离子通道开放状态检测法的计算机模拟研究(Ⅱ)

本文（Ⅱ）在文（Ⅰ）的基础上进一步探讨了将多指数函数检测法和连续平均开放时间检测法相结合的离子通道开放状态检测法．侧重分析了两开放状态平均开放时间的比值和同一开放状态连续出现的次数对自相关函数的影响,并给出了实施流程.本文提供的计算机模拟研究方法对研究生物医学的其它问题也有参考价值.     

离子通道开放状态检测法的计算机模拟研究（Ⅰ）

本文概述了离子通道马尔科夫模型的计算机模拟方法,通过对两个门控模型模拟数据的多指数函数法与本文作者提出的连续分组开放时间法检测的对比研究,探讨了新方法在检测通道开放状态时的效应和适用范围,以及有关的一些问题,本文提供的模拟研究方法对研究生物医学的其它问题也有参考价值。     

端粒酶活性检测的几种方法

端粒酶活性检测方法的不断发展改进为癌症的诊断治疗以及人们对衰老的进一步研究提供了新途径和新思路。近年来端粒酶活性的检测方法有：(1)基本方法。(2)TRAP法。(3)改良的TRAP法。(4)间接检测法等。     

一种有效的复杂疾病基因定位的检测法

连锁不平衡（LD）应用于某些复杂疾病基因的定位,近年来发展了许多LD定位方法,除TDT外,大多数LD定位方法须先假定无人群混和,人群混合可增大在疾病基因定位时犯Ⅰ类错误的机率,产生无效结果。此方法利用LD来检测标记位点和疾病敏感位点（DSL）的连锁（有连锁不平衡）相关（有连锁）。分析时采用不相关样本,已知其父母基因型和至少父母之一为杂合子,再将随机样本依基因型不同分类,然后对来自不同类的数据应用有力的统计方法进行单独和联合分析。此LD定位法不仅适用于患病和正常个体,而且有效消除据父母基因分类的样本定位时人群混合的影响,分析结果和模拟结果也表明此方法解决了在检测标记位点和疾病敏感位点之间的连锁和相关时人群混和的问题,但与TDT比,此法在检测的位点为DSL时丙能有效和充分地利用矫正数据,检测位点不是DSL时,此法和TDT法可相互补充更有效地检测连锁的DSL。     

PC—12细胞离子单通道开放状态的检测

本文对用片膜钳技术记录的PC-12细胞钾通道数据进行了连续分组平均开放时间分析,检测到通道活动的三种开放状态．并用正态密度函数对通道平均开放时间的频数分布进行了拟合,估计了各状态的开放持续时间的均值、方差等参数．本文提供的方法,在通道的数据分析中具有广泛的应用价值．     

应用改良流式法检测猕猴和食蟹猴体内血型抗体水平的分布情况

绝大部分灵长类动物存在与人类相似的ABO血型系统,该研究采用改良流式法(flow cytometry method,FCM)检测猕猴及食蟹猴血清中血型抗体水平的分布情况。以流式细胞术为基础,使用商品化人源红细胞为靶细胞,并通过加入特异性荧光标记的抗人IgM或IgG二抗,对收集的实验用猕猴及食蟹猴的血清样本进行检测,以人类健康受试者的血清样本为对照,比较两者血型抗体水平的差异。结果显示:预先用人O型浓缩红细胞吸附猴血清中所含种属间非特异性抗体后,FCM法能够准确检测其血型抗体水平及分型,并且发现猴血清中天然血型抗体的水平明显低于健康人(P<0.05)。由此得出:通过预处理清除非特异性抗体的干扰后,FCM法同样适用于灵长类动物血清中血型抗体的检测,也为构建灵长类动物模拟人ABO血型不合器官移植模型提供了技术保障和实验数据。     

农作物虫害的机器检测与监测技术研究进展

在早期发现并准确定位害虫, 对其未来的发展趋势作出评价, 可提高施药处方决策和综合防治的针对性和准确性。在作物虫害信息的获取中, 传统的检测和监测方法不但耗时、费力, 而且导致的预报滞后会进一步增加损失程度, 很难较好地满足现代农业的精准生产要求。本文介绍了国内外学者在田间作物上开展害虫及其危害状况的机器检测和监测技术研究取得的进展, 包括声特征检测法、雷达观测法、图像识别法以及光谱监测法等, 讨论了现有技术的局限性, 指出了未来作物虫害机器检测和监测技术的可能发展方向是采用多种技术相结合的组合式检测和监测方法, 从多个角度获取特定虫害的相关信息, 相互进行实证检验, 以提高作物虫害机器检测和监测的精度及效率。     

肺炎球菌1型荚膜多糖多克隆抗体制备与夹心ELISA法建立及其在多糖浓度测定中的应用

目的：利用肺炎球菌1型全菌体制备多克隆抗体,并且利用该抗体建立肺炎1型荚膜多糖夹心酶联免疫吸附分析法（ Enzyme-linked immunosorbent assay ,ELISA）,用于检测发酵和纯化过程中的多糖浓度。方法用灭活的1型肺炎链球菌免疫家兔6周,获得高滴度的抗多糖血清,经过亲和层析纯化,获得高纯度的兔抗肺炎1型多糖抗体IgG。以纯化IgG作为包被抗体,加入多糖样品,再以生物素化的抗体作为检测抗体,建立夹心ELISA法检测肺炎1型多糖浓度。确定标准曲线的最佳线性范围,并对该方法进行特异性、准确性和精密度验证。结果兔免疫血清经过双向免疫扩散检测抗体滴度可达1∶32;该方法的线性检测范围为1．56～50 ng/mL;最低检测限为3．13 ng/mL。在标准品中混入其他型别多糖或培养基,回收率分别为102％和108％;该方法批内精密度和批间精密度分别为6．08％和7．01％。结论建立的夹心ELISA方法,其特异性、准确性和精密度均良好,可以特异地检测肺炎球菌1型多糖浓度。     

综述与专论: 河鲀毒素的检测技术与应用

河鲀毒素（tetrodotoxin， TTX）是毒性极强的小分子生物碱类毒素，包括中国在内的亚洲沿海国家因误食TTX污染食品而中毒的事件时有发生，其发病迅速且无特效解毒剂，对环境安全、食品安全与社会安全造成极大的威胁。通过检测食品与环境中的TTX含量可以实现TTX的风险预警，可有效防范TTX中毒事件的发生。本文梳理了4类TTX的检测技术，分析比较了传统的生物检测法、化学检测法、免疫检测法之间的优势、不足与实际应用进展，介绍了基于适配体技术的新型检测技术的兴起、发展与广阔的应用前景，对生物安全领域中TTX风险的管理与控制有现实意义。     

杉木生长的起伏型时间序列模拟研究

起伏型时间序列法是一种新的时间序列分析法,本文介绍了起伏型时间序列林木模拟与预测方法,以杉木人工林生长为研究对象,对杉木胸径生长进行建模模拟,模拟工达到98．3％,结果令人满意,且比逐步回归法,多维时间序列分析法模拟效果更优,说明起伏型时间序列分析方法可应用于林木生长模拟与预测,从而丰富了林木生长预测与预报方法。     

Identifying kinetic gating mechanisms for ion channels by using two-dimensional distributions of simulated dwell times.

Ion channels are integral membrane proteins that regulate ionic flux through cell membranes by opening and closing (or gating) their pores. The gating can be monitored by observing step changes in the current flowing through single channels. Analysis of the durations of the open and closed intervals and of the correlations among the interval durations can give insight into the gating mechanism. Although it is well known that the correlation information can be essential to distinguish among possible gating mechanisms, it has been difficult to use this information because it has not been possible to correct the predicted correlations for the distortion of the single-channel data because of filtering and noise. To overcome this limitation we present a method based on a comparison of simulated and experimental two-dimensional dwell-time distributions constructed by analysing simulated and experimental single-channel currents in an identical manner. The simulated currents incorporate the true effects of filtering and noise, the two-dimensional distributions retain the correlation information, and the identical analysis allows direct maximum-likelihood comparison of the simulated and experimental two-dimensional distributions. We show that the two-dimensional simulation method has a greatly increased ability to distinguish among models, compared with methods that use one-dimensional distributions.     

Total Charge Movement per Channel : The Relation between Gating Charge Displacement and the Voltage Sensitivity of Activation

One measure of the voltage dependence of ion channel conductance is the amount of gating charge that moves during activation and vice versa. The limiting slope method, introduced by Almers (Almers, W. 1978. Rev. Physiol. Biochem. Pharmacol. 82:96–190), exploits the relationship of charge movement and voltage sensitivity, yielding a lower limit to the range of single channel gating charge displacement. In practice, the technique is plagued by low experimental resolution due to the requirement that the logarithmic voltage sensitivity of activation be measured at very low probabilities of opening. In addition, the linear sequential models to which the original theory was restricted needed to be expanded to accommodate the complexity of mechanisms available for the activation of channels. In this communication, we refine the theory by developing a relationship between the mean activation charge displacement (a measure of the voltage sensitivity of activation) and the gating charge displacement (the integral of gating current). We demonstrate that recording the equilibrium gating charge displacement as an adjunct to the limiting slope technique greatly improves accuracy under conditions where the plots of mean activation charge displacement and gross gating charge displacement versus voltage can be superimposed. We explore this relationship for a wide variety of channel models, which include those having a continuous density of states, nonsequential activation pathways, and subconductance states. We introduce new criteria for the appropriate use of the limiting slope procedure and provide a practical example of the theory applied to low resolution simulation data.     

Application of the ensemble nonequilibrium response spectroscopy to shaker potassium channel gating

Standard electrophysiology techniques study relaxation transients in voltage-gated ion channels generated by discrete voltage steps. The nonequilibrium response spectroscopy involves analyzing responses to fluctuating potentials. We apply the ensemble NRS method to gating kinetics of Shaker potassium ion channels. We evaluate various proposed Markov models of channel gating from the nonequilibrium response viewpoint. These new NRS protocols can be used to test otherwise indistinguishable models or improve estimates for parameters of channel kinetics models.     

Proton Countertransport and Coupled Gating in the Sarcoplasmic Reticulum Calcium Pump

The calcium pump of the sarcoplasmic reticulum (SERCA) is an ATP-driven active transporter of Ca²⁺ ions that functions via an “alternating-access” cycle mechanism. In each cycle, SERCA transports two Ca²⁺ ions toward the lumen of the sarcoplasmic reticulum and two to three protons to the cytoplasm. How the latter conformational transition is coupled to cytoplasmic release of protons remains poorly understood. The present computational study shows how the mechanism of proton countertransport is coupled to the alternating access gating process in SERCA. Molecular dynamics simulation trajectories are generated starting from a series of configurations taken along the E2 to E1 transition pathway determined by the string method with swarms-of-trajectories. Simulations of different protonation configurations at the binding sites reveal how deprotonation events affect the opening of the cytoplasmic gate. The results show that there is a strong coupling between the chronological order of deprotonation, the entry of water molecules into the TM region, and the opening of the cytoplasmic gate. Deprotonation of E309 and E771 is sequential with E309 being the first to lose the proton. The deprotonation promotes the opening of the cytoplasmic gate but leads to a productive gating transition only if it occurs after the transmembrane domain has reached an intermediate conformation. Deprotonation of E309 and E771 is unproductive when it occurs too early because it causes the re-opening of the luminal gate.     

Estimating kinetic parameters for single channels with simulation. A general method that resolves the missed event problem and accounts for noise.

Analysis of currents recorded from single channels is complicated by the limited time resolution (filtering) of the data which can prevent the detection of brief intervals. Although a number of approaches have been used to correct for the undetected intervals (missed events) when identifying kinetic models and estimating parameters, none of them provide a general method which takes into account the true effects of noise and limited time resolution. This paper presents such a method. The approach is to use simulated single-channel currents to incorporate the true effects of filtering and noise on missed events and interval durations. The simulated currents are then analyzed in a manner identical to that used to analyze the experimental currents. An iterative search process using likelihood comparison of two-dimensional dwell-time distributions obtained from the simulated and experimental single-channel currents then allows the most likely rate constants to be determined. The large errors and false solutions that can result from the more typically applied assumptions of no noise and an absolute dead time (idealized filtering) are excluded by the iterative simulation method, and the correlation information contained in the two-dimensional distributions should increase the ability to distinguish among different gating mechanisms. The iterative simulation method is generally applicable to channels which typically open to a single conductance level. For these channels the method places no restrictions on the proposed gating mechanism or the form of the predicted dwell-time distributions.     

PC12细胞钾离子通道门控动力学随机建模与参数估计(Ⅰ)

本文以PC12细胞钾离子通门控机理分析为例证,探索离子通道门控动力学马尔科夫模型的建模方法,并详述建模过程,为通过研究者提供启示与借鉴．在此基础上提出PC12细胞钾离子通道门控动力学的三个备择方案,然后试用Ball等人建议的适用于片膜中只含一个通道记录的极大似然算法估计速率参数,根据AIC及SC准则将模型作了比较,选择较适宜的模型刻划PC12细胞钾离子通道门控动力学．     

A note on modeling mechano-chemical transduction with an application to a skin receptor

Strain-sensitive (also called stretch-sensitive) ionic channels are thought to be present in various mechanoreceptors. The gating of these channels is precipitated by mechanical strains, as opposed to the usual activation processes of changes in membrane potential or other ligands. Below we present a class of models for the strain-activated mechanism, compare our approach to one of Sachs and Lecar (1991), and apply the gating mechanism to a model of a specific mechanoreceptor, namely a Pacinian corpuscle neurite model. The simulation experiment suggests the activation energy of the channel depends linearly, rather than nonlinearly, on the (hoop) strain in the receptor membrane.     

线性规划法在植物生态学数学模型参数估计中的应用初探

本文提出了一种植物生态学数学模型参数估计的新方法——线性规划法,并结合最小二乘法对模型参数的估计进行了实例分析。认为某种程度上前者更优于后者。是一种值得进一步研究的方法。     

Ion-channel gating mechanisms: model identification and parameter estimation from single channel recordings

Patch-clamp data may be analysed in terms of Markov process models of channel gating mechanisms. We present a maximum likelihood algorithm for estimation of gating parameters from records where only a single channel is present. Computer simulated data for three different models of agonist receptor gated channels are used to demonstrate the performance of the procedure. Full details of the implementation of the algorithm are given for an example gating mechanism. The effects of omission of brief openings and closings from the single-channel data on parameter estimation are explored. A strategy for discriminating between alternative possible gating models, based upon use of the Schwarz criterion, is described. Omission of brief events is shown not to lead to incorrect model identification, except in extreme circumstances. Finally, the algorithm is extended to include channel gating models exhibiting multiple conductance levels.     

Gating of MscL studied by steered molecular dynamics

Steered molecular dynamics simulations of the mechanosensitive channel of large conductance, MscL, were used to investigate how forces arising from membrane tension induce gating of the channel. A homology model of the closed form of MscL from Escherichia coli was subjected to external forces of 35-70 pN applied to residues near the membrane-water interface. The magnitude and location of these forces corresponded to those determined from the lateral pressure profile computed from a lipid bilayer simulation. A fully expanded state was obtained on the 10-ns timescale that revealed the mechanism for transducing membrane forces into channel opening. The expanded state agrees well with proposed models of MscL gating, in that it entails an irislike expansion of the pore accompanied by tilting of the transmembrane helices. The channel was most easily opened when force was applied predominantly on the cytoplasmic side of MscL. Comparison of simulations in which gating progressed to varying degrees identified residues that pose steric hindrance to channel opening.