温压结合的超高压技术对虾原肌球蛋白抗原性与结构的影响

近年来食源性虾过敏的事件时有发生。为深入研究超高压技术改变虾类致敏性的机理,从凡纳滨对虾中分离纯化出最主要过敏原原肌球蛋白,利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)对原肌球蛋白进行了鉴定,并运用间接酶联免疫吸附法(间接ELISA)和圆二色光谱法(CD)以及荧光光谱法分析了温压结合的超高压技术中温度因素对原肌球蛋白抗原性与结构的影响。结果表明,原肌球蛋白在300 MPa 15 min处理条件下,经35与45 ℃处理,其抗原性增强,而经55,65与75 ℃处理,其抗原性降低。原肌球蛋白二级结构随温度上升,出现α-螺旋与β-折叠、β-转角以及无规则卷曲结构之间的转换;三级结构发生了由伸展状态到折叠状态,再有部分伸展而后折叠的动态变化。由此推断,温压结合的超高压技术中通过调节温度条件,可引起虾原肌球蛋白构象发生变化,进而影响其抗原性的改变。研究结果对于开发低敏性虾制品的新方法、新工艺具有理论指导意义。     

序列脉冲激光在不同大气模型下的热晕效应研究

考虑到大气中吸收系数随高度变化,通过内插法得到不同大气模型一定高度下大气的吸收系数,建立序列脉冲激光在地对空垂直传输的热晕模型。通过数值计算方法分析风场渡越时间内脉冲数为2时的序列脉冲在中纬度地区夏季和冬季、热带地区、近北极地区夏季和冬季5种大气模型下的热晕效应。结果表明:当光束孔径为0.25 m,初始功率为200 kW,波形为高斯状的激光束在近北极冬季传输10 km后沿横轴方向光强峰值偏移0.056 9 m,而热带光强峰值强度偏移最远为0.224 m,为近北极冬季时的4倍;脉冲激光在热带传输10 km后的靶面功率仅为15.06 kW,近北极冬季靶面功率为热带的10.5倍。热带地区的热晕效应最明显,中纬度地区次之,近北极地区最弱。激光在夏季传输要比冬季传输的热晕效应严重,而且近北极地区夏季要比中纬度地区冬季的热晕效应更严重。     

利用单粒子追踪技术研究活细胞内肌球蛋白的运输模式

利用单个粒子追踪技术研究?1A肾上腺素受体在活细胞内的转运．?1A肾上腺素受体在细胞内沿微丝骨架的转运表现为步进式模式，平均步长与体外对肌球蛋白的研究相一致．对不同时间分辨率下数据的模拟分析结果表明，肌球蛋白在细胞内运输内涵体的单步动力学与单个肌球蛋白在体外的报道相一致．     

肌球蛋白Ⅵ分子马达周期势场下的弹性扩散模型

肌球蛋白Ⅵ分子马达因其特殊的结构及胞内功能,其动力学原理成为研究的热点. 从肌球蛋白Ⅵ自身结构和实验现象出发,建立其弹性扩散模型,并通过Monte Carlo方法分析了肌球蛋白Ⅵ满足朗之万方程的随机动力学行为. 结果表明,在环境噪声作用下,具有弹性势能和轨道周期势能的肌球蛋白Ⅵ可以进行梯跳运动和有效的输运,但负载力会减弱分子马达系统的输运能力;当弹性系数一定时,弹性链越长平均速度越小,当弹性链长度一定时,合理选择弹性系数平均速度可达到最大值;另外,负载力的存在使肌球蛋白Ⅵ在接触位点的平均驻留时间呈指数增加. 关键词： 分子马达 肌球蛋白Ⅵ 朗之万方程 弹性扩散模型     

干风冷水型转轮空调系统全寿命特性研究

干风冷水型转轮空调系统是一种新型的转轮除湿空调系统,它基于吸附除湿和再生式蒸发冷却技术能够在夏季同时输出干空气和较低温度的冷冻水,克服了现有转轮除湿空调显热处理能力的不足。本文采用全寿命周期研究方法,对此种新型转轮空调系统在上海夏季和冬季运行的模式下进行全寿命的能耗分析、全寿命经济分析及碳信用分析,并与传统蒸汽压缩式空调系统进行对比。     

动态瞬时高压作用对膳食纤维酶解速度的影响

 对纤维素酶对动态瞬时高压处理后膳食纤维的酶解速度进行了研究。豆渣膳食纤维（Dietary Fiber,DF）经微射流均质机的瞬时高压作用（Instantaneous High Pressure,IHP）,在不同的压力以及同一压力的不同作用次数下产生不同粒径和密度,它们对应的酶解速度是不同的。纤维素酶水解膳食纤维产生纤维二糖和葡萄糖等还原糖,通过测定水解后还原糖的含量判断不同粒径和密度的膳食纤维的酶解速度。结果表明,在40～90 MPa的压力范围内随压力的增大物料粒径呈显著下降,物料密度随之增大,酶解速度增大,在90 MPa的均质压力下物料粒径达到最小,为202.4 nm;当压力继续增大时,物料因膨化作用,物料粒径呈增大趋势,但其密度开始减小,酶解速度继续增大,在140 MPa的均质压力下物料密度达到最小,为1.027 g/mL,此时酶解后还原糖含量最高,酶解速度最快。压力超过140 MPa后,由于超微颗粒间的团聚,物料的密度和粒径均增大,酶解速度减小。在90 MPa和140 MPa下对分别对物料进行多次的瞬时高压处理,发现随处理次数的增多物料粒径增大,体系密度先减小后增大,酶解速度减小。     

新能源汽车空调负荷特性的研究

通过热平衡法对新能源汽车的稳态负荷特性进行了计算研究,对比了不同季节和不同行驶工况下负荷的变化,并得出了影响负荷的主要因素。结果表明:在研究工况下,随着车速增加,春季、夏季和秋季车身总负荷逐渐减少,而冬季车身总负荷则逐渐增加;春季主要负荷为乘员人体散发的总热量,其占比为32.9%;夏季主要负荷为门窗玻璃传入的总的热量,其占比为26.23%;秋季主要负荷为乘员人体散发的总热量,其占比为30.88%;而冬季主要负荷为新风所传入的总热量,其占比为47.16%。此外,为了改善整车负荷,针对各季节主要占比的负荷,提出了相应的优化方法。     

串联酶解耦合超微粉碎法对蔗渣酶解的促进作用

报道了一种新颖的串联酶解(先酶I后酶II)耦合超微粉碎方法,并系统研究了其对蔗渣酶水解的促进作用. 通过对三种酶解方法(单酶I、单酶II、先酶I后酶II) 的比较发现,串联酶解对蔗渣酶解最为有效. 超微粉碎能破坏蔗渣细胞结构,使纤维素充分暴露出来便于酶解.在串联酶解耦合超微粉碎模式下,蔗渣在反应温度50 oC,pH=4.8,酶I(7.5 FPU/g底物)和酶II(5.0 FPU/g底物),摇床速率1200 r/min条件下反应72 h后,酶水解能得到65%的还原糖浓度,其中葡萄糖选择性为90.1%.     

全内反射双通道观察双标记荧光染色的非洲绿猴肾细胞

在像增强型电荷耦合器件(ICCD)荧光显微成像装置上用双通道成像方法观察了非洲绿猴肾细胞(COS-7)中由EGFP转染的肌球蛋白Myosin 15a,以及Rhodamine标记的细胞微丝。为观察微丝尖端的肌球蛋白Myosin 15a,采用了高灵敏、低损伤的全内反射激发荧光显微成像技术,并在双通道中选用合适滤光片组合消除两种荧光染料间的光谱串扰。实验观测到非洲绿猴肾细胞内过表达的肌球蛋白Myosin 15a和伸长的微丝的分布情况,尤其是清晰观察到Myosin 15a在微丝上的分布。为全内反射双通道荧光成像技术在生命科学中的应用展示了广阔的前景。     

高压和酶解对热变性大米蛋白溶解及结构特征的影响(英文)

研究了100MPa高压和酶解处理对热变性大米蛋白溶解性和分子结构特征的影响。结果显示:高压处理后再用Alcalase(碱性蛋白酶)酶解可使大米蛋白的溶解性由单纯酶解时的58.9%提高到75.33%;SE-HPLC(高效液相凝胶色谱法)分析显示,高压处理可使57~105ku的大分子蛋白质溶出,且随着酶解时间延长,该组分消失,4.4和2.0ku组分含量增加,而非高压处理者没有大分子的溶解;FTIR(傅里叶红外光谱)分析显示,高压处理的样品中β-折叠和β-转角结构占主导地位;SEM(扫描电子显微镜)分析表明,高压处理使致密的大米蛋白体结构变得疏松。以上结果表明,高压处理改变了蛋白的空间结构,进而改变蛋白的酶解位点,从而提高了大米蛋白的溶解性。     

Muscle‘s Motion in an Overdamped Regime

Based on the stochastic inclined rods model proposed by H.Matsuura et al., we study the motion of actin myosin system in an overdamped regime.Our model is composed of an inclined spring (rod),a myosin head and a myosin filament.The results of calculation show that the model can convert the random motion to one-directional motion,and the myosin head works as a resonator of random noise,which absorbs the energy through a stochastic resonance.The results show that the inclined rod and the intermolecular potential are very important for the system to move.     

Quenching of Myosin Intrinsic Fluorescence Unravels the Existence of a High Affinity Binding Site for Decavanadate

Decavanadate, one of the aggregated species of vanadate, is a potent inhibitor of several enzymes, including skeletal muscle myosin. However, its putative binding sites in myosin are largely unknown. Titration of the intrinsic fluorescence of myosin, purified from rabbit skeletal muscle, have been carried out in 0.3 M KCl, 5 mM CaCl₂ and 25 mM Tris-HCl (pH 7.0), with 0.1 mg/ml myosin. In the 0-200 M total vanadate concentration range, decavanadate produced approximately 25% quenching of the intrinsic fluorescence of myosin, with an apparent dissociation constant in the micromolar range. This effect was found to be specific of decavanadate, because titration with metavanadate up to 200 M did not produce a significant quenching of the intrinsic fluorescence of myosin. This quenching was accompanied by a parallel decrease of the accessibility of myosin tryptophans to the water-soluble collisional quencher KI, with an apparent dissociation constant also in the micromolar range. It is concluded that the binding of decavanadate to high-affinity sites in myosin produces local conformational change(s) near the tryptophans more accessible to water in the three-dimensional structure of this protein.     

分子马达与布朗马达

综述了有关分子马达,主要是肌球蛋白马达和动蛋白马达的实验研究进展情况,并对理论模型,特别是近年来广为流行的布朗马达模型作了介绍和评论。最后展望了这一领域的发展前景及其所面临的挑战性问题。     

Model for processive movement of myosin V and myosin VI

Myosin V and myosin VI are two classes of two-headed molecular motors of the myosin superfamily that move processively along helical actin filaments in opposite directions. Here we present a hand-over-hand model for their processive movements. In the model, the moving direction of a dimeric molecular motor is automatically determined by the relative orientation between its two heads at free state and its head‘s binding orientation on track filament.This determines that myosin V moves toward the barbed end and myosin VI moves toward the pointed end of actin.During the moving period in one step, one head remains bound to actin for myosin V whereas two heads are detached for myosin VI: the moving manner is determined by the length of neck domain. This naturally explains the similar dynamic behaviours but opposite moving directions of myosin VI and mutant myosin V (the neck of which is truncated to only one-sixth of the native length). Because of different moving manners, myosin VI and mutant myosin V exhibit significantly broader step-size distribution than native myosin V. However, all the three motors give the same mean step size of -36nm (the pseudo-repeat of actin helix). All these theoretical results are in agreement with previous experimental ones.     

Myostatin preferentially down-regulates the expression of fast 2x myosin heavy chain in cattle

Myostatin is involved in an inhibitor of muscular growth and differentiation. Myoblasts derived from double-muscled Japanese shorthorn cattle (DM myoblasts) with absence of functional myostatin had higher abilities to proliferate and differentiate than myoblasts derived from normal-muscled cattle (NM myoblasts). In DM myoblasts, mRNA expressions of fetal myosin heavy chain (MyHC) in growth medium and of fast 2a and 2x MyHC in fusion medium were significantly greater than that in NM myoblasts. No significant difference existed in expressions of embryonic and slow MyHC mRNA between DM and NM myoblasts. The expression of MyoD mRNA was suppressed in myoblasts by administration of myostatin. Two cloned DM myoblast strains (DMc) were established. Addition of myostatin for DMc resulted in less myotube formation and suppression of mRNA expression of fast 2x MyHC. These findings suggest that the endogenous myostatin preferentially down-regulates the expression of the fast 2x MyHC and participates in differentiation of myofiber types during early bovine myogenesis.     

Simultaneous Measurement of Individual ATPase and Mechanical Reactions by a Single Myosin Molecule at Work

Based on techniques for single molecule imaging and nanomanipulation by optical tweezers, we have developed a new technique that allows simultaneous measurement of individual ATPase and mechanical reactions from a single myosin molecule during force generation. We show how the ATPase reaction couples to the mechanical reaction directly at the single molecule level. The results show that the myosin head can produce force even after releasing the bound nucleotide, probably ADP, suggesting that the chemical energy driven by ATP hydrolysis can be hysteretically stored in the myosin molecule. This view does not support a widely accepted hypothesis in which the force generation is tightly coupled to ligand dissociation.This paper was originally presented as an invited paper at the seventh Meeting on Near Field Optics, which was held on July 1, 1998 at Nagoya University, Nagoya, organized by the Research Group on Near Field Optics of the Optical Society of Japan, an affiliate of the Japan Society of Applied Physics.     

Characteristics of light chains of Chara myosin revealed by immunological investigation

Chara myosin is plant myosin responsible for cytoplasmic streaming and moves actin filaments at 60 µm/s, which is the fastest of all myosins examined. The neck of the myosin molecule has usually mechanical and regulatory roles. The neck of Chara myosin is supposed to bind six light chains, but, at present, we have no knowledge about them. We found Ca⁺⁺-calmodulin activated Chara myosin motility and its actin-activated ATPase, and actually bound with the Chara myosin heavy chain, indicating calmodulin might be one of candidates for Chara myosin light chains. Antibody against essential light chain from Physarum myosin, and antibodies against Chara calmodulin and chicken myosin light chain from lens membranes reacted with 20 kDa and 18 kDa polypeptides of Chara myosin preparation, respectively. Correspondingly, column purified Chara myosin had light chains of 20 kDa, and 18 kDa with the molar ratio of 0.7 and 2.5 to the heavy chain, respectively.     

Motor proteins transporting cargos

Processive motor proteins such as kinesin and myosin-V are enzymes that use the energy of ATP hydrolysis to travel along polar cytoskeletal filaments. One of the functions of these proteins is the transport of vesicles and protein complexes that are linked to the light chains of the motors. Modeling the light chain by a linear elastic spring, and using the two-state model for one- and two-headed molecular motors, we study the influence of thermal fluctuations of the cargo on the motion of the motor-cargo complex. We solve numerically the Fokker-Planck equations of motor motion, and find that the mean velocity of the motor-cargo complex decreases monotonously as the spring becomes softer. This effect is due to the random force of thermal fluctuations of the cargo disrupting the operation of the motor. Increasing the size (thus, the friction coefficient) of the cargo also decreases the velocity. Surprisingly, we find that for a given size of the cargo, the velocity has a maximum for a certain friction of the motor. We explain this effect by the interplay between the characteristic length of thermal fluctuations of the cargo on a spring, the motor diffusion length, and the filament period. Our results may be relevant for the interpretation of single-molecule experiments with molecular motors (bead assays), where the motor motion is observed by tracking of a bead attached to the motor.     

Dynamics and mechanics of motor-filament systems

Motivated by the cytoskeleton of eukaryotic cells, we develop a general framework for describing the large-scale dynamics of an active filament network. In the cytoskeleton, active cross-links are formed by motor proteins that are able to induce relative motion between filaments. Starting from pair-wise interactions of filaments via such active processes, our framework is based on momentum conservation and an analysis of the momentum flux. This allows us to calculate the stresses in the filament network generated by the action of motor proteins. We derive effective theories for the filament dynamics which can be related to continuum theories of active polar gels. As an example, we discuss the stability of homogenous isotropic filament distributions in two spatial dimensions.     

Collective alignment of polar filaments by molecular motors

We study the alignment of polar biofilaments, such as microtubules and actin, subject to the action of multiple molecular motors attached simultaneously to more than one filament. Focusing on a paradigm model of only two filaments interacting with multiple motors, we were able to investigate in detail the alignment dynamics. While almost no alignment occurs in the case of a single motor, the filaments become rapidly aligned due to the collective action of the motors. Our analysis shows that the alignment time is governed by the number of bound motors and the magnitude of the motors’ stepping fluctuations. We predict that the time scale of alignment is in the order of seconds, much faster than that reported for passive crosslink-induced bundling. In vitro experiments on the alignment of microtubules by multiple-motor covered beads are in qualitative agreement. We also discuss another mode of fast alignment of filaments, namely the cooperation between motors and passive crosslinks.