边界处电偶联对1D生物起搏器的作用 — 一种仿真研究

现在生物起搏器越来越引起学者们的关注. 本文旨在研究边界处的电偶联对生物起搏器起搏及驱动能力的影响. 首先利用各向异性的反应扩散方程,建立了包含生物起搏器的1D心室组织模型. 基于该模型,仿真了不同边界电偶联对应的起搏器初次起搏时间、特殊位置细胞的动作电位、心电的传导过程等参考项,发现减弱边界处的电偶联对生物起搏器的起搏能力具有一定的增强作用;然而,当电偶联足够小时,起搏器的电兴奋却不能有效传出,导致其驱动心室组织失败. 另外,本文探讨了边界电偶联的大小与起搏器最小尺寸之间的关系,发现电偶联越小,起搏器成功起搏所需的细胞数量越少,但是细胞数量变化并不明显. 因此,仅仅减弱电偶联对生物起搏器有一定的增强作用,但如果生成高效的起搏器,仍需辅助其它措施.     

边界处电偶联对1D生物起搏器的作用——一种仿真研究（英文）

生物起搏器越来越引起学者们的关注.本文旨在研究边界处的电偶联对生物起搏器起搏及驱动能力的影响.首先利用各向异性的反应扩散方程,建立了包含生物起搏器的1D心室组织模型.基于该模型,仿真了不同边界电偶联对应的起搏器初次起搏时间、特殊位置细胞的动作电位、心电的传导过程等参考项,发现减弱边界处的电偶联对生物起搏器的起搏能力具有一定的增强作用;然而,当电偶联足够小时,起搏器的电兴奋却不能有效地传出,导致其驱动心室组织失败.另外,本文探讨了边界电偶联的大小与起搏器最小尺寸之间的关系,发现电偶联越小,起搏器成功起搏所需的细胞数量越少,但是细胞数量变化并不明显.因此,仅仅减弱电偶联对生物起搏器有一定的增强作用,但如果生成高效的起搏器,仍需辅助其他措施.     

腾格里沙漠东南缘不同类型生物土壤结皮对土壤有机碳矿化的影响

生物土壤结皮(BSCs)是荒漠生态系统的重要组成部分,是该区土壤碳循环及碳平衡的关键影响因素。研究了腾格里沙漠东南缘不同类型生物土壤结皮覆盖下土壤碳矿化过程及其对温度(10℃、25℃和35℃)和水分(土壤含水量10%和25%)变化响应特征,分析了土壤碳矿化过程与土壤理化性质的关系。结果表明:(1)结皮的形成和发育显著影响土壤有机碳矿化过程,藻类、地衣和藓类结皮覆盖的土壤碳矿化速率和CO_2-C累积释放量均显著高于去除结皮的土壤,不同类型BSCs覆盖土壤和去除结皮土壤之间均表现为藓类结皮土壤地衣结皮土壤藻类结皮。(2)含结皮层土壤的平均和最大矿化速率均随温度升高和水分增加而逐渐增大,有结皮覆盖的土壤和去除结皮的土壤对温度和水分变化的响应规律相同。(3)有结皮土壤和去除结皮土壤碳矿化速率的温度敏感性(Q_(10))与结皮类型密切相关,均表现为藓类结皮地衣结皮藻类结皮。结果表明生物土壤结皮由以藻类为主向以藓类为主的演变进一步促进了土壤碳矿化过程,结皮对土壤碳循环的调控作用受水热等环境因子的共同影响。     

测定线粒体细胞色素c,c1,b,aa3含量的简单方法

线粒体是细胞中的重要细胞器,能量代谢的主要场所.在呼吸链中,细胞色素c,c_1,b,aa_3起着电子传递中间体的作用,其任何一种含量的减少或缺失将影响细胞的呼吸功能.由于细胞色素是一类带有卟啉环的蛋白质,在可见光区域有明显的吸收,可以利用这一性质进行比色定量。J.N.Williams首先建立了测定线粒体还原-氧化差光谱计算细胞色素含量的方     

基于空腔填充效应构建伯克霍尔德菌脂肪酶LipA的热稳定性突变体

【目的】中温伯克霍尔德菌胞外脂肪酶LipA在工业领域具有重要的应用价值。利用蛋白质工程技术来提高其热稳定性,对开发脂肪酶LipA酶制剂及提高其应用范围及应用效果,具有重要的意义。【方法】利用生物信息学软件Castp、Voronoia和Cave分析LipA分子中存在的空腔及其组成氨基酸残基;利用FoldX软件构建上述氨基酸残基的突变体电子文库,并基于空腔效应(体积变小)、自由能变化值(降低)和空间结构特点等对前述突变体电子文库进行筛选。从突变体电子文库中选择具有代表性的突变体,通过基因工程技术,引入突变。经诱导表达后,实验验证并筛选出热稳定性的突变体。【结果】构建了一个由58个突变体组成的电子文库;并对其中17个代表性的突变体进行了实验验证;筛选到2个热稳定性有明显提高的突变体LipA-His15Pro和LipA-Ala210Val;其叠加突变体LipA-His~(15)Pro/Ala~(210)Val的T50~(12)较野生型LipA提高了8°C,在55°C下的半衰期较野生型脂肪酶LipA提高了23.1倍。【结论】基于空腔填充技术构建热稳定性伯克霍尔德菌胞外脂肪酶LipA突变体,是一种行之有效的策略。     

hpc2研究进展

生物个体的胚胎发育以及细胞的增殖、分化,都同时受到多种基因的严格调控,PcG基因家族就是一类重要的发育相关基因.而hPc2基因是人PcG基因家族中的一个重要成员,其编码的HPC2蛋白,不仅可以和HPH、BMI-1以及RING1等其他人类PcG蛋白结合形成HPC/HPH PcG复合体,以蛋白复合体的形式参与对homeotic基因的表达抑制,以维持机体的正常发育以及细胞的增殖和定向分化,还发现它能与其他多种蛋白质相结合,提示HPC2可能具有多种功能.因此,对hPc2的深入研究不仅有助于进一步阐明PcG基因家族的作用机理,扩展人们对基因表达调控的认识,还有助于发现PcG基因家族与其他信号转导通路的联系,更好地理解细胞信号网络系统.     

地黄C3H基因的克隆及生物信息学分析

香豆酸-3-羟化酶属于植物中最大的蛋白酶细胞色素P450家族之一,在植物生命活动中发挥着重要作用。为了解地黄香豆酸-3-羟化酶基因RgC3H合成毛蕊花糖苷的功能,该研究基于地黄代谢组学分析获得KEGG途径中的C3H,采用多重比对在NCBI中获得同源基因的一个保守序列,并基于该保守序列和地黄SRA数据库,采用电子克隆和RT-PCR克隆技术获得地黄C3H基因全长CDS(RgC3H),对其进行生物信息学分析。结果表明:RgC3H基因全长为1 530 bp,且编码一个含509个氨基酸、分子量为57.91 kD、无信号肽的蛋白质; 基于氨基酸序列的结构分析显示,RgC3H有一个保守区域-P450结构域; 系统进化分析结果显示,RgC3H与芝麻和猴面花的C3H基因具有很高的同源性。上述结果为进一步研究RgC3H基因在地黄毛蕊花糖苷生物合成途径中的作用奠定了基础。     

利用Tol2转座子介导的增强子诱捕技术获得血管相关转基因斑马鱼系

心血管系统形成于胚胎发育极早期并为其他器官的发育、维持、修复所必需,血管生长异常可造成多种疾病.然而,由于研究对象所限,胚胎血管的发育机制尚未完全阐明,调控血管发育的基因也所知有限.通过Tol2转座子介导的大规模增强子诱捕筛选到26个血管特异表达绿色荧光蛋白(EGFP)报告基因的转基因斑马鱼系,其中有一些品系在胚胎的某些特异血管结构中表达绿色荧光.通过linker-mediated PCR克隆到22个鱼系中Tol2插入位点附近的斑马鱼基因组序列,其中有17个鱼系的Tol2插入可定位到现有的斑马鱼基因组中的单一位点.通过整体胚胎原位杂交对插入位点附近的基因进行表达谱分析,得到8个表达谱与转基因鱼系一致的基因,涵盖了9个鱼系,其中dusp5基因对应于2个不同的鱼系.这8个基因中包括hhex、ets1a和dusp5等3个功能已知的基因,但是大部分(5个)基因在斑马鱼中尚无功能研究,分别为zvsg1、micall2a、arl8b(1of2)、zgc:73355以及hecw2(1of2).hhex和ets1a基因对血管与血细胞前体的发育具有重要作用,所获得的EGFP报告基因受hhex或ets1a基因增强子控制的转基因斑马鱼(mp378b和mp430c-2)为国际首例,为深入研究这两个基因在血管与血液发育中的作用机制提供了新的机遇.筛选到的功能未知基因可以用来进一步研究其在血管发育中的功能;同时,利用所获得的转基因鱼系,可以实现实时、动态观察成血管细胞的起源、分化与基因表达调控,并可用于高通量小分子药物筛选等重要研究.     

7种秋海棠叶片斑纹结构及遗传特性分析

以7种(品种)秋海棠为材料,观察叶片斑区和非斑区组织结构、测定叶绿素含量及叶绿素荧光参数F_v/F_m值,分析叶片斑纹的形成原因及银点秋海棠点状斑的遗传特性。结果显示：(1)银点秋海棠、铺地秋海棠、假厚叶秋海棠、‘皮卡’和‘非洲丛林’叶片斑区的上表皮细胞与栅栏组织细胞间存在空隙,非斑区则没有空隙,彩纹秋海棠和‘虎斑’的斑区与非斑区上表皮细胞和栅栏组织细胞间均紧密相连。(2)7种(品种)秋海棠叶片斑区和非斑区都具有完整的叶绿体超微结构,类囊体膜丰富,基质和基粒片层清晰;银点秋海棠、假厚叶秋海棠、‘皮卡’和‘非洲丛林’斑区的叶绿素a、b及总叶绿素含量均低于非斑区,而铺地秋海棠斑区和非斑区差别不大;除假厚叶秋海棠的斑区叶绿素荧光参数F_v/F_m值小于非斑区外,其余6种秋海棠均为斑区高于非斑区。(3)银点秋海棠与无斑种类杂交,杂交后代叶片有斑和无斑的植株约为1∶1,而其自交后代中有斑和无斑的植株比例近3∶1。研究发现,银点秋海棠、铺地秋海棠、假厚叶秋海棠、‘皮卡’和‘非洲丛林’的叶斑属于空隙结构型,彩纹秋海棠和‘虎斑’叶斑属于色素型。银点秋海棠点状叶斑与无斑是1对可遗传的相对性状,白色点状斑为显性性状。     

化能自养硫氧化细菌Halothiobacillus sp.LS2介导的以乙炔为电子受体的硫氧化反应

【目的】探究化能自养硫氧化细菌Halothiobacillus sp. LS2介导的以乙炔为电子受体的厌氧硫氧化反应。【方法】稀释涂布法测定细胞生长情况,离子色谱仪测试硫氧化动力学中SO_4~(2–)和S_2O_3~(2–)以及基于相对荧光定量法的基因表达分析。【结果】尽管菌株LS2在以氧气为电子受体时的最大反应速率V_(max)更高,但在厌氧条件下且以乙炔为电子受体时,菌株LS2的生长量是氧气为电子受体时的2倍,且硫氧化酶基因soxB的表达量显著高于氧气作为电子受体时。【结论】菌株LS2不仅可以以乙炔为电子受体完成厌氧硫氧化反应,且这一代谢过程的产能效率较有氧硫氧化过程更高。本研究首次发现了微生物介导的以乙炔为电子受体的厌氧硫氧化反应,对丰富硫的生物地球化学循环理论有积极意义。     

The vicissitudes of the pacemaker current <Emphasis Type="Italic">I</Emphasis><Subscript>Kdd</Subscript> of cardiac purkinje fibers

Summary The mechanisms underlying the pacemaker current in cardiac tissues is not agreed upon. The pacemaker potential in Purkinje fibers has been attributed to the decay of the potassium current I _Kdd. An alternative proposal is that the hyperpolarization-activated current I _f underlies the pacemaker potential in all cardiac pacemakers. The aim of this review is to retrace the experimental development related to the pacemaker mechanism in Purkinje fibers with reference to findings about the pacemaker mechanism in the SAN as warranted. Experimental data and their interpretation are critically reviewed. Major findings were attributed to K⁺ depletion in narrow extracellular spaces which would result in a time dependent decay of the inward rectifier current I _K1. In turn, this decay would be responsible for a “fake” reversal of the pacemaker current. In order to avoid such a postulated depletion, Ba²⁺ was used to block the decay of I _K1. In the presence of Ba²⁺ the time-dependent current no longer reversed and instead increased with time and more so at potentials as negative as −120 mV. In this regard, the distinct possibility needs to be considered that Ba²⁺ had blocked I _Kdd (and not only I _K1). That indeed this was the case was demonstrated by studying single Purkinje cells in the absence and in the presence of Ba²⁺. In the absence of Ba²⁺, I _Kdd was present in the pacemaker potential range and reversed at E _K. In the presence of Ba²⁺, I _Kdd was blocked and I _f appeared at potentials negative to the pacemaker range. The pacemaker potential behaves in a manner consistent with the underlying I _Kdd but not with I _f. The fact that I _f is activated on hyperpolarization at potential negative to the pacemaker range makes it suitable as a safety factor to prevent the inhibitory action of more negative potentials on pacemaker discharge. It is concluded that the large body of evidence reviewed proves the pacemaker role of I _Kdd (but not of I _f) in Purkinje fibers.     

Bifurcation Analysis of Genetically Engineered Pacemaking in Mammalian Heart

Genetically engineered pacemaking in ventricular cells has been achieved by down-regulation of the time independent inward rectifying current (I _K1), or insertion of the hyperpolarisation-activated funny current (I _f). We analyse the membrane system (i.e. ionic concentrations clamped) of an epicardial Luo-Rudy dynamic cell model using continuation algorithms with the maximum conductance () of I _K1 and I _f as bifurcation parameters. Pacemaker activity can be induced either via Hopf or homoclinic bifurcations. As _K1 is decreased by ≈74%, autorhythmicity emerged via a homoclinic bifurcation, i.e., the periodicity first appear with infinitely large periods. In contrast, the insertion of _f induced periodicity via a subcritical Hopf bifurcation at _f≈ 0.25 mSμF⁻¹. Stable autorhythmic action potentials occurred at _f > 0.329 mSμF⁻¹.     

Reciprocal interaction between IK1 and If in biological pacemakers: A simulation study

Pacemaking dysfunction (PD) may result in heart rhythm disorders, syncope or even death. Current treatment of PD using implanted electronic pacemakers has some limitations, such as finite battery life and the risk of repeated surgery. As such, the biological pacemaker has been proposed as a potential alternative to the electronic pacemaker for PD treatment. Experimentally and computationally, it has been shown that bio-engineered pacemaker cells can be generated from non-rhythmic ventricular myocytes (VMs) by knocking out genes related to the inward rectifier potassium channel current (I_K1) or by overexpressing hyperpolarization-activated cyclic nucleotide gated channel genes responsible for the “funny” current (I_f). However, it is unclear if a bio-engineered pacemaker based on the modification of I_K1- and I_f-related channels simultaneously would enhance the ability and stability of bio-engineered pacemaking action potentials. In this study, the possible mechanism(s) responsible for VMs to generate spontaneous pacemaking activity by regulating I_K1 and I_f density were investigated by a computational approach. Our results showed that there was a reciprocal interaction between I_K1 and I_f in ventricular pacemaker model. The effect of I_K1 depression on generating ventricular pacemaker was mono-phasic while that of I_f augmentation was bi-phasic. A moderate increase of I_f promoted pacemaking activity but excessive increase of I_f resulted in a slowdown in the pacemaking rate and even an unstable pacemaking state. The dedicated interplay between I_K1 and I_f in generating stable pacemaking and dysrhythmias was evaluated. Finally, a theoretical analysis in the I_K1/I_f parameter space for generating pacemaking action potentials in different states was provided. In conclusion, to the best of our knowledge, this study provides a wide theoretical insight into understandings for generating stable and robust pacemaker cells from non-pacemaking VMs by the interplay of I_K1 and I_f, which may be helpful in designing engineered biological pacemakers for application purposes.     

Cesium,Na+-K+ pump and pacemaker potential in cardiac purkinje fibers

The mechanisms of the hyperpolarizing and depolarizing actions of cesium were studied in cardiac Purkinje fibers perfused in vitro by means of a microelectrode technique under conditions that modify either the Na⁺-K⁺ pump activity or I_f. Cs⁺ (2 mM) inconsistently increased and then decreased the maximum diastolic potential (MDP); and markedly decreased diastolic depolarization (DD). Increase and decrease in MDP persisted in fibers driven at fast rate (no diastolic interval and no activation of I_f). In quiescent fibers, Cs⁺ caused a transient hyperpolarization during which elicited action potentials were followed by a markedly decreased undershoot and a much reduced DD. In fibers depolarized at the plateau in zero [K⁺]_o (no I_f), Cs⁺ induced a persistent hyperpolarization. In 2 mM [K⁺]_o, Cs⁺ reduced the undershoot and suppressed spontaneous activity by hyperpolarizing and thus preventing the attainment of the threshold. In 7 mM [K⁺]_o, DD and undershoot were smaller and Cs⁺ reduced them. In 7 and 10 mM [K⁺]_o, Cs⁺ caused a small inconsistent hyperpolarization and a net depolarization in quiescent fibers; and decreased MDP in driven fibers. In the presence of strophanthidin, Cs⁺ hyperpolarized less. Increasing [Cs⁺]_o to 4, 8 and 16 mM gradually hyperpolarized less, depolarized more and abolished the undershoot. We conclude that in Purkinje fibers Cs⁺ hyperpolarizes the membrane by stimulating the activity of the electrogenic Na⁺-K⁺ pump (and not by suppressing I_f); and blocks the pacemaker potential by blocking the undershoot, consistent with a Cs⁺ block of a potassium pacemaker current.     

Effects of pressure overload-induced hypertrophy on TTX-sensitive inward currents in guinea pig left ventricle

We investigated the effects of pressure overload hypertrophy on inward sodium (I _Na) and calcium currents (I _Ca) in single left ventricular myocytes to determine whether changes in these current systems could account for the observed prolongation of the action potential. Hypertrophy was induced by pressure overload caused by banding of the abdominal aorta. Whole-cell patch clamp experiments were used to measure tetrodotoxin (TTX)-sensitive inward currents. The main findings were that I _Ca density was unchanged whereas I _Na density after stepping from –80 to –30 mV was decreased by 30% (–9.0 ± 1.16 pA pF^–1 in control and –6.31 ± 0.67 pA pF^–1 in hypertrophy, p < 0.05, n= 6). Steady-state activation/inactivation variables of I _Na, determined by using double-pulse protocols, were similar in control and hypertrophied myocytes, whereas the time course of fast inactivation of I _Na was slowed (p < 0.05) in hypertrophied myocytes. In addition, action potential clamp experiments were carried out in the absence and presence of TTX under conditions where only Ca²⁺ was likely to enter the cell via TTX-sensitive channels. We show for the first time that a TTX-sensitive inward current was present during the plateau phase of the action potential in hypertrophied but not control myocytes. The observed decrease in I _Na density is likely to abbreviate rather than prolong the action potential. Delayed fast inactivation of Na⁺ channels was not sustained throughout the voltage pulse and may therefore merely counteract the effect of decreased I _Na density so that net Na⁺ influx remains unaltered. Changes in the fast I _Na do not therefore appear to contribute to lengthening of the action potential in this model of hypertrophy. However, the presence of a TTX-sensitive current during the plateau could potentially contribute to the prolongation of the action potential in hypertrophied cardiac muscle. (Mol Cell Biochem 261: 217–226, 2004)     

The Influences of I h on Temporal Summation in Hippocampal CA1 Pyramidal Neurons: A Modeling Study

Recent experimental and theoretical studies have found that active dendritic ionic currents can compensate for the effects of electrotonic attenuation. In particular, temporal summation, the percentage increase in peak somatic voltage responses invoked by a synaptic input train, is independent of location of the synaptic input in hippocampal CA1 pyramidal neurons under normal conditions. This independence, known as normalization of temporal summation, is destroyed when the hyperpolarization-activated current, I _h, is blocked [Magee JC (1999a), Nature Neurosci. 2: 508–514]. Using a compartmental model derived from morphological recordings of hippocampal CA1 pyramidal neurons, we examined the hypothesis that I _h was primarily responsible for normalization of temporal summation. We concluded that this hypothesis was incomplete. With a model that included I _h, the persistent Na⁺ current (I _NaP), and the transient A-type K⁺ current (I _A), however, we observed normalization of temporal summation across a wide range of synaptic input frequencies, in keeping with experimental observations.     

On the integration of subthreshold inputs from Perforant Path and Schaffer Collaterals in hippocampal CA1 pyramidal neurons

Using a realistic model of a CA1 hippocampal pyramidal neuron, we make experimentally testable predictions on the roles of the non-specific cation current, I _h , and the A-type Potassium current, I _A , in modulating the temporal window for the integration of the two main excitatory afferent pathways of a CA1 neuron, the Schaffer Collaterals and the Perforant Path. The model shows that the experimentally observed increase in the dendritic density of I _h and I _A could have a major role in constraining the temporal integration window for these inputs, in such a way that a somatic action potential (AP) is elicited only when they are activated with a relative latency consistent with the anatomical arrangement of the hippocampal circuitry.     

Simulation analysis of intracellular Na and Cl homeostasis during β1-adrenergic stimulation of cardiac myocyte

To quantitatively understand intracellular Na⁺ and Cl⁻ homeostasis as well as roles of Na⁺/K⁺ pump and cystic fibrosis transmembrane conductance regulator Cl⁻ channel (I_CFTR) during the β1-adrenergic stimulation in cardiac myocyte, we constructed a computer model of β1-adrenergic signaling and implemented it into an excitation-contraction coupling model of the guinea-pig ventricular cell, which can reproduce membrane excitation, intracellular ion changes (Na⁺, K⁺, Ca²⁺ and Cl⁻), contraction, cell volume, and oxidative phosphorylation. An application of isoproterenol to the model cell resulted in the shortening of action potential duration (APD) after a transient prolongation, the increases in both Ca²⁺ transient and cell shortening, and the decreases in both Cl⁻ concentration and cell volume. These results are consistent with experimental data. Increasing the density of I_CFTR shortened APD and augmented the peak amplitudes of the L-type Ca²⁺ current (I_CaL) and the Ca²⁺ transient during the β1-adrenergic stimulation. This indirect inotropic effect was elucidated by the increase in the driving force of I_CaL via a decrease in plateau potential. Our model reproduced the experimental data demonstrating the decrease in intracellular Na⁺ during the β-adrenergic stimulation at 0 or 0.5 Hz electrical stimulation. The decrease is attributable to the increase in Na⁺ affinity of Na⁺/K⁺ pump by protein kinase A. However it was predicted that Na⁺ increases at higher beating rate because of larger Na⁺ influx through forward Na⁺/Ca²⁺ exchange. It was demonstrated that dynamic changes in Na⁺ and Cl⁻ fluxes remarkably affect the inotropic action of isoproterenol in the ventricular myocytes.     

In silico risk assessment for drug-induction of cardiac arrhythmia

The main components of repolarization reserve for the ventricular action potential (AP) are the rapid (I_Kr) and slow (I_Ks) delayed outward K⁺ currents. While many drugs block I_Kr and cause life-threatening arrhythmias including torsades de pointes, the frequency of arrhythmias varies between different I_Kr-blockers. Different types of block of I_Kr cause distinct phenotypes of prolongation of action potential duration (APD), increase in transmural dispersion of repolarization (TDR) and, accordingly, occurrence of torsades de pointes. Therefore the assessment of a drug's proarrhythmic risk requires a method that provides quantitative and comprehensive comparison of the effects of different forms of I_Kr-blockade upon APDs and TDR. However, most currently available methods are not adapted to such an extensive comparison. Here, we introduce I_Kr–I_Ks two-dimensional maps of APD and TDR as a novel risk-assessment method. Taking the kinetics of I_Kr-blockade into account, APDs can be calculated upon a ventricular AP model which systematically alters the magnitudes of I_Kr and I_Ks. The calculated APDs are then plotted on a map where the x axis represents the conductance of I_Kr while the y axis represents that of I_Ks. TDR is simulated with models corresponding to APs in epicardial, midcardial and endocardial myocardium. These two-dimensional maps of APD and TDR successfully account for differences in the risk resulting from three distinct types of I_Kr-blockade which correspond to the effects of dofetilide, quinidine and vesnarinone. This method may be of use to assess the arrhythmogenic risk of various I_Kr-blockers.