Laminins and human disease

The laminin protein family has diverse tissue expression patterns and is involved in the pathology of a number of organs, including skin, muscle, and nerve. In the skin, laminins 5 and 6 contribute to dermal-epidermal cohesion, and mutations in the constituent chains result in the blistering phenotype observed in patients with junctional epidermolysis bullosa (JEB). Allelic heterogeneity is observed in patients with JEB: mutations that results in premature stop codons produce a more severe phenotype than do missense mutations. Gene therapy approaches are currently being studied in the treatment of this disease. A blistering phenotype is also observed in patients with acquired cicatricial pemphigoid (CP). Autoantibodies targeted against laminins 5 and 6 destabilize epithelial adhesion and are pathogenic. In muscle cells, laminin alpha 2 is a component of the bridge that links the actin cytoskeleton to the extracellular matrix. In patients with laminin alpha 2 mutations, the bridge is disrupted and mature muscle cells apoptose. Congenital muscular dystrophy (CMD) results. The role of laminin in diseases of the nervous system is less well defined, but the extracellular protein has been shown to serve an important role in peripheral nerve regeneration. The adhesive molecule influences neurite outgrowth, neural differentiation, and synapse formation. The broad spatial distribution of laminin gene products suggests that laminin may be involved in a number of diseases for which pathogenic mechanisms are still being unraveled.     

Laminins: structure and genetic regulation

The laminins form a large family of modular proteins found in basement membranes, but also elsewhere. They function as structural components and are essential for morphogenesis, but in addition interact with cell surface receptors such as integrins and alpha-dystroglycan. By virtue of their receptor interactions, they initiate intracellular signalling events that regulate cellular organization and differentiation. The many interactions of laminins are mediated by binding sites, often contributed by single domains, which may differ between different forms of laminin. In the present article, we describe how the diversity of laminins and the genetic regulation of the expression of different laminin forms lead to the formation of extracellular matrices with variable laminin composition and thereby different biological properties.     

Laminins and their roles in mammals

Laminins are alpha-beta-gamma heterotrimeric components of all basement membranes. Laminins are now known to play the central role in organizing and establishing the basement membrane. The diversity of laminins allows them to impart special structural and signaling properties to the basement membrane. Of the 12 known laminin chain genes, 10 have been either found to be mutated in humans or experimentally mutated in mice. This has led to great progress over the last several years towards understanding both the functions of laminins and the reasons for their great diversity. In this review, I will summarize the in vivo studies in mice and humans that have contributed to this new knowledge.     

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.     

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 spectrin-based skeleton at the postsynaptic membrane of the neuromuscular junction

Membrane skeletons, in particular the spectrin-based skeleton, are thought to participate in the organization of specialized membrane domains by restricting integral proteins to specific membrane sites. In the neuromuscular junction, discrete isoforms of spectrin and ankyrin, the peripheral protein that links spectrin to the membrane, colocalize with voltage-dependent sodium channels and N-CAM at the troughs of the postsynaptic membrane folds. Moreover, beta-spectrin, N-CAM, and sodium channels become clustered at the endplate during a period of time coincident with postsynaptic fold formation and synapse maturation. These observations suggest a role of the spectrin skeleton in directing and maintaining postsynaptic accumulations of sodium channels and N-CAM. In addition, the coexistence of spectrin and dystrophin at the troughs of the junctional folds raises the question of their respective functions in this membrane domain, where both cytoskeletal proteins have the potential to associate with sodium channels via ankyrin and syntrophin, respectively. Possible scenarios are discussed here with respect to accumulating evidence from studies of assembly of similar membrane domains in neurons.     

EEG controlled neuromuscular electrical stimulation of the upper limb for stroke patients

This paper describes the Brain Computer Interface (BCI) system and the experiments to allow post-acute (<3 months) stroke patients to use electroencephalogram (EEG) to trigger neuromuscular electrical stimulation (NMES)-assisted extension of the wrist/fingers, which are essential pre-requisites for useful hand function. EEG was recorded while subjects performed motor imagery of their paretic limb, and then analyzed to determine the optimal frequency range within the mu-rhythm, with the greatest attenuation. Aided by visual feedback, subjects then trained to regulate their mu-rhythm EEG to operate the BCI to trigger NMES of the wrist/finger. 6 post-acute stroke patients successfully completed the training, with 4 able to learn to control and use the BCI to initiate NMES. This result is consistent with the reported BCI literacy rate of healthy subjects. Thereafter, without the loss of generality, the controller of the NMES is developed and is based on a model of the upper limb muscle (biceps/triceps) groups to determine the intensity of NMES required to flex or extend the forearm by a specific angle. The muscle model is based on a phenomenological approach, with parameters that are easily measured and conveniently implemented.     

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.     

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.     

Synaptic target recognition at Drosophila neuromuscular junctions

Every synaptogenesis begins with "synaptic target recognition," a cell-cell recognition event in which a neuron and its target stably adhere. Despite its importance in developing nervous systems, synaptic target recognition has been difficult to study in complex systems. The relatively simple and genetically accessible Drosophila NMJ model system provides a repertoire of target recognition cues. We describe how these molecules control the targeting of specific growth cones in either a positive (synaptogenic) or negative (anti-synaptogenic) manner. We also propose two alternate signaling paradigms to explain how these initial cell recognition events are coupled to the intracellular signaling pathways that begin the process of synapse maturation.     

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.     

不同剂量顺式阿曲库铵对小儿肌松作用比较

目的 :比较不同剂量顺式阿曲库铵在小儿外科手术中的肌松作用,探讨其临床使用中最适剂量。方法 :48位患儿随机分为3组,分别应用2倍ED95,3倍ED95和4倍ED95的顺式阿曲库铵进行随机对照研究,观察3组患儿肌松药起效时间、有效作用时间、插管条件、血流动力学等指标。结果 :3组间注射顺式阿曲库铵前及注射1⁵min后心率、平均动脉压差异无统计学意义。高剂量组(3倍ED95和4倍ED95)顺式阿曲库铵起效时间、有效作用时间、体内作用时间均短于和久于2倍ED95剂量组,差异有统计学意义。3组间恢复指数差异无统计学意义。高剂量组能获得较好的插管条件,与低剂量组比较差异具有统计学意义。但3倍ED95和4倍ED95剂量组在起效时间、插管条件等方面差异无统计学意义。结论 :小儿应用3倍ED95和4倍ED95顺式阿曲库铵有着更快速的神经肌松作用,且持续时间更长、血流动力学更稳定。3倍ED95为最合适的诱导剂量。     

Freeze-Fracture of physiologically identified neuromuscular junctions from single frog muscle fibers

We developed a technique for freeze-fracturing single physiologically identified neuromuscular junctions. This technique permits direct comparison of quantal content with morphological variables such as active zone length per unit terminal length for the same cell. The technique was developed to elucidate the structural basis for variability in transmitter release at the neuromuscular junction. The procedures were as follows: (1) record quantal content by conventional intracellular recordings; (2) mark cells for identification by fluorescent dye injection; (3) fix and stain endplate cholinesterase; (4) glycerinate and remove single fibers from the muscle; (5) draw endplate morphology; (6) freeze-fracture single muscle fibers; (7) examine in a transmission electron microscope; and (8) photograph and measure nerve terminal membrane ultrastructure. We found that approximately 15% of freeze-fractured single muscle fibers exhibited nerve terminal active zones. To demonstrate the usefulness of this technique, physiological and morphological information from an identified junction is presented. Freeze-fracture of identified cells has several advantages over thin sections, which cannot accurately show such things as active zone length, spacing, or intramembrane particles. This technique also has applications to the study of active zone ultrastructure in situations where neurotransmitter release is known to differ from normal levels. In addition, direct correlations between membrane structure and function can be studied in other preparations by this method.     

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.     

ControlLogix在大型输煤控制系统中的应用

ControlLogix是Rockwell公司的第三代主流产品,具有可靠性高、指令集丰富、联网通讯能力强、I／O模板丰富、开放性好等优点,是一个真正的集成控制平台。它利用统一的编程软件和一个公共系统数据库,将顺序控制、过程控制、传动和运动控制集成在一个框架内。介绍了ControlLogix系统在输煤系统改造中的成功应用,使该输煤系统的自动化程度得到大幅度提高。     

基于专家系统的制粉系统故障诊断的研究

根据制粉系统运行特性及故障特点,提出了制粉系统故障诊断系统总体框架,建立了制粉系统故障诊断知识库,包括专家知识的转换、录入与存储等,开发了良好的人机界面。以VC++6.0和CLIPS为开发工具研制了一个适用于复杂工业生产过程运行故障诊断的模糊专家系统。     

激光跟踪运动目标伺服系统设计

激光跟踪测量系统实现对运动目标位置检测,要求跟踪机构响应快、动态性能好、精度高.本论文设计了伺服跟踪系统,采用直流力矩电机控制反射镜跟踪目标,实现了对运动目标的高精度快速跟踪.