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The formation of neuronal networks depends critically on the growth cone, a motile ameboid structure at the tip of elongating axon. Graded distributions of extracellular guidance cues attract or repel the growth cone by generating asymmetric cytosolic Ca2+ signals across the growth cone. We showed that the directional polarity of growth cone guidance is determined by the source of Ca2+ signals. We also demonstrated that, downstream of Ca2+ signals, asymmetric exocytosis and endocytosis drive growth cone attraction and repulsion, respectively. These findings provide a novel concept that polarized membrane trafficking acts as an instructive mechanism to spatially localize the steering apparatus such as cytoskeletal components and adhesion molecules. [ABSTRACT FROM AUTHOR]

Recent studies clarified a dynamic regulation of the intracellular trafficking of GABA(A) receptors and its involvement in the pathophysiology of epilepsy. GABA(A) synaptic inhibition decreased in the hippocampal CA1 area of patients with intractable temporal lobe epilepsy (TLE). The reduction of GABAergic inhibition was accompanied by a decrease in the expression of gephyrin, a scaffolding protein, and GABA(A) receptor gamma2 subunit. These findings indicate that the reduction of gephyrin impairs the clustering and fixation of GABA(A) receptors in postsynaptic membranes, leading to a decrease in number of GABA(A) receptor subunits and GABA(A) synaptic inhibition. In contrast, the GABA(A) synaptic inhibition was lastingly potentiated in the dentate gyrus of kindled animals and the expression of GABA(A) receptor subunits(especially alpha2) was significantly increased in TLE patients. It is plausible that the potentiation of dentate GABAergic inhibition counteracts a hyperexcitability of granule cells as a defense mechanism in epilepsy. In status epilepticus, furthermore, the hippocampal GABA(A) receptor beta3 subunits were significantly disphosphorylated, resulting in a facilitation of the endocytosis of GABA(A) receptors and reduced benzodiazepine sensitivity.

Coronaviruses contain positive-stranded RNA with ca. 30 kb as a genome, which is wrapped by the envelope, and constitute Nidovirales together with Arteriviridae. The feature of viruses in Nidovirales is the unique structure of the mRNA set, called 3' co-terminal nested set. Coronaviruses have several to more than 10 different species of subgenomic mRNA and generally only the OFR located in the 5' end of each mRNA is translated. The 5' 20 kb of the coronavirus genome or mRNA-1 consists of two ORFs, 1a and 1b, between that there is a unique RNA structure called pseudoknot. From mRNA-1, 1a as well as 1a+1b are translated; the latter 1a+1b results from the translation due to ribosomal frame-shifting facilitated by the pseudoknot structure. From those two proteins, totally 16 proteins are produced as a result of auto-cleavage by the proteases included in la protein. Those proteins exhibit different functions, such as RNA-dependent RNA polymerase, helicase, proteases and proteins that regulate cellular functions, mRNAs smaller than mRNA-2 translate in general the structural proteins, nucleocapsid (N) protein, spike (S) protein, integrated membrane (M) protein and envelope (E) proteins. Those proteins assemble to the vesicles located from ER to Golgi (ER Golgi intermediate compartment) and virions bud into the vesicles. Those virions are released from infected cells via exocytosis.

Synaptic transmission is regulated by vesicular exocytosis and subsequent recycling at the presynaptic terminals. These vesicular dynamics were quantified by measuring the synaptop Hluorin fluorescence from individual large mossy fiber boutons in the hippocampus of a TV-42 transgenic mouse in which synaptopHluorin is specifically expressed in the mossy fiber boutons. We found that there are 2 distinct vesicle pools: a resting pool, which is resistant to exocytosis, and a releasable pool. The initially docked vesicles are easily depleted and the readily releasable pool (RRP) is replenished by the reserve subpopulation of the releasable pool ("reserve" releasable pool, RsvRP). When some of the silent synapses, which are pre-existing but are incapable of transmission, are recruited for transmission, a large number of alternative neuronal circuits are created in an off/on-switching manner. Here, we show that protein kinase C (PKC) activation facilitates presynaptic unsilencing at certain release sites, provided by the large mossy fiber boutons in the mouse hippocampus, by redirecting the synaptic vesicles from the resting pool to the RsvRP. At other sites PKC also facilitates the replenishment of the RRP from the RsvRP. Synaptic transmission was also potentiated through a mechanism involving non-PKC C1 domain-containing receptors, which would increase the size of the RRP. Thus, PKC and non-PKC mechanisms differentially reorganize vesicular dynamics and synergistically potentiate transmission.

Exocytosis is a common process for the secretion of physiologically active substances such as neurotransmitter, hormone, and inflammatory mediators. Exocytosis is triggered by an increase in intracellular calcium ion concentration. At the nerve terminal, voltage dependent calcium channels (VDCCs) are responsible for this calcium increase. There are several types of VDCC which are different from each other in their electro-physiological and pharmacological characteristics. In order to identify the types of VDCC at the cholinergic nerve terminal, acetylcholine (ACh) release from the electric organ synaptosomes was measured in the presence of type-specific channel blockers. At least three types of VDCC were involved in the ACh release, and N- and P/Q-type VDCC had a major contribution. Adenosine receptor A1 was coupled with N-type VDCC and had negative feedback regulation of ACh release, while A2 receptor coupled with P/Q-type VDCC enhanced the ACh release. Investigation of the inhibitory effects of antibodies from patients of autoimmune disease Lambert-Eaton syndrome on ACh release revealed that P/Q-type channel was a target for the autoantibodies. Unlike the nerve terminal, little is known about the mechanism and molecules involved in the exocytosis of immune cells. Ion channel activities of secretory granule proteins of mast cells were observed. The calcium dependency of the open probability of the channel was similar to that of histamine release from mast cells. We also showed the expression of some SNARE proteins in RBL-2H3 cells. Localization and dynamics of VAMP-7 and syntaxin-3 after antigen stimulation suggested the involvement of SNARE proteins in the exocytosis of mast cells.

Insulin is the only 'hypoglycemic' hormone synthesized in and secreted from the pancreatic beta cell. Type 2 diabetes results from both secretory failure in the beta cell and insulin resistance in the target tissues for insulin. Attempts to develop anti-diabetic drugs that induce insulin secretion from residual beta cells in type 2 diabetic patients originate from the serendipitous discovery of sulphonylureas as hypoglycemic agents 60 years ago. Generally, secretion is carried out by sequential processes such as granule formation, intracellular traffic, granule docking/priming and the final step, exocytosis (secretory cascade). In the beta cell, recent progress in cell biology enables us to analyze each step in the secretory cascade and to reveal controlling mechanisms. This review describes regulatory mechanisms of insulin release by distinct nutrients, hormones and neurotransmitters, and roles of second messengers and protein phosphorylation in the insulin secretory cascade. Possible development of insulinotropic drugs for the treatment of type 2 diabetes has been also discussed.

Growth cones have been regarded to play central roles in forming complex neural network through target tract selection and target cell recognition. We have found that the vesicle fusion molecular system in growth cones is similar to that in synapses. The cleavage of SNAP25, a protein involved in neurotransmitter release in synapse, resulted in inhibition of neurite elongation. Inversely the overexpression of VAMP2/synaptobrevin, another protein involved in neurotransmitter release in synapse, in PC12 cells resulted in promotion of neurite elongation. We have recently isolated a novel components at cadherin-based cell-cell adherens junctions, named neurabin I, neurabin II, and afadin. Neurabin I is specifically expressed in nervous tissues and localized at synapses. Afadin is uviquitously expressed in various tissues and highly concentrated at cell-cell adherens junctions of small intestine epithelial cells, cardiac muscles, synapses. We have identified the transmembrane protein binding to afadin, and named it nectin, which belongs to immunoglobulin superfamily. Nectin, afadin, neurabin I, and neurabin II may be key molecules to find out various unknown guidance cues which can induce regenerating growth cones to repair the functional neuronal connections in central nervous system after injury.

Oxytocin and vasopressin (AVP), which are octapeptides with a molecular mass of approximately 1.1 kd, are posterior pituitary hormones. AVP produced by magnocellular neurons in the supraoptic and paraventricular nuclei of the hypothalamus is in a form bound to the specific peptide neurophysin II, in the neurosecretory granula (NSG). Cleavage of this prohormone occurs within the NSGs during its transport via the axons to the neurohypophysis. Neurohypophyseal secretion of these peptides is believed to occur by exocytosis, which might be regulated by intracellular Ca++. The biosynthesis and secretion of AVP are mainly regulated by plasma osmolality (osmotic regulation) and blood pressure or blood volume (nonosmotic regulation). Some drugs, neurotransmitters, and other chemical agents modulate the regulation of AVP release.

To clarify the physiological significance of g-s connection (intimate apposition of the glomus cell to the smooth muscle), vascular responses of the carotid labyrinth to both catecholamines and chemoreceptor stimulants were investigated using Xenopus laevis. The results obtained were as follows. Density of dense-cored vesicles in the glomus cell was significantly varied in three different parts of the cytoplasm (N, M, P). In the part of the cytoplasm containing the nucleus (N) the density was lowest, and in the processes (P) it was highest. This bias in distribution was intensified by efferent stimulation of the glossopharyngeal nerve. In the artificially perfused labyrinth, the administration of catecholamines (adrenaline, noradrenaline and dopamine) decreased the outflow of the internal carotid artery in many cases. In a few cases it increased the internal outflow. Phentolamine changed the decrease of the outflow to an increase, while propranolol changed the increase to decrease. Acetylcholine strongly reduced the internal outflow. This response was depressed by atropine, hexamethonium and phentolamine, while intensified by propranolol. Sodium cyanide reduced the internal outflow. This effect was depressed by phentolamine. The possibility that the glomus cell participates in controlling the blood flow of the labyrinth as a result of the secretion of catecholamine through the g-s connection is discussed.