Axonal proteins of presynaptic neurons during synaptogenesis

Changes occur in the synthesis and axonal transport of neuronal proteins in dorsal-root ganglia axons as a result of contact with cells from the spinal cord during synapse formation. Dorsal-root ganglia cells were cultured in a compartmental cel culture system that allows separate access to neuronal cell bodies and their axons. When cells from the ventral spinal cord were cultured with the dorsal-root ganglia axons, synapses were established within a few days. Metabolic labeling and two-dimensional electrophoresis revealed that four of more than 300 axonal proteins had changed in their expression by the time synapses were established. The highly selective nature of these changes suggests that the proteins involved may be important in the processes of axon growth and synapse formation and their regulation by the regional environment.     

Microtubule gelation-contraction: essential components and relation to slow axonal transport

Preparations of microtubule proteins isolated by assembly and disassembly undergo gelation-contraction after addition of adenosine triphosphate (ATP). A particulate fraction from these preparations that is required, along with purified tubulin, to produce ATP-dependent microtubule gelation-contraction in vitro has been isolated. The particulates exhibited microtubule-stimulated adenosine triphosphatase activity and moved slowly (about 1 micrometer per minute) along microtubule walls in the presence of ATP. The particulates contained tubulin, neurofilament, and spectrin polypeptides. The composition, solubility, and motility of the particulates are consistent with those of slow component a of axonal transport.     

Synaptic connections in vitro: modulation of number and efficacy by electrical activity

The functional architecture of synaptic circuits is determined to a crucial degree by the patterns of electrical activity that occur during development. Studies with an in vitro preparation of mammalian sensory neurons projecting to ventral spinal cord neurons slow that electrical activity induces competitive processes that regulate synaptic efficacy so as to favor activated pathways over inactive convergent pathways. At the same time, electrical activity initiates noncompetitive processes that increase the number of axonal connections between these sensory and spinal cord neurons.     

An ATP gate controls tubulin binding by the tethered head of kinesin-1

Kinesin-1 is a two-headed molecular motor that walks along microtubules, with each step gated by adenosine triphosphate (ATP) binding. Existing models for the gating mechanism propose a role for the microtubule lattice. We show that unpolymerized tubulin binds to kinesin-1, causing tubulin-activated release of adenosine diphosphate (ADP). With no added nucleotide, each kinesin-1 dimer binds one tubulin heterodimer. In adenylyl-imidodiphosphate (AMP-PNP), a nonhydrolyzable ATP analog, each kinesin-1 dimer binds two tubulin heterodimers. The data reveal an ATP gate that operates independently of the microtubule lattice, by ATP-dependent release of a steric or allosteric block on the tubulin binding site of the tethered kinesin-ADP head.     

Axonal transport: each major rate component reflects the movement of distinct macromolecular complexes

The proteins of the three major rate components of axonal transport in guinea pig retinal ganglion cells were analyzed by one- and two-dimensional gel electrophoresis. Each rate component consisted of a different set of proteins that remained associated with each other during transport. This suggests that each rate component represents a distinct macromolecular complex and that these complexes may be definable organelles such as microtubules, microfilaments, and smooth endoplasmic reticulum. Thus, the transport of radiolabeled proteins in the axon reflects the movement of complete subcellular rather than the movement of individual proteins.     

Posttranslational glutamylation of alpha-tubulin

The high degree of tubulin heterogeneity in neurons is controlled mainly at the posttranslational level. Several variants of alpha-tubulin can be posttranslationally labeled after incubation of cells with [3H]acetate or [3H]glutamate. Peptides carrying the radioactive moiety were purified by high-performance liquid chromatography. Amino acid analysis, Edman degradation sequencing, and mass spectrometric analysis of these peptides led to the characterization of a posttranslational modification consisting of the successive addition of glutamyl units on the gamma-carboxyl group of a glutamate residue (Glu445). This modification, localized within a region of alpha-tubulin that is important in the interactions of tubulin with microtubule-associated proteins and calcium, could play a role in regulating microtubule dynamics.     

Retrograde axonal transport of endogenous proteins in sciatic nerve demonstrated by covalent labeling in vivo

Extracellularly applied N-succinimidyl [2,3-3H]propionate was used in vivo to covalently label intra-axonal proteins in the rat sciatic nerve. This technique permitted a unique view of axonal transport of proteins independent of biosynthesis. The proteins detected in slow anterograde transport (1 to 2 millimeters per day) correspond to cytoskeletal proteins described in previous papers. The slowly retrogradely transported component (3 to 6 millimeters per day) was composed primarily of a single protein with a molecular weight of 68,000.     

Receptor-mediated transport of insulin across endothelial cells

Hormones such as insulin are transported from the interior to the exterior of blood vessels. Whether endothelial cells, which line the inner walls of blood vessels have a role in this transport of hormones is not clear, but it is known that endothelial cells can internalize and release insulin rapidly with little degradation. The transport of iodine-125-labeled insulin was measured directly through the use of dual chambers separated by a horizontal monolayer of cultured bovine aortic endothelial cells. In this setting, endothelial cells took up and released the labeled insulin, thereby transporting it across the cells. The transport of insulin across the endothelial cells was temperature sensitive and was inhibited by unlabeled insulin and by antibody to insulin receptor in proportion to the ability of these substances to inhibit insulin binding to its receptor. More than 80 percent of the transported insulin was intact. These data suggest that insulin is rapidly transported across endothelial cells by a receptor-mediated process.     

Chaos, symmetry, and self-similarity: exploiting order and disorder in mixing processes

Fluid mixing is a successful application of chaos. Theory anticipates the coexistence of order and disorder-symmetry and chaos-as well as self-similarity and multifractality arising from repeated stretching and folding. Experiments and computations, in turn, provide a point of confluence and a visual analog for chaotic behavior, multiplicative processes, and scaling behavior. All these concepts have conceptual engineering counterparts: examples arise in the context of flow classification, design of mixing devices, enhancement of transport processes, and controlled structure formation in two-phase systems.     

Glial membranes at the node of Ranvier prevent neurite outgrowth

Nodes of Ranvier are regularly placed, nonmyelinated axon segments along myelinated nerves. Here we show that nodal membranes isolated from the central nervous system (CNS) of mammals restricted neurite outgrowth of cultured neurons. Proteomic analysis of these membranes revealed several inhibitors of neurite outgrowth, including the oligodendrocyte myelin glycoprotein (OMgp). In rat spinal cord, OMgp was not localized to compact myelin, as previously thought, but to oligodendroglia-like cells, whose processes converge to form a ring that completely encircles the nodes. In OMgp-null mice, CNS nodes were abnormally wide and collateral sprouting was observed. Nodal ensheathment in the CNS may stabilize the node and prevent axonal sprouting.     

Fast axonal transport in squid giant axon

Video-enhanced contrast-differential interference contrast microscopy has revealed new features of axonal transport in the giant axon of the squid, where no movement had been detected previously by conventional microscopy. The newly discovered dominant feature is vast numbers of "submicroscopic" particles, probably 30- to 50-nanometer vesicles and other tubulovesicular elements, moving parallel to linear elements, primarily in the orthograde direction but also in a retrograde direction, at a range of steady velocities up to +/- 5 micrometers per second. Medium (0.2 to 0.6 micrometer) and large (0.8 micrometer) particles move more slowly and more intermittently with a tendency at times to exhibit elastic recoil. The behavior of the smallest particles and the larger particles during actual translocation suggests that the fundamental processes in the mechanisms of organelle movement in axonal transport are not saltatory but continuous.     

Promotion of tubulin assembly by aluminum ion in vitro

It has been proposed that aluminum ion is a contributing factor in a variety of neurological diseases. In many of these diseases, aberrations in the cytoskeleton have been noted. The effects of aluminum ion on the in vitro assembly of tubulin into microtubules has been examined by determining the association constants for the metal ion-guanosine triphosphate-tubulin ternary complex required for polymerization. The association constant for aluminum ion was approximately 10(7) times that of magnesium ion, the physiological mediator of microtubule assembly. In addition, aluminum ion at 4.0 X 10(-10) mole per liter competed effectively with magnesium ion for support of tubulin polymerization when magnesium ion falls below 1.0 millimole per liter. The microtubules produced by aluminum ion were indistinguishable from those produced by magnesium ion when viewed by electron microscopy, and they showed identical critical tubulin concentrations for assembly and sensitivities to cold-induced depolymerization. However, the rate of guanosine triphosphate hydrolysis and the sensitivity to calcium ion-induced depolymerization, critical regulatory processes of microtubules in vivo, were markedly lower for aluminum ion microtubules than for magnesium ion microtubules.     

Methylation increases sodium transport into A6 apical membrane vesicles: possible mode of aldosterone action

When isolated apical membrane vesicles prepared from cultured A6 epithelia were incubated in vitro with the methyl donor S-adenosylmethionine, the control rate of amiloride-inhibitable sodium transport was doubled. The methylation inhibitors 3-deazaadenosine and S-adenosyl homocysteine returned the S-adenosyl-methionine-stimulated sodium transport to control levels. Neither these agents nor adenosine affected sodium transport into control vesicles. In vesicles incubated with S-adenosyl-[3H-methyl]methionine, both membrane phospholipids and proteins were labeled, and this labeling was inhibited by deazaadenosine. In vesicles prepared from A6 cells treated with aldosterone, sodium transport was twice the control value and S-adenosylmethionine did not cause any further stimulation of transport. In those vesicles, both lipid and protein methylation were increased. These results suggest that methylation, which increases the rate of amiloride-sensitive sodium transport is involved in the action of aldosterone at the apical membrane level in epithelia.     

Methionine sulfoximine--resistant mutants of tobacco

Selecting mutants from populations of haploid plant cells cultured in vitro may provide a rapid method for recovering agriculturally useful variants. Mutants of Nicotiana tabacum were recovered which were resistant to methionine sulfoximine, an analog structurally similar to methionine. Induction of chlorosis was prevented or less evident in mutant plants that were inoculated withPseudomonas tabaci, a bacterial pathogen which produces a toxin that is a structural analog of methioning. Several mutants show a specific increase in the level of free methionine.     

The role of chloride transport in postsynaptic inhibition of hippocampal neurons

Hippocampal inhibitory postsynaptic potentials are depolarizing in granule cells but hyperpolarizing in CA3 neurons because the reversal potentials and membrane potentials of these cells differ. Here the hippocampal slice preparation was used to investigate the role of chloride transport in these inhibitory responses. In both cell types, increasing the intracellular chloride concentration by injection shifted the reversal potential of these responses in a positive direction, and blocking the outward transport of chloride with furosemide slowed their recovery from the injection. In addition, hyperpolarizing and depolarizing inhibitory responses and the hyperpolarizing and depolarizing responses to the inhibitory neurotransmitter gamma-aminobutyric acid decreased in the presence of furosemide. These effects of furosemide suggest that the internal chloride activity of an individual hippocampal neuron is regulated by two transport processes, one that accumulates chloride and one that extrudes chloride.     

Vasoactive intestinal peptide alters membrane potential and cyclic nucleotide levels in retinal horizontal cells

Vasoactive intestinal peptide stimulated the synthesis of adenosine 3',5'-monophosphate in fractions of isolated carp horizontal cells. When applied extracellularly to isolated and cultured horizontal cells, the peptide also induced a slow depolarization (30 to 40 millivolts) accompanied by a decrease in membrane resistance. However, analogs of adenosine 3',5'-monophosphate applied extracellularly or intracellularly, and forscolin applied extracellularly, had no effect on the membrane potential of cultured horizontal cells, indicating that the induced depolarization was not related to the accumulation of adenosine 3',5'-monophosphate in these cells.     

猪成纤维细胞的简易分离培养

比较了不同来源、不同状态组织和不同分离方法对猪成纤维细胞的影响,结果表明,离体组织长时间乙醇预处理和长时间储运会降低细胞活力,使生长速度减慢;初始原代细胞种类比较多,生长不均匀,只有传代培养3代以上,才能得到比较纯的成纤维细胞。     

Axonopathy and transport deficits early in the pathogenesis of Alzheimer's disease

We identified axonal defects in mouse models of Alzheimer's disease that preceded known disease-related pathology by more than a year; we observed similar axonal defects in the early stages of Alzheimer's disease in humans. Axonal defects consisted of swellings that accumulated abnormal amounts of microtubule-associated and molecular motor proteins, organelles, and vesicles. Impairing axonal transport by reducing the dosage of a kinesin molecular motor protein enhanced the frequency of axonal defects and increased amyloid-beta peptide levels and amyloid deposition. Reductions in microtubule-dependent transport may stimulate proteolytic processing of beta-amyloid precursor protein, resulting in the development of senile plaques and Alzheimer's disease.     

Selective inhibition of tubulin synthesis by amiprophos methyl during flagellar regeneration in Chlamydomonas reinhardi

Amiprophos methyl (APM) is a strong, readily reversible and highly selective inhibitor of tubulin synthesis in Chlamydomonas reinhardi. The extensive induction of tubulin synthesis that accompanies flagellar regeneration in this organism is prevented by 3 to 10 micrometerAPM. When applied after induction has begun, APM causes a rapid cessation of tubulin synthesis. Translation studies in vitro indicate that the lack of tubulin production in APM-treated cells is not due to a direct inhibition of tubulin messenger RNA translation but rather to a selective depletion of tubulin messenger RNA.     

Axonal transport of gangliosides in the goldfish optic nerve

Radioactive glucosamine and N-acetylmannosamine injected into the goldfish eye are incorporated into gangliosides that undergo rapid axonal transport to the optic nerve terminals. All ganglioside fractions are labeled. These data provide the first evidence that axonal transport has a role in neuronal ganglioside function and metabolism.