F nuclear magnetic resonance screening of glucokinase activators

Human hexokinase enzyme IV (EC 2.7.1.1) catalyzes the phosphorylation of glucose and regulates the level of glucose. This enzyme exhibits strong positive cooperativity due to an allosteric transition between an inactive form and a closed active form. This form can be stabilized by activators and, thus, can increase its turnover by a kinetic memory effect characterized by a slow decay to the inactive state. The structural details of this kinetic allostery are known. Several synthetic activators have been reported. We present a preliminary nuclear magnetic resonance (NMR) screening of a chemical library in search of molecules with some affinity for glucokinase (GK). The library, composed of eight molecules with known activity as well as molecules that display no interaction, has been tested using the FAXS (fluorine chemical shift anisotropy and exchange for screening) method, based on monitoring the R₂ relaxation of the ¹⁹F spin. To ensure a valid interaction measurement, the enzyme was placed in the presence of glucose and magnesium. The binding signal of one known fluorinated ligand was measured by determining the displacement of the known ligand. This simple measure of the ¹⁹F signal intensity after an 80-ms spin echo correlates nicely with the EC₅₀, opening a route for NMR screening of GK activators.     

Perinatal essential fatty acid deficiency influences body weight and bone parameters in adult male rats

Electropermeabilization designates the use of electric pulses to overcome the barrier of the cell membrane. This physical method is used to transfer anticancer drugs or genes into living cells. Its mechanism remains to be elucidated. A position-dependent modulation of the membrane potential difference is induced, leading to a transient and reversible local membrane alteration. Electropermeabilization allows a fast exchange of small hydrophilic molecules across the membrane. It occurs at the positions of the cell facing the two electrodes on an asymmetrical way. In the case of DNA transfer, a complex process is present, involving a key step of electrophoretically driven association of DNA only with the destabilized membrane facing the cathode. We report here at the membrane level, by using fluorescence microscopy, the visualization of the effect of the polarity and the orientation of electric pulses on membrane permeabilization and gene transfer. Membrane permeabilization depends on electric field orientation. Moreover, at a given electric field orientation, it becomes symmetrical for pulses of reversed polarities. The area of cell membrane where DNA interacts is increased by applying electric pulses with different orientations and polarities, leading to an increase in gene expression. Interestingly, under reversed polarity conditions, part of the DNA associated with the membrane can be removed, showing some evidence for two states of DNA in interaction with the membrane: DNA reversibly associated and DNA irreversibly inserted.     

All signaling is local?

The mechanism of signal transmission following ligand stimulation of receptor tyrosine kinases in living cells is poorly understood. Recent studies have visualized the spatio-temporal pattern of EGF signaling, indicating that receptor density is an important factor in the mechanism of lateral propagation of local EGF signaling.     

Protein kinase‐mediated signalling in priming: Immune signal initiation,propagation, and establishment of long‐term pathogen resistance in plants

“Priming” in plant phytopathology describes a phenomenon where the “experience” of primary infection by microbial pathogens leads to enhanced and beneficial protection of the plant against secondary infection. The plant is able to establish an immune memory, a state of systemic acquired resistance (SAR), in which the information of “having been attacked” is integrated with the action of “being prepared to defend when it happens again.” Accordingly, primed plants are often characterized by faster and stronger activation of immune reactions that ultimately result in a reduction of pathogen spread and growth. Prerequisites for SAR are (a) the initiation of immune signalling subsequent to pathogen recognition, (b) a rapid defence signal propagation from a primary infected local site to uninfected distal parts of the plant, and (c) a switch into an immune signal‐dependent establishment and subsequent long‐lasting maintenance of phytohormone salicylic acid‐based systemic immunity. Here, we provide a summary on protein kinases that contribute to these three conceptual aspects of “priming” in plant phytopathology, complemented by data addressing the role of protein kinases crucial for immune signal initiation also for signal propagation and SAR.     

Release of acetylcholinesterase (AChE) from beta-amyloid plaques assemblies improves the spatial memory impairments in APP-transgenic mice

The major protein constituent of amyloid deposits in Alzheimer's disease (AD) is the amyloid-beta-peptide (Abeta). Amyloid deposits contain "chaperone molecules" which play critical roles in amyloid formation and toxicity. In the present work, we test an analog of hyperforin (IDN 5706) which releases the AChE from both the Abeta fibrils and the AChE-Abeta burdens in transgenic mice. Hyperforin is an acylphloroglucinol compound isolated from Hypericum perforatum (St. John's Wort), which is able to prevent the Abeta-induced spatial memory impairments and Abeta neurotoxicity. Altogether this gathered evidence indicates the important role of AChE in the neurotoxicity of Abeta plaques and finding new compounds which decrease the AChE-Abeta interaction may be a putative therapeutic agent to fight the disease.     

The liver fatty acid binding protein - comparison of cavity properties of intracellular lipid-binding proteins

The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common -barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited portal region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound.     

Alternate costimulatory molecules in T cell activation: differential mechanisms for directing the immune response

T cells are required for an effective immune response against a wide range of pathogens and for the generation of immunological memory. T cell activation can be divided into two phases: an antigen-specific signal delivered through the T cell antigen receptor, and a costimulatory signal delivered through accessory molecules on the T cell surface. Following activation, T cells differentiate to acquire distinct effector functions depending on the costimulatory signal, cytokine environment, and the pathogen itself. Although CD28 has been identified as the dominant costimulatory molecule, several other molecules have been described as having a costimulatory function. This review will focus on recent evidence for the existence of alternate costimulatory molecules, and the differential roles they might play in the activation, development, and survival of T cells.     

"Chemical analogues" of HLA-DM can induce a peptide-receptive state in HLA-DR molecules

We had recently identified small molecular compounds that are able to accelerate the ligand exchange reactions of HLA-DR molecules. Here we show that this acceleration is due to the induction of a "peptide-receptive" state. Dissociation experiments of soluble HLA-DR2.CLIP (class II-associated invariant chain peptide) complex and peptide-binding studies with "nonreceptive" empty HLA-DR1 and -DR2 molecules revealed that the presence of a small phenolic compound carrying an H-bond donor group (-OH) results in the drastic increase of both off- and on-rates. The rate-limiting step for ligand exchange, the transition of the major histocompatibility complex molecule from a nonreceptive into the receptive state, is normally mediated by interaction with the chaperone HLA-DM. In this respect, the effect of small molecules resembles that of the natural catalyst, except that they are still active at neutral pH. These "chemical analogues" of HLA-DM can therefore modulate the response of CD4+ T cells by editing the antigen composition of surface-bound class II major histocompatibility complex on living antigen-presenting cells.     

Plant memory: a tentative model

All memory functions have molecular bases, namely in signal reception and transduction, and in storage and recall of information. Thus, at all levels of organisation living organisms have some kind of memory. In plants one may distinguish two types. There are linear pathways from reception of signals and propagation of effectors to a type of memory that may be described by terms such as learning, habituation or priming. There is a storage and recall memory based on a complex network of elements with a high degree of integration and feedback. The most important elements envisaged are calcium waves, epigenetic modifications of DNA and histones, and regulation of timing via a biological clock. Experiments are described that document the occurrence of the two sorts of memory and which show how they can be distinguished. A schematic model of plant memory is derived as emergent from integration of the various modules. Possessing the two forms of memory supports the fitness of plants in response to environmental stimuli and stress.     

Study of water-protein interaction by high resolution NMR

The interaction between carbonic anhydrase B in the molten globule state and water molecules was studied by high-resolution NMR spectroscopy. NMR spin diffusion experiments revealed spin diffusion propagation from the protein to waters. This is a process of complex bioexponential kinetics presented in spin diffusion spectra as a change in water signal intensity dependent on the post-excitation time of protein molecules. Its reverse, spin diffusion propagation from waters to the protein, was also found. These phenomena are protein concentration- and temperature-dependent and shown to be possibly explained with the assumption that there exist water-protein complexes provoking the formation of large branched associations. At a temperature above 309 K, a stepwise increase in the interaction between waters and proteins occurs in these complexes. The formation of water-protein associations is induced by increasing temperature and/or protein concentration. In these associations, at normal temperature, the protein mobility is close to that of carbonic anhydrase B dimers.     

Ligand‐induced formation of transient dimers of mammalian 12/15‐lipoxygenase: A key to allosteric behavior of this class of enzymes?

Mammalian lipoxygenases (LOXs) have been implicated in cellular defense response and are important for physiological homeostasis. Since their discovery, LOXs have been believed to function as monomeric enzymes that exhibit allosteric properties. In aqueous solutions, the rabbit 12/15-LOX is mainly present as hydrated monomer but changes in the local physiochemical environment suggested a monomer-dimer equilibrium. Because the allosteric character of the enzyme can hardly be explained using a single ligand binding-site model, we proposed that the binding of allosteric effectors may shift the monomer-dimer equilibrium toward dimer formation. To test this hypothesis, we explored the impact of an allosteric effector [13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid] on the structural properties of rabbit 12/15-LOX by small-angle X-ray scattering. Our data indicate that the enzyme undergoes ligand-induced dimerization in aqueous solution, and molecular dynamics simulations suggested that LOX dimers may be stable in the presence of substrate fatty acids. These data provide direct structural evidence for the existence of LOX dimers, where two noncovalently linked enzyme molecules might work in unison and, therefore, such mode of association might be related to the allosteric character of 12/15-LOX. Introduction of negatively charged residues (W181E + H585E and L183E + L192E) at the intermonomer interface disturbs the hydrophobic dimer interaction of the wild-type LOX, and this structural alteration may lead to functional distortion of mutant enzymes.     

Fluctuations and Fractal Noise in Biological Membranes

Our understanding of cell structure and function derives from applications of a variety of physical and life science disciplines, methods and models to an important physiological process, namely, the exchange and transport of ions and molecules across biological membranes. We know that ion transport through membranes arises from a diversity of interrelated and interactive physical and chemical phenomena over a wide range of spatial and temporal scales. Among these phenomena common to all cellular structure and function include metabolism, kinetics of molecules, chemically mediated alteration of cell membrane electrical potential, membrane ion conductance, electrical signal propagation, and modulation by chemo- and mechanoreceptive mechanisms. This review focuses on the unique information contained in fluctuations in electrical properties associated with cell membrane ion transport. Received: 19 May 2000/Revised: 10 July 2000     

珊瑚共生体中“细菌-虫黄藻-宿主”三角关系的通讯交流

“细菌-虫黄藻-珊瑚”是生态系统中一对经典的三角关系,其中包含着复杂的物质流、信息流和能量流,三者的平衡与稳定是维护珊瑚礁生态系统健康的重要保障。过去20年里针对共生体交互关系进行了大量研究,并取得了一些重要成果,明确了“细菌-虫黄藻-宿主”三者之间的物质代谢、营养交换以及与环境的交互关系。然而,基于共生系统的复杂性,一些现象背后的机制仍然未被充分揭示,尤其是共生体之间的通讯交流。信号分子介导的相互作用是珊瑚共生体稳态维持和高效运转的内在驱动力。本文以珊瑚共生体系中化学信号为重点,尝试梳理最新的研究进展,包括细菌与细菌、细菌与珊瑚、细菌与虫黄藻以及虫黄藻与珊瑚之间的通讯方式,重点关注了群体感应信号(QS)、二甲基巯基丙酸盐(DMSP)、糖类信号、脂类信号以及非编码RNA。选择性例举了QS信号介导的微生物协作和竞争、DMSP调节下的细菌和宿主的相互作用,以及环境胁迫下珊瑚和虫黄藻对非编码RNA的响应过程,强调了它们在共生体中的作用模式和生态意义。并对今后的研究重点和可能方向进行了提炼,包括研究维度的扩充、新技术-新方法的应用以及生态模型的构建等,旨在提升对三角关系互作方式的认识,增进对珊瑚共生体的理解,探索基于通讯语言的操纵方式为珊瑚礁生态系统的恢复和保护提供新思路。     

Determinants of histone H1 mobility and chromatin binding in living cells

The dynamic interaction of chromatin-binding proteins with their nucleosome binding sites is an important element in regulating the structure and function of chromatin in living cells. Here we review the major factors regulating the intranuclear mobility and chromatin binding of the linker histone H1, the most abundant family of nucleosome-binding proteins. The information available reveals that multiple and diverse factors modulate the interaction of H1 with chromatin at both a local and global level. This multifaceted mode of modulating the interaction of H1 with nucleosomes is part of the mechanism that regulates the dynamics of the chromatin fiber in living cells.     

Analysing a magnetic molecule detection system--computer simulation

The detection of single molecules, e.g. in biology is possible by marking the interesting molecules with magnetic beads and detect the influence of the beads on giant magnetoresistance (GMR)/tunnel magnetoresistance (TMR)/spin valve (SV) sensors. The development of suitable multilayers has been studied experimentally as well as theoretically in order to optimize the sensor parameters. A finite difference (FD) method including the usually used contributions to the total energy [exchange, antiferromagnetically (af) coupling, anisotropy and magnetostatic] is used for the simulation with additional contributions to the local field according to the stray fields of the beads. In this work, we will show the results of micromagnetic calculations of the magnetization behavior of GMR/TMR sensors considering also the interaction between the domains in the magnetic layers of the sensor and the bead area. We can present first calculations where the bead particles (signal source) and the magnetic layers (sensor device) are considered as a whole magnetic ensemble.     

Guanine nucleotide exchange factors operate by a simple allosteric competitive mechanism

Guanine nucleotide exchange factors are essential components of the mode of action of GTPases involved in signal transduction. Their fundamental mechanism is generally accepted to derive from stabilization of the nucleotide-free form of GTPases, which is reflected in an increase in the rate of GDP dissociation when such an exchange factor is bound to a GTPase. The known kinetic properties of exchange factors can be explained on the basis of this simple allosteric competitive mechanism. Here, we describe experiments designed to distinguish this mechanism from a newer model, which invokes an active role for the incoming (i.e., displacing) nucleotide, implying the transient formation of a quaternary complex consisting of an exchange factor, a GTPase, and two nucleotides, one which is being displaced while the other stimulates this displacement. We show that for a well-known system (the small GTPase Ras and its exchange factor Cdc25) there is no evidence for an effect of the concentration or the nature (i.e., GDP or GTP) of the displacing nucleotide on the rate constant of GDP release from the Cdc25.Ras.GDP complex, consistent with the simple allosteric competitive model, and in disagreement with the newer suggestion. In addition, we present arguments, which demonstrate how the erroneous conclusions leading to the alternative model were derived.     

植物与病原菌互作的分子机制研究进展

植物的先天免疫主要包括模式识别受体对保守的微生物病原相关分子模式的识别和抗病蛋白对效应蛋白的识别。植物与病原体互作过程中存在广泛的信号交流,信号分子在植物与病原体的互作攻防中发挥了重要的调控作用,决定了二者的竞争关系。当前,大量植物与病原体互作中的信号分子被定位和克隆,其作用方式被揭示。本文总结了这些信号分子及其在植物免疫过程中的作用机制,主要包括植物细胞表面的模式识别受体分子对病原相关分子模式的识别与应答,植物抗病蛋白对病原体效应蛋白的识别与应答,以及免疫反应下游相关信号分子及其在植物抗病中的作用。此外,本文对未来相关研究提出了展望。