分子拥挤条件下生物大分子性质研究

细胞中成千上万种生物分子以不同的浓度发挥着各自的生理作用,但是体外研究中常常忽略细胞内的拥挤环境,随着分子拥挤理论的提出,体外添加大分子拥挤试剂被越来越多的生物学家和化学家所重视,众多的研究结果显示分子拥挤试剂的加入对细胞内的生物大分子的性质造成了一定的影响。从分子拥挤试剂对蛋白质折叠与聚集以及核酸分子结构与性质的影响等方面探讨了拥挤环境下生物大分子的性质和功能,为以后大分子拥挤条件下生物分子的研究提供一定的参考依据。     

大分子拥挤对DNA结构的影响

大分子拥挤(macromolecular crowding effect)代表了细胞内高度拥挤状态,其源于非特异性容积排斥效应,是细胞内与pH、离子强度等同等重要的生理因素。生物大分子介导的拥挤环境对于DNA-DNA、DNA-蛋白质的相互作用以及DNA高级结构、细胞核或核区结构的稳定具有重要作用。在拥挤环境中,大分子总浓度的增加将增强溶质的浓缩倾向,从而降低溶液的自由能。拥挤效应是胞内大分子环境的总体反映,具有高度的缓冲性,保证了胞内反应的稳定进行及细胞功能的正常行使。     

超离心法及其在放射损伤研究中的应用

超离心机是研究高分子化合物如生物大分子蛋白质、核酸和病毒等的一个重要工具。在分析方面主要用以测定这些生物大分子的沉降系数与分子量。在制备方面可用以分离这些生物大分子和有关的亚细胞成分。在研究电离辐射对细胞原发损伤的规律时,观察射线对生物大分子的损伤和亚细胞成分的破坏,及其对生化功能的影响等问题,超离     

膜蛋白的表达：在无细胞体系中实现功能性表达、折叠和组装

膜蛋白在人类和其他物种的生命活动中都起着至关重要的作用.在已完成测序的基因组中,膜蛋白占据30%.药物作用靶向位点、细胞之间的信号传递以及对外界环境的探测等功能,大多是通过生物膜上的特殊膜受体蛋白实现的.膜蛋白研究在工业、环境、国防、医学等领域具有重大意义.嗅觉受体蛋白是一类典型的膜蛋白,属于G蛋白偶联受体家族.嗅觉受体蛋白调控着生物的食物寻找、危险趋避和求偶行为.其主要分布于脊椎动物鼻腔和昆虫触角.嗅觉受体蛋白可以直接识别气味分子,将生物信号转化为电信号,最终传递至神经中枢,进而做出相关应答.膜蛋白的获取并不容易.天然组织中的膜蛋白含量太低不足以支撑学术研究.异源表达难以实现膜蛋白整合上膜,这给膜蛋白的结构和功能研究带来很大挑战.无细胞蛋白质合成系统是一个开放体系,且不依赖细胞活性,是体外表达蛋白质的有效方法.通过无细胞蛋白合成体系,在体外实现膜蛋白二聚体的自组装,将为膜蛋白研究带来全新突破.本文总结了用于无细胞表达的膜蛋白研究进展.     

无细胞蛋白质合成系统的研究进展

无细胞蛋白质合成系统是一种以外源mRNA或DNA为模板 ,通过在细胞抽提物的酶系中补充底物和能源物质来合成蛋白质的体外系统 .与传统的体内重组表达系统相比 ,体外无细胞合成系统具有多种优点 ,如可表达对细胞有毒害作用或含有非天然氨基酸 (如D 氨基酸 )的特殊蛋白质 ,能够直接以PCR产物作为模板同时平行合成多种蛋白质 ,开展高通量药物筛选和蛋白质组学的研究等 .本文综述了无细胞蛋白质合成系统的发展历史、系统中合成蛋白质所需的能量供应、遗传模板的稳定性和微型无细胞生物反应器等多方面的研究 ,并探讨了无细胞蛋白质合成系统中存在的难点、研究方向和广泛的应用前景     

生物大分子从头合成和设计的研究进展

生物大分子指生物体内存在的DNA、蛋白质、多糖等物质,其对生物体正常生命活动至关重要.从头合成和设计技术在生物大分子的合成和结构设计上具有自由度高、前体简单等特点,能够按照特定研究目的对生物大分子进行全新设计和高效合成.近年来,从头合成与设计技术在人造基因组合成、新型蛋白质类药物设计、糖缀合物合成等领域已开始受到重视.基于生物大分子从头合成和设计技术,可以定向制备全新设计的DNA或全新的基因表达产物,以及具有识别功能的糖链或糖缀合物,将大大推进诸如细胞因子模拟物、基因治疗递送载体等生物活性物质的开发,为人工生物系统的构建、罕见疾病的治疗等提供新的解决方法.本文就DNA、蛋白质和多糖的从头合成和设计进行了综述,阐述了相关方法及应用,最后概括分析了三者之间的关系.     

激光拉曼光谱在蛋白质构象研究中的应用和进展

激光拉曼光谱法被公认为是研究生物大分子的结构、动力学和功能的有效方法。近年来拉曼光谱在蛋白质构象研究中的最新进展,涉及到拉曼光谱在非折叠蛋白质、蛋白质装配的特征描述,拉曼晶体学在实时监控蛋白质单晶中化学变化等方面的应用。另外,介绍了蛋白质拉曼光谱分析在生物技术中的应用现状。并对拉曼光谱技术在蛋白质等生物大分子领域中的研究前景做了进一步的展望。     

蛋白质检测的方法学研究概述

蛋白质作为生命活动中起重要作用的生物大分子,与一切揭开生命奥秘的重大研究课题都有密切的关系.蛋白质的分离与检测是蛋白质研究的主要技术之一.我们就蛋白质检测的常规方法、电化学检测方法、分子生物学检测方法、电泳法和质谱法等进行了简要综述.     

生物质谱技术在蛋白质组学研究中的应用

随着技术的进步,蛋白质组学的研究重心由最初旨在鉴定细胞或组织内基因组所表达的全部蛋白质转移到从整个蛋白质组水平上阐述包括蛋白翻译后修饰、生物大分子相互作用等反映蛋白质功能的层次。多种质谱离子化技术的突破使质谱技术成为蛋白质组学研究必不可少的手段。质谱技术联合蛋白质组学多角度、深层次探索生命系统分子本质成为现阶段生命科学研究领域的主旋律之一。本文简要综述了肽和蛋白质等生物大分子质谱分析的原理、方式和应用,并对其发展前景做出展望。     

Large cosolutes,small cosolutes,and dihydrofolate reductase activity

Protein enzymes are the main catalysts in the crowded and complex cellular interior, but their activity is almost always studied in dilute buffered solutions. Studies that attempt to recreate the cellular interior in vitro often utilize synthetic polymers as crowding agents. Here, we report the effects of the synthetic polymer cosolutes Ficoll, dextran, and polyvinylpyrrolidone, and their respective monomers, sucrose, glucose, and 1‐ethyl‐2‐pyrrolidone, on the activity of the 18‐kDa monomeric enzyme, Escherichia coli dihydrofolate reductase. At low concentrations, reductase activity increases relative to buffer and monomers, suggesting a macromolecular effect. However, the effect decreases at higher concentrations, approaching, and, in some cases, falling below buffer values. We also assessed activity in terms of volume occupancy, viscosity, and the overlap concentration (where polymers form an interwoven mesh). The trends vary with polymer family, but changes in activity are within threefold of buffer values. We also compiled and analyzed results from previous studies and conclude that alterations of steady‐state enzyme kinetics in solutions crowded with synthetic polymers are idiosyncratic with respect to the crowding agent and enzyme.     

Effects of molecular crowding by saccharides on alpha-chymotrypsin dimerization

Given the importance of protein complexes as therapeutic targets, it is necessary to understand the physical chemistry of these interactions under the crowded conditions that exist in cells. We have used sedimentation equilibrium to quantify the enhancement of the reversible homodimerization of alpha-chymotrypsin by high concentrations of the osmolytes glucose, sucrose, and raffinose. In an attempt to rationalize the osmolyte-mediated stabilization of the alpha-chymotrypsin homodimer, we have used models based on binding interactions (transfer-free energy analysis) and steric interactions (excluded volume theory) to predict the stabilization. Although transfer-free energy analysis predicts reasonably well the relatively small stabilization observed for complex formation between cytochrome c and cytochrome c peroxidase, as well as that between bobtail quail lysozyme and a monoclonal Fab fragment, it underestimates the sugar-mediated stabilization of the alpha-chymotrypsin dimer. Although predictions based on excluded volume theory overestimate the stabilization, it would seem that a major determinant in the observed stabilization of the alpha-chymotrypsin homodimer is the thermodynamic nonideality arising from molecular crowding by the three small sugars.     

Perception of residential crowding,classroom experiences,and student health

The present research is based on a typology of crowding experiences incorporating two main dimensions: neutral-personal thwartings and primary-secondary environments. The thwarting dimension concerns the degree to which crowding experiences are associated with spatial inconveniences, alone, or with spatial as well as social constraints. The environmental dimension relates to the type of setting in which crowding experiences occur. A major assumption of this typology is that crowding experiences involving social conflict will be more intense and disruptive to the individual than those in which interpersonal conflict is minimal. The reported study examined the relationship between college students' evaluations of the physical amenity, social climate, and crowdedness of their residential environments, on the one hand, and their sensitivity to crowding in a classroom situation, their academic performance, and the frequency of their visits to the campus health center, on the other. Results indicated that perceived residential crowding and negative perceptions of residential social climate were strongly associated with increased sensitivity to crowding in a classroom situation, impaired course performance, and visits to the student health center. The implications of these correlational findings for future field-experimental research are discussed.     

Local adaptation to developmental density does not lead to higher mating success in Drosophila melanogaster

In this study, we investigate the effect of local adaptation to developmental density on male mating success in laboratory populations of Drosophila melanogaster. Mating success is known to be influenced by body condition which can in turn be influenced by local adaptation. We test the hypothesis that males adapted to a given environment have higher mating success when assayed in that environment. We used males selected for adaptation to high larval density and their controls which are reared at low larval density. We grew assay males in low and high densities whereas the focal females (raised at low larval density) used for the experiment belonged to the common ancestor of selected and control populations. We considered selected males grown at high density and control males grown at low density as ‘adapted’. Similarly, we considered selected males grown at low density and control males grown at high density as ‘nonadapted’. Selected male belonging to a given treatment (larval density) was made to compete with control male of the same treatment for mating with ancestral female. We quantified components of reproductive fitness: mating latency, copulation duration, mating success and number of progeny sired by the ‘adapted’ and ‘nonadapted’ males. The results show that local adaptation does not lead to higher mating success in populations adapted to their own larval rearing environment.     

Protein self-association in the cell: a mechanism for fine tuning the level of macromolecular crowding?

A new role for protein self-association in the cell is discussed. An argument is advanced that when cellular protein is in its associated state the excluded volume component of the solution is minimized. Conversely, when cellular protein is in its dissociated state the excluded volume component of the solution is maximized. For proteins that make up a substantial fraction of the intracellular protein concentration, control of the self-association event thus presents itself as a means of regulating cellular processes that are influenced by different levels of volume exclusion. In this communication we examine how the control of protein association/dissociation might influence one such important process, namely the folding of a protein to a compact state.     

Optimal cytoplasmatic density and flux balance model under macromolecular crowding effects

Macromolecules occupy between 34% and 44% of the cell cytoplasm, about half the maximum packing density of spheres in three dimension. Yet, there is no clear understanding of what is special about this value. To address this fundamental question we investigate the effect of macromolecular crowding on cell metabolism. We develop a cell scale flux balance model capturing the main features of cell metabolism at different nutrient uptakes and macromolecular densities. Using this model we show there are two metabolic regimes at low and high nutrient uptakes. The latter regime is characterized by an optimal cytoplasmatic density where the increase of reaction rates by confinement and the decrease by diffusion slow-down balance. More important, the predicted optimal density is in the range of the experimentally determined density of Escherichia coli.     

Iwao’s patchiness regression through the origin: biological importance and efficiency of sampling applications

Iwao’s mean crowding-mean density relation can be treated both as a linear function describing the biological characteristics of a species at a population level, or a regression model fitted to empirical data (Iwao’s patchiness regression). In this latter form its parameters are commonly used to construct sampling plans for insect pests, which are characteristically patchily distributed or overdispersed. It is shown in this paper that modifying both the linear function and statistical model to force the intercept or lower functional limit through the origin results in more intuitive biological interpretation of parameters and better sampling economy. Firstly, forcing the function through the origin has the effect of ensuring that zero crowding occurs when zero individuals occupy a patch. Secondly, it ensures that negative values of the intercept, which do not yield an intuitive biological interpretation, will not arise. It is shown analytically that sequential sampling plans based on regression through the origin should be more efficient compared to plans based on conventional regression. For two overdispersed data sets, through-origin based plans collected a significantly lower sample size during validation than plans based on conventional regression, but the improvement in sampling efficiency was not large enough to be of practical benefit. No difference in sample size was observed when through-origin and conventional regression based plans were validated using underdispersed data. A field researcher wishing to adopt a through-origin form of Iwao’s regression for the biological reasons outlined above can therefore be confident that their sampling strategies will not be affected by doing so.     

第二恒磨牙正锁[牙合]畸形相关因素的研究

目的探讨引发下颌第二磨牙正锁(牙合)的相关因素,为口腔正畸临床诊断和治疗提供参考。方法随机抽取800例符合条件的病例作为研究对象。男349例,女451例,平均年龄21.7±3.31岁,其中45例发生第二磨牙正锁(牙合)。测量上颌后段牙弓间隙,下颌后段牙弓间隙,ANB角,上颌第一磨牙区和上颌第二磨牙区的牙槽弓宽度和下颌第一磨牙区和下颌第二磨牙区的牙槽弓宽度,对测量值进行统计分析。结果第二磨牙正锁(牙合)的发生率为5.6%,其性别构成差异无显著性(P>0.05)。回归方程:第二磨牙正锁(牙合)=22.9748 2.0257×上颌后段牙弓拥挤度 1.5372×下颌后段牙弓拥挤度 1.0071×ANB角度 0.3785×上颌第二磨牙处牙槽弓宽度-0.4897×下颌第二磨牙处牙槽弓宽度。结论第二磨牙正(牙合)与上颌后段牙弓拥挤有显著的相关关系,与下颌后段牙弓拥挤度、ANB角度和上颌第二磨牙处牙槽弓宽度呈正相关关系,与下颌第二磨牙处的牙槽弓宽度呈负相关关系。