Clustering of sodium channels at the neuromuscular junction

Voltage-gated sodium channels (NaChs) are highly concentrated in the postsynaptic region of the neuromuscular junction, especially in the depths of postsynaptic folds and in the perijunctional region. The formation of the high NaCh density occurs during synapse maturation, approximately 2 weeks after initial synaptic contact in the rodent. The concentration of NaChs and their localization in the troughs of the folds increase the safety factor for neuromuscular transmission by reducing the threshold for initiation of the action potential. There is evidence that agrin plays a role in the formation of NaCh aggregation. Molecules such as ankyrin and syntrophin that bind NaChs may be important for maintenance of the high channel density at the endplate.     

The torpedo electrocyte: a model system to study membrane-cytoskeleton interactions at the postsynaptic membrane

Many aspects of the organization of the electromotor synapse of electric fish resemble the nerve-muscle junction. In particular, the postsynaptic membrane in both systems share most of their proteins. As a remarquable source of cholinergic synapses, the Torpedo electrocyte model has served to identify the most important components involved in synaptic transmission such as the nicotinic acetylcholine receptor and the enzyme acetylcholinesterase, as well as proteins associated with the subsynaptic cytoskeleton and the extracellular matrix involved in the assembly of the postsynaptic membrane, namely the 43-kDa protein-rapsyn, the dystrophin/utrophin complex, agrin, and others. This review encompasses some representative experiments that helped to clarify essential aspects of the supramolecular organization and assembly of the postsynaptic apparatus of cholinergic synapses.     

Atomic force microscopy of the erythrocyte membrane skeleton

The atomic force microscope was used to examine the cytoplasmic surface of untreated as well as fixed human erythrocyte membranes that had been continuously maintained under aqueous solutions. To assess the effects of drying, some membranes were examined in air. Erythrocytes attached to mica or glass were sheared open with a stream of isotonic buffer, which allowed access to the cytoplasmic membrane face without exposing cells to non‐physiological ionic strength solutions. Under these conditions of examination, the unfixed cytoplasmic membrane face revealed an irregular meshwork that appeared to be a mixture largely of triangular and rectilinear openings with mesh sizes that varied from 35 to 100 nm, although few were at the upper limit. Fixed ghosts were similar, but slightly more contracted. These features represent the membrane skeleton, as when the ghosts were treated to extract spectrin and actin, these meshworks were largely removed. Direct measurements of the thickness of the membrane skeleton and of the lateral dimensions of features in the images suggested that, especially when air dried, spectrin can cluster into large, quite regularly distributed aggregates. Aggregation of cytoskeletal components was also favoured when the cells were attached to a polylysine‐treated substrate. In contrast, the membrane skeletons of cells attached to substrates rendered positively charged by chemical derivatization with a cationic silane were much more resistant to aggregation. As steps were taken to reduce the possibility of change of the skeleton after opening the cells, the aggregates and voids were eliminated, and the observed structures became shorter and thinner. Ghosts treated with Triton X‐100 solutions to remove the bilayer revealed a meshwork having aggregated components resembling those seen in air. These findings support the proposition that the end‐to‐end distance of spectrin tetramers in the cell in the equilibrium state is much shorter than the contour length of the molecule and that substantial rearrangements of the spectrin‐actin network occur when it is expanded by low ionic strength extraction from the cell. This study demonstrates the applicability of AFM for imaging the erythrocyte membrane skeleton at a resolution that appears adequate to identify major components of the membrane skeleton under near‐physiological conditions.     

Cytoskeletal architecture of neuromuscular junction: Localization of vinculin

Cytoskeletons underneath the postsynaptic membrane of neuromuscular junctions were studied by using a quick-freeze deep-etched method and immunoelectron microscopy of ultrathin frozen sections. In a quick-freeze deep-etched replica of fresh, unfixed muscles, 8.9 ± 1.5-nm particles were present on the true postsynaptic membrane surface. Underneath this receptor-rich postsynaptic membrane, networks of fine filaments were observed. These cytoskeletal networks were more clearly observed in extracted samples. In these samples, diameters of the filaments which formed networks were measured. In the platinum replica, three kinds of filament were recognized—12 nm, 9 nm, and 7 nm in diameter. The 12-nm filament seemed to correspond to the intermediate filament. The other two filaments formed meshworks between intermediate filaments and plasma membrane. In ultrathin frozen sections vinculin label was localized just beneath the plasma membrane. Thirty-six percent of the label was within 18 nm from the cytoplasmic side of the plasma membrane and 50% was within 30 nm. Taking the size of the vinculin molecule into account, it was concluded that vinculin is localized just beneath the plasma membrane and might play some role in anchoring filaments which formed meshworks underneath the plasma membrane.     

Structural and functional correlates of synaptic transmission in the vertebrate neuromuscular junction

Because vertebrate neuromuscular junctions are readily accessible for experimental manipulation, they have provided a superb model in which to examine and test functional correlates of chemical synaptic transmission. In the neuromuscular synapse, acetylcholine receptors have been localized to the crests of the junctional folds and visualized by a variety of ultrastructural techniques. By using ultrarapid freezing techniques with a temporal resolution of less than 1 msec, quantal transmitter release has been correlated with synaptic vesicle exocytosis at discrete sites called “active zones.” Mechanisms for synaptic vesicle membrane retrieval and recycling have been identified by using immunological approaches and correlated with endocytosis via coated pits and coated vesicles. In this review, available ultrastructural, physiological, immunological, and biochemical data have been used to construct an ultrastructural model of neuromuscular synaptic transmission that correlates structure and function at the molecular level.     

Drosophila larval neuromuscular junction: molecular components and mechanisms underlying synaptic plasticity

Understanding the mechanisms that mediate synaptic plasticity is a primary goal of molecular neuroscience. The Drosophila larval neuromuscular junction provides a particularly useful model for investigating the roles of synaptic components in both structural and functional plasticity. The powerful molecular genetics of this system makes it possible to uncover new synaptic components and signaling molecules, as well as their function in the intact organism. Together with the mouse hippocampus and Aplysia dissociated cell culture, the Drosophila larval neuromuscular junction has been among the most valuable model systems for examining the molecular and cellular basis of neuronal plasticity.     

Rodent nerve-muscle cell culture system for studies of neuromuscular junction development: refinements and applications

Understanding of vertebrate neuromuscular junction (NMJ) development has been advanced by experimentation with cultures of dissociated embryonic nerve and skeletal muscle cells, particularly those derived from Xenopus and chick embryos. We previously developed a rodent (rat) nerve-muscle coculture system that is characterized by extensive induction of acetylcholine receptor (AChR) aggregation at sites of axonal contact with myotubes (Dutton et al., 1995). In this article, we report modifications of this culture system and examples of its application to the study of NMJ development: (1) We describe improved methods for the enrichment of myoblasts to give higher yields of myotubes with equal or greater purity. (2) We demonstrate lipophilic dye labeling of axons in cocultures by injection of dye into neuron aggregates and show the feasibility of studying the growth of living axons on myotubes during synapse formation. (3) We describe the preparation of a better-defined coculture system containing myotubes with purified rat motoneurons and characterize the system with respect to axon-induced AChR aggregation. (4) We demonstrate dependence of the pattern of axon-induced AChR aggregation on muscle cell species, by the use of chick-rat chimeric co-cultures. (5) We provide evidence for the role of alternatively-spliced agrin isoforms in synapse formation by using single cell RT-PCR with neurons collected from co-cultures after observation of axon-induced AChR aggregation. Microsc. Res. Tech. 49:26-37, 2000. Published 2000 Wiley-Liss, Inc.     

Regulation and functional significance of utrophin expression at the mammalian neuromuscular synapse

Duchenne muscular dystrophy (DMD) is caused by the absence of full-length dystrophin molecules in skeletal muscle fibers. In normal muscle, dystrophin is found along the length of the sarcolemma where it links the intracellular actin cytoskeleton to the extracellular matrix, via the dystrophin-associated protein (DAP) complex. Several years ago, an autosomal homologue to dystrophin, termed utrophin, was identified and shown to be expressed in a variety of tissues, including skeletal muscle. However, in contrast to the localization of dystrophin in extrajunctional regions of muscle fibers, utrophin preferentially accumulates at the postsynaptic membrane of the neuromuscular junction in both normal and DMD adult muscle fibers. Since it has recently been suggested that the upregulation of utrophin might functionally compensate for the lack of dystrophin in DMD, considerable interest is now directed toward the elucidation of the various regulatory mechanisms presiding over expression of utrophin in normal and dystrophic skeletal muscle fibers. In this review, we discuss some of the most recent data relevant to our understanding of the impact of myogenic differentiation and innervation on the expression and localization of utrophin in skeletal muscle fibers.     

Laminins of the neuromuscular system

The mammalian neuromuscular system expresses seven laminin genes (alpha 1, alpha 2, alpha 4, alpha 5, beta 1, beta 2, and gamma 1), produces seven isoforms of the laminin trimer (laminins 1, 2, 4, 8, 9, 10, and 11), and distributes these trimers to at least seven distinct basal laminae (perineurial, endoneurial, terminal Schwann cell, myotendinous junction, synaptic cleft, synaptic fold, and extrajunctional muscle). The patterns of expression, assembly, and distribution are regulated during development, and primary and secondary changes in laminin expression occur in several neuromuscular genetic disorders. Functional studies using knockout and transgenic mice, and purified laminins and cell types, demonstrate that laminins are required components of basal laminae in the neuromuscular system. Collectively, laminins have both structural and signaling functions; individually, laminin isoforms have unique roles in regulating the behavior of nerve, muscle, and Schwann cell. Among them, laminin-2 (alpha 2 beta 1 gamma 1) plays an important structural role in supporting the muscle plasma membrane, laminin-4 regulates adhesion and differentiation of the myotendinous junction, and laminin-11 regulates nerve terminal differentiation and Schwann cell motility. Together, these observations reveal remarkable diversity in the formation and function of laminins and basal laminae, and suggest avenues for addressing some neuromuscular diseases.     

屯子衡器中接线盒的标准化

本文较为详细地介绍了由应变式传感器组成的电子衡器中所使用接线盒而构成的并联融合电路,并进一步定性、定量的理论分析、演绎而给出电路函数表达式、元器件的选用条件及公式等内容,可为制定接线盒标准化提供先前的、价值性的依据。     

Transmembrane alpha-helices in the gap junction membrane channel: systematic search of packing models based on the pair potential function

Recent progress in the field of electron cryo-microscopy and image analysis has shown that there is an overwhelming need to interpret medium resolution (5 to 10 A) three-dimensional maps. Traditional methods of fitting amino acid residues into electron density using molecular modeling programs must be supplemented with further analysis. We have used a potential of mean force (PMF) method, derived from Boltzmann statistics in protein structure, to generate models for the packing of alpha-helices, using pairwise potentials between amino acid residues. The approach was tested using the three-dimensional map of a recombinant cardiac gap junction membrane channel provided by electron cryo-crystallography (Unger et al., 1997; 1999a, 1999b) which had a resolution of 7.5 A in the membrane plane and 21 A in the vertical direction. The dodecameric channel was formed by the end-to-end docking of two hexamers, each of which displayed 24 rods of density in the membrane interior, which was consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. Based on the three-dimensional map and the amino acid sequence for the 4 transmembrane domains determined by hydropathy analysis, we used the modeling utility SymServ (Macke et al., 1998) to build hexameric connexons with 24 transmembrane alpha-helices. Canonical alpha-helices were aligned to the axes of the rods of density and translated along the density so that the center of masses coincided. The PMF function was used to evaluate 162,000 conformations for each of the 24 possible alpha-helical packing models. Since the different packing models yielded different energy distributions, the pair potential function appears to be a promising tool for evaluating the packing of alpha-helices in membrane proteins. The analysis will be refined by energy calculations based on the expectations that the outer boundary of the channel will be formed by hydrophobic residues in contact with the lipids.     

管接头的加工工艺改进及模具设计

介绍用冷挤压工艺代替以机械加工工艺来成形管接头的优点及模具结构。对类似零件的工艺改进具有一定的参考价值。     

Experimental analysis of junction growth with a junction model

A study has been carried out on the junction growth mechanism of a one-piece specimen which may be assumed to be an ideally adhered junction model. Specimen materials were mild steel, aluminium, phosphor-bronze and brass. The real contact area in the junction growth process was measured using an optical microscope and the relation between the nominal normal stress and the nominal shear stress was obtained.The junction growth process varied greatly depending on the workhardening rate of the metal and the initial contact pressure; the junction growth of the contact area increased with increasing initial contact pressure and decreasing work-hardening coefficient. Complete adhesion was not always sufficient for high friction.     

Cellulose microfibril deposition: coordinated activity at the plant plasma membrane

Plant cell wall production is a membrane-bound process. Cell walls are composed of cellulose microfibrils , embedded inside a matrix of other polysaccharides and glycoproteins. The cell wall matrix is extruded into the existing cell wall by exocytosis. This same process also inserts the cellulose synthase complexes into the plasma membrane. These complexes, the nanomachines that produce the cellulose microfibrils, move inside the plasma membrane leaving the cellulose microfibrils in their wake. Cellulose microfibril angle is an important determinant of cell development and of tissue properties and as such relevant for the industrial use of plant material. Here, we provide an integrated view of the events taking place in the not more than 100 nm deep area in and around the plasma membrane, correlating recent results provided by the distinct field of plant cell biology. We discuss the coordinated activities of exocytosis, endocytosis, and movement of cellulose synthase complexes while producing cellulose microfibrils and the link of these processes to the cortical microtubules.     

Radiation damage of purple membrane at low temperature.

The intensities of diffracted electron beams for the purple membrane of Halobacterium halobium are found to decay exponentially as a function of the accumulated electron exposure, both at room temperature and at -120 degrees C. This permits us to define the "critical dose" Ne(h,k) for the (h,k) diffracted beam, as being the electron exposure (electrons/A2) at which the diffracteed intensity has fallen to e-1 of its initial value. The critical of purple membrane is found to increase from the room temperature value by at least a factor of four when the specimen is maintained at a temperature of -120 degrees C on a liquid-nitrogen-cooled stage. A relationship derived between the critical dose, Ne, and the dose for optimum imaging, Nopt. Both Ne and Nopt depend, of course, upon the spatial frequency, or resolution. The derivation is valid only for the case in which all sources of noise other than quantum fluctuations are neglected. In this case, Nopt approximately equal to 2.5Ne. Finally, Nuclear Track Emulsion plates have been shown to be advantageous for recording high resolution electron diffraction patterns of small (1 micrometer 2) patches of crystalline biological materials.     

基于现代设计方法的豪华客车车身骨架分析

在全新造型和样车设计的基础上,对GDW6850H车身骨架及蒙皮的结构作合理的简化,根据实际的边界条件建立了有限元分析模型,对整车骨架和部分蒙皮进行了强度、刚度及模态分析,根据分析结果对整车骨架蒙皮进行轻量化设计,在确保客车性能满足要求和经过试验验证的基础上,优化了骨架的结构,有效地减轻了车身质量,达到了满意的工程效果.     

Matlab软件在叠片联轴器叠片参数优化设计中的应用

首次将Matlab软件用于叠片联轴器的叠片参数优化。得出了叠片在综合载荷作用下，为使叠片的应力达到取小而优化出叠片的最佳尺寸并分析某一因素的变化对叠片应力的影响。为今后该型号的叠片联轴器的进一步设计研究奠定了理论基础，同时也为各类叠片联轴器设计提供理论参考。     

基于Pro/E骨架模型的浆体泵泵体的参数化设计

自上而下的设计模式符合产品的设计思路,Pro/E软件装配模式下的骨架模型设计是自上而下设计的方法之一.设计了浆体泵泵体的参数化骨架模型,并通过骨架模型创建了泵体零件,通过改变泵体骨架模型参数,实现了泵体的参数化设计和系列化设计.Pro/E软件骨架模型反映了产品的总体结构和设计方案,保证了产品设计合理性和装配的准确性,对...