of G(K). Second, external Na(+) fosters the drop of G(K) by binding to a site with a K(d) = 3.3 mM. External K(+) competes, in a mutually exclusive manner, with Na(o)(+) for binding to this site, with an estimated K(d) = 80 microM. Third, NMG and choline are relatively inert regarding the stability of G(K); fourth, with [K(o)(+)] = 0, the energy required to relieve Na(i)(+) block of Shaker (French, R.J., and J.B. Wells. 1977. J. Gen. Physiol. 70:707-724; Starkus, J.G., L. Kuschel, M. Rayner, and S. Heinemann. 2000. J. Gen. Physiol. 110:539-550) decreases with the molar fraction of Na(i)(+) (X(Na,i)), in an extent not accounted for by the change in Delta(mu)(Na). Finally, when X(Na,i) = 1, G(K) collapses by the binding of Na(i)(+) to two sites, with apparent K(d)s of 2 and 14.3 mM.     

Loss of caveolin-1 impairs retinal function due to disturbance of subretinal microenvironment

Caveolin-1 (Cav-1), an integral component of caveolar membrane domains, is expressed in several retinal cell types, including photoreceptors, retinal vascular endothelial cells, Müller glia, and retinal pigment epithelium (RPE) cells. Recent evidence links Cav-1 to ocular diseases, including autoimmune uveitis, diabetic retinopathy, and primary open angle glaucoma, but its role in normal vision is largely undetermined. In this report, we show that ablation of Cav-1 results in reduced inner and outer retinal function as measured, in vivo, by electroretinography and manganese-enhanced MRI. Somewhat surprisingly, dark current and light sensitivity were normal in individual rods (recorded with suction electrode methods) from Cav-1 knock-out (KO) mice. Although photoreceptor function was largely normal, in vitro, the apparent K(+) affinity of the RPE-expressed α1-Na(+)/K(+)-ATPase was decreased in Cav-1 KO mice. Cav-1 KO retinas also displayed unusually tight adhesion with the RPE, which could be resolved by brief treatment with hyperosmotic medium, suggesting alterations in outer retinal fluid homeostasis. Collectively, these findings demonstrate that reduced retinal function resulting from Cav-1 ablation is not photoreceptor-intrinsic but rather involves impaired subretinal and/or RPE ion/fluid homeostasis.     

Spatial,Cellular, and Intracellular Localization of Na+/K+-ATPase in the Sterically Disposed Renal Tubules of Japanese Eel

The kidney plays a crucial role in the regulation of water and ion balances in both freshwater and seawater fishes. However, the complicated structures of the kidney hamper comprehensive understanding of renal functions. In this study, to investigate the structure of sterically disposed renal tubules, we examined spatial, cellular, and intracellular localization of Na⁺/K⁺-ATPase in the kidney of the Japanese eel. The renal tubule was composed of the first (PT-I) and second (PT-II) segments of the proximal tubule and the distal tubule (DT), followed by the collecting ducts (CDs). Light microscopic immunocytochemistry detected Na⁺/K⁺-ATPase along the renal tubules and CD; however, the subcellular distribution of the Na⁺/K⁺-ATPase immunoreaction varied among different segments. Electron microscopic immunocytochemistry further revealed that Na⁺/K⁺-ATPase was distributed on the basal infoldings of PT-I, PT-II, and DT cells. Three-dimensional analyses showed that the renal tubules meandered in a random pattern through lymphoid tissues, and then merged into the CD, which was aligned linearly. Among the different segments, the DT and CD cells showed more-intense Na⁺/K⁺-ATPase immunoreaction in freshwater eel than in seawater-acclimated eel, confirming that the DT and CD segments are important in freshwater adaptation, or hyperosmoregulation. (J Histochem Cytochem 58:707–719, 2010)     

Interaction of the BKCa channel gating ring with dendrotoxins

Two classes of small homologous basic proteins, mamba snake dendrotoxins (DTX) and bovine pancreatic trypsin inhibitor (BPTI), block the large conductance Ca²⁺-activated K⁺ channel (BK_Ca, KCa1.1) by production of discrete subconductance events when added to the intracellular side of the membrane. This toxin-channel interaction is unlikely to be pharmacologically relevant to the action of mamba venom, but as a fortuitous ligand-protein interaction, it has certain biophysical implications for the mechanism of BK_Ca channel gating. In this work we examined the subconductance behavior of 9 natural dendrotoxin homologs and 6 charge neutralization mutants of δ-dendrotoxin in the context of current structural information on the intracellular gating ring domain of the BK_Ca channel. Calculation of an electrostatic surface map of the BK_Ca gating ring based on the Poisson-Boltzmann equation reveals a predominantly electronegative surface due to an abundance of solvent-accessible side chains of negatively charged amino acids. Available structure-activity information suggests that cationic DTX/BPTI molecules bind by electrostatic attraction to site(s) on the gating ring located in or near the cytoplasmic side portals where the inactivation ball peptide of the β2 subunit enters to block the channel. Such an interaction may decrease the apparent unitary conductance by altering the dynamic balance of open versus closed states of BK_Ca channel activation gating.     

Overexpression of SeNHX1 improves both salt tolerance and disease resistance in tobacco

Recently, we found NHX1, the gene encoding a Na⁺/H⁺ exchanger, participated in plant disease defense. Although NHX1 has been confirmed to be involved in plant salt tolerance, whether the NHX1 transgenic plants exhibit both salt tolerance and disease resistance has not been investigated. The T1 progenies of Nicotiana tabacum L. lines expressing SeNHX1 (from Salicornia europaea) were generated for the present study. Compared with PBI-type control plants, SeNHX1 transgenic tobaccos exhibited more biomass, longer root length, and higher K⁺/Na⁺ ratio at post germination or seedling stage under NaCl treatment, indicating enhanced salt tolerance. The vacuolar H⁺ efflux in SeNHX1 transgenic tobacco was increased after treatment of NaCl with different concentration. Meanwhile, the SeNHX1 transgenic tobaccos showed smaller wilted spot area, less H₂O₂ accumulation in leaves after infection of Phytophthora parasitica var. nicotianae. Further investigation demonstrated a larger NAD(P)(H) pool in SeNHX1 transgenic tobacco. These evidences revealed that overexpression of SeNHX1 intensified the compartmentation of Na⁺ into vacuole under salt stress and improved the ability of eliminating ROS after pathogen attack, which then enhanced salt tolerance and disease resistance simultaneously in tobacco. Our findings indicate NHX1 has potential value in creating crops with both improved salt tolerance and disease resistance.     

Na+/K+-ATPase resistance and cardenolide sequestration: basal adaptations to host plant toxins in the milkweed bugs (Hemiptera: Lygaeidae: Lygaeinae)

Despite sequestration of toxins being a common coevolutionary response to plant defence in phytophagous insects, the macroevolution of the traits involved is largely unaddressed. Using a phylogenetic approach comprising species from four continents, we analysed the ability to sequester toxic cardenolides in the hemipteran subfamily Lygaeinae, which is widely associated with cardenolide-producing Apocynaceae. In addition, we analysed cardenolide resistance of their Na⁺/K⁺-ATPases, the molecular target of cardenolides. Our data indicate that cardenolide sequestration and cardenolide-resistant Na⁺/K⁺-ATPase are basal adaptations in the Lygaeinae. In two species that shifted to non-apocynaceous hosts, the ability to sequester was secondarily reduced, yet Na⁺/K⁺-ATPase resistance was maintained. We suggest that both traits evolved together and represent major coevolutionary adaptations responsible for the evolutionary success of lygaeine bugs. Moreover, specialization on cardenolides was not an evolutionary dead end, but enabled this insect lineage to host shift to cardenolide-producing plants from distantly related families.     

Activation of Ca(2+)-dependent K(+) channels contributes to rhythmic firing of action potentials in mouse pancreatic beta cells

We have applied the perforated patch whole-cell technique to beta cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K(+) conductance. The current was dependent on Ca(2+) influx but unaffected by apamin and charybdotoxin, two blockers of Ca(2+)-activated K(+) channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K(+) channels) but partially (>60%) blocked by high (10-20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca(2+)-activated K(+) current plays an important role in the generation of oscillatory electrical activity in the beta cell.     

Altered intracellular pH regulation in cells with high levels of P-glycoprotein expression

P-glycoprotein is an ATP-binding-cassette transporter that pumps many structurally unrelated drugs out of cells through an ATP-dependent mechanism. As a result, multidrug-resistant cells that overexpress P-glycoprotein have reduced intracellular steady-state levels of a variety of chemotherapeutic agents. In addition, increased cytosolic pH has been a frequent finding in multidrug-resistant cells that express P-glycoprotein, and it has been proposed that this consequence of P-glycoprotein expression may contribute to the lower intracellular levels of chemotherapeutic agents. In these studies, we measured intracellular pH and the rate of acid extrusion in response to an acid load in two cells with very different levels of P-glycoprotein expression: V79 parental cells and LZ-8 multidrug resistant cells. Compared to the wild-type V79 cells, LZ-8 cells have a lower intracellular pH and a slower recovery of intracellular pH after an acid load. The data also show that LZ-8 cells have reduced ability to extrude acid, probably due to a decrease in Na⁺/H⁺ exchanger activity. The alterations in intracellular pH and acid extrusion in LZ-8 cells are reversed by 24-h exposure to the multidrug-resistance modulator verapamil. The lower intracellular pH in LZ-8 indicates that intracellular alkalinization is not necessary for multidrug resistance. The reversal by verapamil of the decreased acid-extrusion suggests that P-glycoprotein can affect other membrane transport mechanism.