兔骨骼肌ryanodine受体/钙释放通道的AFM研究

Ryanodine受体（RyR)是位于细胞内质网膜上的钙释放通道,在肌细胞的兴奋－收缩偶联中发挥重要作用。RyR难于结晶,以往主要是用电镜和计算机三维重组相结合的方法来获得其高级结构的信息。由于RyR结构庞大,易于水解,在提取的过程中需要加入外源性磷脂以保护其结构,而磷脂的存在对电镜的分辨率有影响,所以电镜研究的RyR在提 过程中一般不添加外源性磷脂,这样得到的结构信息并没有反映RyR在生理状况下的真实情况,我们用原子力显微镜（AFM）对RyR进行研究,经过了空气中RyR成像,液体中RyR成像,重组到脂双层中的RyR研究几个阶段,提供了RyR在接近生理条件下的结构信息,并为进一步研究其结构和功能的关系打下了基础。     

Isradipine interacts with the open state of the L-type calcium channel at high concentrations

Triple mutation of Tyr1485, Met1486 and Ile1493 in the IVS6 segment of alpha 1C-b subunit of the L-type calcium channel results in a loss of the high affinity inhibition by isradipine. The mutant channel (Ch30) yet exhibits a concentration-dependent inhibition by isradipine with a 110-fold lower affinity. The mechanisms underlying the remaining low affinity block were investigated. Isradipine accelerated the current decay in Ch30 but not in wild type channel in a concentration dependent manner. Dependence of the current amplitude inhibition on holding potential was parallel in Ch30 and in wild type channels, while the acceleration of current decay in Ch30 was independent of the membrane potential. The recovery from voltage-dependent inactivation was biphasic in both channels and was slowed down by isradipine in the wild type but not in the Ch30 channel. The change of the charge carrier (Ba2+ or Ca2+) and calcium chelator (EGTA or BAPTA) did not affect the acceleration of current decay indicating that isradipine did not interact with the Ca(2+)-inactivated state of the channel. These results demonstrate that the mutations of Ch30 affect selectively the high affinity inhibition of an inactivated channel and unmask a low affinity interaction of isradipine with an open state of the channel.     

PKA信号通路与ryanodine受体介导的心肌肌浆网钙泄漏关系的研究

目的：研究β肾上腺素激活的PKA信号通路与成年大鼠心肌细胞RyR2介导的肌浆网Ca^2＋泄漏之间的关系,探讨治疗心力衰竭的新方法。方法：NiCl2（5mmol/L）和毒胡萝卜素（TG,100nmol/L）分别阻断成年大鼠心室肌细胞的钠钙交换体和钙泵,胞外灌流异丙肾上腺素（ISO,1μmol/L）。采用激光扫描共聚焦显微镜记录灌流前后细胞内钙火花及[Ca2＋]i的变化,并使用Ca^2＋通道阻断剂钌红（RR,5μmol/L）对β肾上腺素刺激作用进行逆转,进而阻断RyR2介导的肌浆网内钙泄漏。结果：ISO胞外灌流后,心肌细胞钙火花发生率和[Ca^2＋]i与对照组相比明显升高（n=10,P〈0.05）;加入RR后,心肌细胞内钙火花发生率与对照组比较显著降低（n=20,P〈0.05）,[Ca^2＋]i两组没有明显差别（n=20,P〉0.05）。结论：ISO激活PKA信号通路后可诱导心肌细胞RyR2介导的钙泄漏,RR可有效地逆转这种钙泄漏。     

Multiscale modeling of calcium dynamics in ventricular myocytes with realistic transverse tubules

Spatial-temporal Ca(2+) dynamics due to Ca(2+) release, buffering, and reuptaking plays a central role in studying excitation-contraction (E-C) coupling in both normal and diseased cardiac myocytes. In this paper, we employ two numerical methods, namely, the meshless method and the finite element method, to model such Ca(2+) behaviors by solving a nonlinear system of reaction-diffusion partial differential equations at two scales. In particular, a subcellular model containing several realistic transverse tubules (or t-tubules) is investigated and assumed to reside at different locations relative to the cell membrane. To this end, the Ca(2+) concentration calculated from the whole-cell modeling is adopted as part of the boundary constraint in the subcellular model. The preliminary simulations show that Ca(2+) concentration changes in ventricular myocytes are mainly influenced by calcium release from t-tubules.     

Cytochemical and electron probe X-ray microanalysis studies on the distribution change of intracellular calcium in columella cells of soybean roots under simulated microgravity

The columella cells of soybean roots grown under gravity and simulated microgravity induced by a clinostat were examined using potassium pyroantimonate (PA) and quantitative X-ray microanalysis of cryosections to determine the role of Ca in the regulation of the gravitropic response. Amyloplasts in the columella cells were localized exclusively at the bottom under gravity, but diffusely distributed in the cytoplasmic matrix under simulated microgravity, thus supporting the statolith theory. In the columella cells, PA precipitates containing Ca were diffusely distributed in the cytoplasmic matrix under gravity. Under simulated microgravity, however, they decreased in number and size in the cytoplasmic matrix, whereas increased only in number in the vacuole, indicating that Ca moved from the cytoplasmic matrix into the vacuole. The vacuole of columella cells contained mostly electron-dense granular structures localized along the inner surface of tonoplasts, which closely resembled the tannin vacuole reported in Mimosa pulvinar motor cells. Under simulated microgravity, their configuration changed dramatically from a granular shape to a flat plate. The quantitative X-ray microanalysis of cryosections showed that the vacuolar electron-dense structures contained a large amount of Ca. Under simulated microgravity, the concentration of Ca increased conspicuously in these vacuolar electron-dense structures, concomitantly with a marked decrease of K in the vacuoles and an increase of K in the cell walls. These results suggest that the release of Ca(2+) from, and uptake by, the vacuolar electron-dense structures is closely related to the signal transmission in the gravitropic response and that Ca movement occurs opposite to that of K.     

Extracellular Ca2+ sensitivity of mGluR1alpha induces an increase in the basal cAMP level by direct coupling with Gs protein in transfected CHO cells

We previously reported that the metabotropic glutamate receptor R1alpha (mGluR1alpha) can be activated not only by applying glutamate but also by raising extracellular Ca2+ (Ca2+o) concentration, and that the constant stimulation by Ca2+o causes morphological change of transfected Chinese Hamster Ovary (CHO) cells (Kubo Y Miyashita T and Murata Y (1998) Science 279, 1722-1725). The physiological role of the Ca2+o-sensing function of mGluR1alpha, however, is not fully clear yet, especially because Ca2+ is constitutively present in the extracellular space unlike other neurotransmitters. In this work, we aimed to elucidate the physiological significance of the Ca2+o-sensing function of mGluR1alpha. The effect of mGluR1alpha activation by Ca2+o on the morphological change of CHO cells was mimicked by forskolin. The effect of mGluR1alpha activation on the morphological change was suppressed by the inhibitors of adenylate cyclase, protein kinase A (PKA) and MAP kinase kinase (MAPKK), and the effect of forskolin was also decreased by the inhibitors of PKA and MAPKK. These results demonstrate the involvement of cAMP, PKA, MAPKK, MAPK pathway in the morphological change. We actually confirmed that the Ca2+o stimulation of mGluR1alpha increased the basal cAMP level of transfected CHO cells. This increase in cAMP was observed even when only the membrane fraction of mGluR1alpha transfected CHO cells were used, and the increase was inhibited by anti-Gs alpha antibody. Taken together, we concluded that the Ca2+o-sensing function of mGluR1alpha and the continuous stimulation by Ca2+o caused the increase in the basal cAMP level by direct coupling with Gs, and triggered the subsequent activation of PKA, MAPKK, and MAPK cascade which resulted in the morphological change of transfected CHO cells.     

Voltage-dependent effects of Ca(2+)/calmodulin on Cl(-) channel in cardiac sarcoplasmic reticulum

A new kind of chloride channels in the cardiac sarcoplasmic reticulum, 116 pS Cl(-) channel (500 mM Cl(-) in the cis and 50 mM Cl(-) in the trans chamber solutions), which is activated by protein-kinase-A-dependent phosphorylation, has been determined to conduct adenine nucleotide as a transporter between cytosol and SR lumen. We investigated the voltage-dependent gating of this Cl(-) channel by recording single-channel activities using the planar lipid bilayer-vesicle fusion technique. The channel activities did not change at different membrane potentials (-100 mV to +50 mV) or different Ca(2+) concentrations (1 nM to 1 mM) in cis solution. In the presence of calmodulin (CaM) (0.1 microM /microg SR vesicles), however, Ca(2+) added to the cis solution at 0 mV inhibited channel openings in a Ca(2+) -concentration-dependent manner. These effects were prevented by the addition of CaM inhibitors. The blocking effects of CaM differed depending on the membrane potentials at negative potentials below -20 mV. With CaM and 3 microM Ca(2+), the values of opening probability were 0 at -80 mV, 0.2 at -40 mV, 0.3 at -20 mV, 0.71 at 0 mV and 0.92 at +20 mV. These results may indicate the membrane potential affects the action of Ca(2+) /CaM complex     

Mechanism of Abnormal Sarcoplasmic Reticulum Calcium Release in Canine Left-Ventricular Myocytes Results in Cellular Alternans

Electrocardiographic alternans are known to predispose to increased susceptibility to life threatening arrhythmias and sudden cardiac death. While deficiencies in Ca²⁺ transport processes have been implicated in the genesis of cellular alternans, the underlying mechanisms have been elusive, and are the goal of this study. A novel reverse engineering approach that applies a simultaneous action potential (AP) and [Ca²⁺ ]i clamp of experimentally obtained data, to a previously described left-ventricular canine myocyte model, is employed to isolate the molecular and cellular mechanisms underlying cardiac alternans. The model-derived sarcoplasmic reticulum (SR) Ca²⁺ in control beats (102.1 plusmn 12.9 nM, n = 639 ), although larger, is not statistically significantly different as compared to beats corresponding to small [Ca²⁺ ]_i (99.3 plusmn 35.4 nM, n = 310, p = NS), but is significantly smaller as compared to beats corresponding to large [Ca²⁺ ]_i (122.6 plusmn 31.0 nM, n = 311, p<0.000001) during alternans. The model indicates that the increased SR Ca²⁺ in these beats triggers multiple ryanodine receptor (RyR) channel openings and delayed Ca²⁺ release that subsequently triggers an inward depolarizing current, a subthreshold early after depolarization, and AP prolongation. In conclusion, the results presented in this study support the idea that aberrant RyR openings on alternate beats are responsible for the [Ca²⁺ ]_i alternans-type oscillations, which, in turn, give rise to AP alternans.     

2-Aminoethoxydiphenyl borate (2-APB) inhibits capacitative calcium entry independently of the function of inositol 1,4,5-trisphosphate receptors

Capacitative calcium entry (CCE), the mechanism that replenishes intracellular calcium stores after depletion, is essential to intracellular calcium signaling. CCE is mediated by the channels in the plasma membrane generally referred to as "store operated channels (SOCs)". However, the molecular identity of the SOCs has never been determined, and the mechanism of the activation of SOCs remains to be elucidated. Recent studies have demonstrated that 2-aminoethoxydiphenyl borate (2-APB), which has been found to be an antagonist of inositol 1,4,5-trisphosphate receptors (IP3Rs), inhibits CCE, suggesting that IP3Rs channel activity is essential to the generation of CCE. However, CCE has also been reported to occur normally in IP3R-deficient cells. In order to resolve this discrepancy, we investigated the effect of 2-APB on CCE in IP3Rs-deficient cells. In response to store depletion with thapsigargin or N,N,N',N'-tetrakis (2-pyridylmethyl) ethylene diamine (TPEN), CCE was generated in IP3Rs-deficient cells the same as in wild-type cells, however, 2-APB abolished CCE in IP3Rs-deficient cells, despite the fact that this cell line does not possess functional IP3Rs. We also examined the effect of 2-APB on several types of TRP Ca2+ channels, which exhibit properties similar to those of SOCs. 2-APB had a different inhibitory effect on spontaneous and thapsigargin-induced Ba2+ influx in cells that transiently expressed individual TRP subtypes. These results suggest that the channel activity of IP3Rs is not essential to the generation of CCE in this cell line and that 2-APB inhibits CCE independently of the function of IP3Rs.     

Cr，Ca：YAG晶体中Cr~（4＋）的吸收和退火特性

用Ｃｚｏｃｈｒａｌｓｋｉ法生长了Ｃｒ４＋：ＹＡＧ可调谐激光晶体，对样品进行氧化气氛高温退火实验，分析比较了退火前后Ｃｒ４＋吸收特性的变化，发现退火后样品中Ｃｒ４＋的吸收显著增强；Ｃｒ３＋的主要吸收峰值红移，并提出相应模型对此现象进行了解释。     

Extracellular Ca2+ sensitivity of mGluR1alpha associated with persistent glutamate response in transfected CHO cells

We previously reported that the metabotropic glutamate receptor 1alpha (mGluR1alpha) can be activated not only by glutamate but also by extracellular Ca2+ (Ca2+o), and that Ser 166 in the extracellular domain determines the sensitivity to Ca2+o. In the present study, we investigated by intracellular Ca2+ (Ca2+i) imaging, the effect of Ca2+o on the glutamate responses of Chinese Hamster Ovary (CHO) cells stably expressing mGluR1alpha wild-type (CHO-wt). As a negative control, we carried out similar experiments using CHO cells expressing Ser166Asp mutant of mGluR1alpha (CHO-S166D) or the substance P receptor (CHO-SPR), which were not activated by Ca2+o application. We observed a remarkable prolongation of the duration of the glutamate response in CHO-wt cells in a Ca2+o concentration dependent manner. In CHO-S166D cells and CHO-SPR cells, only a small sustained component of the glutamate response was observed in the presence of Ca2+o. These sustained components were blocked by SKF-96365, a blocker of receptor-operated Ca2+-influx. Thus, it was concluded that the Ca2+o-sensing function of mGluR1alpha-wt induced the persistent opening of the receptor-operated Ca2+-permeable channels, probably by persistent activation of the receptor by glutamate. We additionally observed that the dose-response relationship of CHO-S166D and CHO-SPR shifted significantly by changing Ca2+o concentration, i.e. Ca2+o was required to maintain the normal ligand responses of these receptors.     

Desensitization of muscarinic receptors

When Chinese hamster ovary cells transfected with the gene for M(3)-muscarinic receptors were stimulated with carbachol continuously for 30 min, the response at the end of the stimulation period was about 20% of the early response (2-3 min after the start of the stimulation). Long-term treatment of the cells with phorbol ester abolished the response completely while desensitization was significantly reduced upon pre-treatment of the cells with GF109203X, antisense oligonucleotide against the alpha-isoform of protein kinase C and wortmannin. We conclude that in the Chinese hamster ovary expression system, desensitization of M(3)-muscarinic receptors is dependent on a fast feedback loop including the alpha-isoform of protein kinase C.     

Characterization of a new type HPV16 E7 variant isolated from cervical cancer highest incidence area in Hubei Province of China

AIM: To investigate the variation and biological properties of HPV16 E7 isolated from cervical cancer biopsy samples from highest incidence area in HuBei province of China. METHODS: HVP16 E7 sequences isolated from the cervical cancer biopsies of 10 local patients were amplified, sequenced and compared with prototype E7 gene. Then the variant gene was cloned into different vectors to study the antigenicity, expression and immunogenicity of its protein by Western blot, immunofluorescence and genetic immunization in vitro or in vivo. RESULTS: The results showed that 7 of 10 samples had the same mutations which led to a nonsense mutation at codon 43 of E7 sequence. The truncated E7 protein could be recognized by standard E7 monoclonal antibody in Western blot and expressed in NIH3T3 cells. In the blood sera of mice immunized intramuscularly by the plasmid DNA expressing the variant E7 gene specific E7 antibodies could be detected at week 2, 3, 5 and 6 after inoculation. However, no specific lymphoproliferation after E7 protein stimulation in vitro was detected by MTT colorimetric assay in comparison to the prototype E7 protein. CONCLUSION: HPV16 E7 gene may show variation in China and the variant protein could be expressed and induce host humoral immune response, but could not elicit special cellular-immune response against it. These data might hold the key for future development of HPV16 vaccine in HuBei province of China.     

Different populations of inositol 1,4,5-trisphosphate receptors expressed in the bovine adrenal cortex

The inositol 1,4,5-trisphosphate (InsP3) receptor forms a tetrameric channel responsible for the release of Ca2+ from intracellular stores. In the present study we showed that the experimental approach used to separate bound and free ligands may discriminate between two populations of InsP3 binding sites in bovine adrenal cortex microsomes. A large population of low affinity sites and a small population of high affinity sites were detected with centrifugation and filtration approaches, respectively. Both populations were found in the supernatant and the cytoskeleton fractions of Triton X-100 solubilized microsomes. After treatment of microsomes with thimerosal, an alkylating reagent known to increase InsP3 receptor affinity, the filtration and the centrifugation approaches yielded identical results. With selective anti-InsP3 receptor antibodies, we showed that types 1, 2 and 3 InsP3 receptors are present in intact microsomes and in the cytoskeleton fraction. Binding studies on immunoprecipitated receptors revealed that anti-type 1 antibody recognizes a large population of low affinity sites whereas anti-type 2 antibody recognizes a small population of high affinity sites. Our results indicate that the three types of InsP3 receptors are expressed at different levels in the bovine adrenal cortex. The presence of different types of InsP3 receptors with different ligand binding affinities and their association with the cytoskeleton offer a convenient way for the cell to simultaneously regulate its intracellular Ca2+ concentration and reorganize the spatial distribution of its Ca2+ stores.     

A “Nano-Courier” for Precise Delivery of Acetylcholine and Melatonin by C5a-Targeted Aptamers Effectively Attenuates Reperfusion Injury of Ischemic Stroke

Overactive inflammatory response and excessive oxidative stress are the main pathophysiological culprits for cerebral ischemia/reperfusion injury (IRI) that arouse neuronal damage . The neurotransmitter acetylcholine (ACh) exerts anti-inflammatory roles by stimulating α7 nicotinic acetylcholine receptor (α7nAChR) on microglia to activate the cholinergic anti-inflammatory pathway. Simultaneously, as a circadian rhythm-dependent hormone , melatonin (MT) possesses promising neuroprotective effects that eliminate reactive oxygen species (ROS) in the ischemic region. Relying on these, a biocompatible fluorescein isothiocyanate (FITC)-labeled SiO₂@PAA-MT/ACh nanospheres are constructed to effectively alleviate oxidative stress and polarize microglial phenotype to suppress inflammatory response in cerebral IRI. Despite of biosafety and curative effects of ACh and MT, the poor aggregation in ischemic penumbra hinders their neuroprotection. To address that, complement component 5a (C5a) is used as a molecular target for delivery of ACh and MT. C5a conspicuously exists at local inflammatory sites of cerebral IRI, recruits immune cells, and mediates further release of inflammatory cytokines. Upon binding of anti-C5a (aC5a) aptamers onto FITC-labeled SiO₂@PAA-MT/ACh nanospheres, they can effectively target the ischemic penumbra and promote neurological recovery. Taken together, the current study suggests that the FITC-labeled SiO₂@PAA-MT/ACh-aC5a nanospheres after intravenous (i.v.) administration can act as an effective targeted nanotherapy to salvage neurons in cerebral IRI.     

Ba2+掺杂对CaCu3Ti4O12陶瓷结构与电性能的影响

采用传统固相法制备了Ca1-xBaxCu3Ti4O12(x=0, 0.005, 0.010, 0.020, 0.030, 0.040, 0.050, 0100,摩尔分数) 陶瓷。用X线衍射仪、扫描电子显微镜、介电温谱测试系统及阻抗测试仪研究了Ba2+掺杂量的变化对Ca1-xBaxCu3Ti4O12陶瓷的相结构、微观形貌及电性能影响。研究结果表明,随着Ba2+掺杂量的增加,陶瓷试样产生了第二相CuO,同时Ba2+掺杂使CaCu3Ti4O12的晶格常数增大。Ca1-xBaxCu3Ti4O12陶瓷的晶粒尺寸随Ba2+掺杂量的增加而减小,气孔率随之降低。掺杂适量的Ba2+可有效降低CaCu3Ti4O12陶瓷的介电损耗,也可降低相对介电常数随温度的变化率。一定量的Ba2+掺杂还能增加CaCu3Ti4O12的晶界电阻。     

Cellular signaling mechanisms for muscarinic acetylcholine receptors

Signaling pathways for muscarinic acetylcholine receptors (mAChRs) include several enzymes and ion channels. Recent studies have revealed the importance of various isoforms of both alpha and betagamma subunits of G proteins in initiation of signaling as well as the role of the small monomeric G protein, Rho, in the activation of phospholipase D. Modulation of adenylyl cyclase activity by mAChRs appears more diverse as the interaction of various receptor subtypes with the many isoforms of the enzyme are studied. Both alpha and beta subunits of G(i/o) may be involved. Some mAChR responses arise through release of nitric oxide from nitrergic nerves, including salivary gland secretion and hippocampal slow wave activity. mAChRs utilize a variety of intracellular pathways to activate various mitogen-activated protein kinases. The kinases are involved in cholinergic regulation of kidney epithelial function, catabolism of amyloid precursor protein, hippocampal long-term potentiation, activation of phospholipase A(2), and gene induction. mAChR activation can also stimulate or inhibit cellular growth and apoptosis, dependent on prior levels of cellular activity. Modulation of ion channels by mAChR agonists appears increasingly complex, based on recent studies. K(+) channels may be activated by M(2) and M(4) mAChR stimulation, although in the rat superior cervical ganglion topographical constraints appear to limit the effect to the M(2) mAChR. Another ganglionic K(+) current, the M current, is inhibited by M(1) mAChR activation, but in murine hippocampus inhibition involves another receptor subtype. R-type Ca(2+) channels are both facilitated and inhibited by M(1) and M(2) mAChRs; facilitation being more pronounced with activation of M(1) mAChRs and inhibition with M(2) mAChRs.     

Neuromuscular transmission on the rebound

Recent work at the zebrafish neuromuscular junction (NMJ) has shown that positively charged acetylcholine (ACh), at the high concentrations reached in the cleft during neuromuscular transmission, blocks acetylcholine receptors (AChRs) as soon as they open. Thus after two ACh molecules bind and open the channel, a third molecule enters and blocks the pore at a site resembling that for block by local anesthetics, suggesting that ACh is the endogenous anesthetic of the NMJ. Recovery from open channel block results in a rebound synaptic current only after ACh is cleared from the cleft. Kinetic modeling of other AChRs suggests that a rebound current is generated at all vertebrate NMJs, from fish to frogs to mammals. Open channel block prolongs the current at fast zebrafish NMJs in order to more effectively spread charge along the fibers, akin to multiple central synapses spread over dendrites. Together these findings indicate the need for a fundamental revision of current thinking about neuromuscular transmission at many levels, including channel structure, function and pharmacology.     

电视机上I2C通信的E2PROM设计稳健性应用研究

在电视机中,E2PROM常用于保存系统关键数据和用户数据,其工作的可靠性对电视系统非常重要.针对I2C接口的E2PROM在电视机上的应用,从硬件、软件两方面系统介绍了提高设计稳健性的关键技术.