Heterodimerization of integrin Mac-1 subunits studied by single-molecule imaging

Heterodimerization of integrin Mac-1 (alpha(M) beta(2)) Subunits plays important role on regulating leukocytes adhesion to extracellular matrix or endothelial cells. Here, using total internal reflection microscopy, we investigated the heterodimerization of integrin Mac-1 subunits at the single-molecule level in live cells. Individual alpha(M) subunit fused to the enhanced yellow fluorescent protein (eYFP) was imaged at the basal plasma membrane of live Chinese hamster ovary (CHO) cells. Through analysis of mean square displacement (MSD), diffusion coefficient, the size of restricted domain and fraction of molecules undergoing restricted diffusion, we found that as compared with the diffusion in the absence of beta(2) subunit, the diffusion of single-molecule of alpha(M)-YFP was suppressed significantly in the presence of beta(2) subunit. Thus, based on the oligomerization-induced trapping model, we suggested that in the presence of beta(2) subunit, the am subunit may form heterodimer with it. (C) 2008 Elsevier Inc. All rights reserved.

Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to beta(2) subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively. (c) 2006 Elsevier Inc. All rights reserved.

Detection of constitutive homomeric associations of the integrins Mac-1 subunits by fluorescence resonance energy transfer in living cells

Integrins alpha(M)beta(2) plays important role on leukocytes, such as adhesion, migration, phagocytosis, and apoptosis. It was hypothesized that homomeric associations of integrin subunits provide a driving force for integrins activation, and simultaneously inducing the formation of integrins clusters. However, experimental reports on homomeric associations between integrin subunits are still controversial. Here, we proved the homomeric associations of the isolated Mac-1 subunits in living cells using three-channel fluorescence resonance energy transfer (FRET) microscopy and FRET spectra methods. We found that the extent of homomeric associations between beta(2) subunits is higher than alpha(M) subunits. Furthermore, FRET imaging indicated that the extent of homomeric associations of the Mac-1 subunits is higher along the plasma membrane than in the cytoplasm. Finally, we suggested that homomeric associations of the transmernbrane domains or/and cytoplasmic domains may provide the driving force for the formation of constitutive homomeric associations between alpha(M) or beta(2) subunits. (c) 2006 Elsevier Inc. All rights reserved.

Interaction between single molecules of Mac-1 and ICAM-1 in living cells: An atomic force microscopy study

The interaction between integrin macrophage differentiation antigen associated with complement three receptor function (Mac-1) and intercellular adhesion molecule-1 (ICAM-1), which is controlled tightly by the ligand-binding activity of Mac-1, is central to the regulation of neutrophil adhesion in host defense. Several "inside-out" signals and extracellular metal ions or antibodies have been found to activate Mac-1, resulting in an increased adhesiveness of Mac-1 to its ligands. However, the molecular basis for Mac-1 activation is not well understood yet. In this work, we have carried out a single-molecule study of Mac-1/ICAM-1 interaction force in living cells by atomic force microscopy (AFM). Our results showed that the binding probability and adhesion force of Mac-1 with ICAM-1 increased upon Mac-1 activation. Moreover, by comparing the dynamic force spectra of different Mac-1 mutants, we expected that Mac-1 activation is governed by the downward movement of its alpha 7 helix. (c) 2007 Elsevier Inc. All rights reserved.

RBAC与MAC在多级关系数据库中的综合模型

多级安全数据库的安全策略需要各种模型来表达,访问控制模型是其中之一.强制访问控制(MAC)模型保证多级数据库中的信息流动符合系统的安全策略.利用基于角色的访问控制(RBAC)来实现MAC能方便多级安全数据库的权限管理.提出了一种MAC与RBAC的综合模型,定义了多级角色与内部角色的概念,并给出了综合模型中经过修改后的操作,使得系统能自动地完成符合强制访问控制策略的用户权限的管理.该模型方便了管理员的权限管理,适合用户较多,安全层次比较复杂的多级关系数据库系统.最后给出了模型的部分实现机制.

XOR-MAC消息认证码的安全性新证明

消息认证码(Message Authentication Codes,MACs)是保证消息完整性的重要工具.Bellare等人提出了称为XOR-MAC的消息认证码,界定了攻击者成功伪造的概率,从而证明了其安全性,但是他们给出的证明方法较为复杂.本文使用Game-Playing技术采用新的安全性定义证明了XOR-MAC的安全性,证明方法简单明了;在底层所使用的分组密码是伪随机置换的假设下,量化了该消息认证码与随机函数之间区分的概率.

Two-Dimensional Kinetics Of Beta(2)-Integrin And Icam-1 Bindings Between Neutrophils And Melanoma Cells In A Shear Flow

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between beta(2)-integrin ( lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of beta(2)-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of beta(2)-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.

Bingham流体与固壁、液面斜撞击的数值模拟

在交错网格中用MAC方法求解二维不可压N S方程 ,对圆截面液柱与固壁、液面斜撞击问题进行了数值模拟 ,得到了自由面随时间演化的图像。主要考察了 :(1)固壁和液面 ;(2 )牛顿、非牛顿流体 ;(3)碰撞入射角θ ;(4 )Bingham流体近似本构式中参数q0 、K对计算结果的影响。结果表明 :液柱与液面碰撞形成的自由面更复杂。碰撞初期 ,Bingham流体和水的自由面相似 ;但碰撞后期 ,Bingham流体的自由面相对简单。θ对液柱与液面碰撞自由面的影响较大。本文条件下 ,当K >2 0时 ,K对自由面的影响不大 ;当q0 增大时 ,自由面变得相对简单

泥浆与液面、阻挡墙撞击的二维数值模拟(英文)

在交错网格中 ,用MAC方法求解非牛顿流体的二维不可压N S方程 .论文分别对圆柱形泥浆撞击液面、泥浆溃坝波撞击阻挡墙问题进行了数值模拟 ,并与水的相应结果作了对照 .结果表明 :当与液面撞击时 ,水和泥浆自由面随时间的演化图象与物理上的定性分析结果是一致的 ;由于本构关系不同 ,两者的侵彻自由面和液面运动均存在较大差异 .当水和泥浆与阻挡墙撞击时 ,水的自由面变化要比泥浆复杂得多 .在相同的时间内 ,左、右挡墙所受的冲击力和冲击次数也不相同

化学反应流中分子扩散的二点封闭MCA模型

在化学反应流的概率密度函数(PDF)方法中，对流项和化学反应项都是封闭的，但分子扩散项必须模拟．现有的分子扩散模型都是唯象的，需要引入外加参数，并难以通过一些基本物理过程的检验．本文发展了随机映射逼近(mapping closure approximation，MCA)方法，解析地从控制方程导出一个封闭的分子扩散模型．该方法考虑两点联合概率密度函数方程，引入空间特征尺度，因此解决了以往映射封闭方法中分子扩散速率无法确定的问题．数值模拟表明该方法能用于预测标量扩散的速度，以及概率密度函数和条件平均扩散等统计量．