Inositol 1,3,4,5-tetrakisphosphate binding activities of neuronal and non-neuronal synaptotagmins. Identification of conserved amino acid substitutions that abolish inositol 1,3,4,5-tetrakisphosphate binding to synaptotagmins III, V, and X

Synaptotagmins I and II are essential for Ca2+-regulated exocytosis of synaptic vesicles from neurons, probably serving as Ca2+ sensors. This Ca2+-sensing function is thought to be disrupted by binding of an inositol 1,3,4,5-tetrakisphosphate (IP4) to the C2B domain of synaptotagmin I or II (Fukuda, M., Moreira, J. E., Lewis, F. M. T., Sugimori, M., Niinobe, M., Mikoshiba, K., and Llinás, R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10708-10712). Recently, several synaptotagmin isoforms, expressed outside the nervous system, have been identified in rats and proposed to be involved in constitutive vesicle traffic. To test whether the inositol high polyphosphates also regulate constitutive vesicle traffic by binding to the non-neuronal synaptotagmins, we examined the IP4 binding properties of the recombinant C2 domains of both neuronal (III, V, X, and XI) and non-neuronal (VI-VIII and IX) synaptotagmins. The C2B domains of synaptotagmins VII-IX and XI had strong IP4 binding activity, but the C2B domain of synaptotagmin VI showed very weak IP4 binding activity. In contrast, there was no significant IP4 binding activity of the C2B domains of synaptotagmins III, V, and X or any of the C2A domains. A phylogenetic tree of the C2 domains of 11 isoforms revealed that synaptotagmins III, V, VI, and X (IP4-insensitive or very weak IP4-binding isoforms) belong to the same branch. Based on the sequence comparison between the IP4-sensitive and -insensitive isoforms, we performed site-directed mutagenesis of synaptotagmin III and identified several amino acid substitutions that abolish IP4 binding activity. Our data suggest that the inositol high polyphosphates might also regulate constitutive vesicle traffic via binding to the IP4-sensitive non-neuronal synaptotagmins.     

Antisense knock out of the inositol 1,3,4,5-tetrakisphosphate receptor GAP1(IP4BP) in the human erythroleukemia cell line leads to the appearance of intermediate conductance K(Ca) channels that hyperpolarize the membrane and enhance calcium influx

To study the role of the inositol 1,3,4,5-trisphosphate-binding protein GAP1(IP4BP) in store-operated Ca2+ entry, we established a human erythroleukemia (HEL) cell line in which the expression of GAP1(IP4BP) was substantially reduced by transfection with a vector containing antisense DNA under control of a Rous Sarcoma virus promoter and the Escherichia coli LacI repressor (AS-HEL cells). Control cells were transfected with vector lacking antisense DNA (V-HEL cells). GAP1(IP4BP) protein, which is a member of the GTPase-activating protein (GAP1) family, was reduced by 85% in AS-HEL cells and was further reduced by 96% by treatment with isopropylthio-beta-D- galactoside to relieve LacI repression. The loss of GAP1(IP4BP) was associated with both a membrane hyperpolarization and a substantially increased Ca2+ entry induced by thrombin or thapsigargin. The activation of intermediate conductance Ca2+-activated K+ channels in AS-HEL cells (not seen in V-HEL cells) was responsible for the membrane hyperpolarization and the enhanced Ca2+ entry, and both were blocked by charybdotoxin. Stimulated V-HEL cells did not hyperpolarize and basal Ca2+ influx was unaffected by charybdotoxin. In V-HEL cells hyperpolarized by removal of extracellular K+, the thapsigargin-stimulated Ca2+ influx was increased. Expression of mRNA for the human Ca2+-activated intermediate conductance channel KCa4 was equivalent in both AS-HEL and V-HEL cells, suggesting that the specific appearance of calcium-activated potassium current (IK(Ca)) in AS-HEL cells was possibly due to modulation of preexisting channels. Our results demonstrate that GAP1(IP4BP), likely working through a signaling pathway dependent on a small GTP-binding protein, can regulate the function of K(Ca) channels that produce a hyperpolarizing current that substantially enhances the magnitude and time course of Ca2+ entry subsequent to the release of internal Ca2+ stores.     

2-Deoxy derivative is a partial agonist of the intracellular messenger inositol 3,4,5,6-tetrakisphosphate in the epithelial cell line T84

We have synthesized the first deoxy analogues of myo-inositol 3,4,5, 6-tetrakisphosphate (1) [Ins(3,4,5,6)P4], rac-2-deoxy-myo-inositol 3, 4,5,6-tetrakisphosphate (rac-2), 2-deoxy-myo-inositol 1,4,5, 6-tetrakisphosphate (ent-2), and rac-1-deoxy-myo-inositol 3,4,5, 6-tetrakisphosphate (rac-3). In order to evaluate the binding properties of the three derivatives to the yet unidentified intracellular binding sites for Ins(3,4,5,6)P4, the analogues were converted to membrane-permeant derivatives. Starting with common inositol precursors, various forms of Barton-McCombie deoxygenation and classical protection/deprotection procedures yielded the desired precursors rac-1-O-butyryl-2-deoxy-myo-inositol (rac-12), ent-3-O-butyryl-2-deoxy-myo-inositol (ent-12), and rac-2-O-butyryl-1-deoxy-myo-inositol (rac-19), respectively. Phosphorylation and subsequent deprotection yielded rac-2, ent-2, and rac-3. Alternatively, phosphorylation followed by alkylation with acetoxymethyl bromide gave the membrane-permeant derivatives 1-O-butyryl-2-deoxy-myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl) ester (rac-5), 3-O-butyryl-2-deoxy-myo-inositol 1,4,5,6-tetrakisphosphate octakis(acetoxymethyl) ester (ent-5), and 2-O-butyryl-1-deoxy-myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl) ester (rac-6), respectively. We examined the potency of the membrane-permeant deoxy derivatives in inhibition of calcium-mediated chloride secretion (CaMCS) in intact T84 cells. Compared to the 1,2-di-O-butyryl-myo-inositol 3,4,5, 6-tetrakisphosphate octakis(acetoxymethyl) ester (4), the membrane-permeant derivative of Ins(3,4,5,6)P4 (1), the 2-deoxy derivative (rac-5) exhibited a slightly weaker inhibitory effect, while the enantiomerically pure 2-deoxy-Ins(1,4,5,6)P4 (ent-5) and the 1-deoxy derivative (rac-6) were inactive. As expected, the effect was stereoselective. Thus, the 1-hydroxyl group is apparently essential for binding and the inhibitory effect of Ins(3,4,5,6)P4 on chloride secretion, whereas the 2-hydroxyl group plays a less important role.     

Autoradiographic characterization of [3H]inositol (1,4,5) trisphosphate and [3H]inositol (1,3,4,5) tetrakisphosphate binding sites in human brain

Autoradiographic techniques were used to investigate the characteristics of tritiated inositol(1,4,5)trisphosphate ([3H]IP3) and inositol (1,3,4,5) tetrakisphosphate ([3H]IP4) binding to human brain. In brain sections [3H]IP3 exhibited a two-site binding with KD values of 87 nM and 9.3 microM respectively for the higher and lower affinity sites. [3H]IP4 also bound to two sites with KD values of 43 nM and 1.4 microM, respectively. With the conditions fixed in this study, [3H]IP3 and [3H]IP4 autoradiography in the cortex, caudate, hippocampus and cerebellum were performed. The most prominent [3H]IP3 binding among these regions was found in the cerebellum, particularly in the molecular layer. Within the hippocampus, the subiculum and the CA1 region showed much more prominent binding than the other subfields. [3H]IP4, binding was fairly homogeneous in the regions studied, with the exception of a slightly higher binding in the molecular layer of the cerebellum.     

Crack Bridging in Polymer Nanocomposites

Carbon nanotube reinforced composites offer enhancements in fracture properties since the reinforcing nanotubes provide a bridging mechanism to resist crack growth. In this paper, a study of crack bridging by nanotubes in a nanotube-reinforced polymer composite is presented. The process of crack bridging is idealized as normal pullout of the participating nanotubes from the polymer matrix. The resistance to crack growth due to bridging is taken as the aggregate of the resistance offered by all the nanotubes, ignoring any interaction among the nanotubes themselves. The pullout of a single nanotube from the polymer matrix is modeled as an axisymmetric, nearly one-dimensional problem. This is done by assuming that fracture along the nanotube–polymer interface is dominated by shear openings, and that the nanotube behaves as a rigid body. When the polymer is a linear elastic material, the force–displacement relation for pullout is obtained as a function of dimensionless variables representing the interfacial fracture energy and the pullout length scale. Applying the correspondence principle, the elastic results are extended to the case where the polymer is a linear viscoelastic material with a single relaxation time. The force–displacement relation is then a function of the viscoelastic properties of the polymer and the pullout velocity as well. Using these results, the apparent enhancement in the fracture energy of the composite is obtained. This provides a guideline to design these composites for desired fracture properties in terms of the interfacial strength of the nanotube–polymer interface and the volume fraction of the nanotubes. Results of numerical simulations of nanotube pullout are compared to the predictions of the analytical model.     

Fertilization stimulates an increase in inositol trisphosphate and inositol lipid levels in Xenopus eggs

Previous experiments from our lab have suggested that the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is required for sperm-induced egg activation in Xenopus laevis. Here we measure the endogenous production of both Ins(1,4,5)P3 and PIP2 during the sperm-induced and ionomycin-induced calcium wave in the egg and find that both increase following fertilization. Ins(1,4,5)P3 increases 3.2-fold from an unfertilized egg level of 0.13 pmole per egg (0.29 microM) to a peak of 0.42 pmole per egg (0.93 microM) as the calcium wave reaches the antipode in the fertilized egg. This continuous production of Ins(1,4,5)P3 during the time that the Ca2+ wave is propagating across the egg suggests the involvement of Ins(1,4,5)P3 in wave propagation. This increase in Ins(1,4,5)P3 is smaller in ionomycin-activated eggs than in sperm-activated eggs, suggesting that the sperm-induced production of Ins(1,4,5)P3 involves a PIP2 hydrolysis pathway that is not simply raising intracellular Ca2+. While one might expect PIP2 levels to fall as a result of hydrolysis, we find that PIP2 actually increases 2-fold. The total lipid fraction in unfertilized egg exhibits 0.8 pmole PIP2 per egg and this increases to 1.5 pmole as the calcium wave reaches the antipode. The PIP2 concentration peaks 2 min after the completion of the calcium wave at 1.8 pmole per egg. The amount of PIP2 in the animal and vegetal hemispheres of the egg was also measured by cutting frozen eggs in half. The vegetal hemisphere contained twice the amount of PIP2 as the animal hemisphere but it also contained twice the amount of lipid. Thus, there was an equivalent amount of PIP2 normalized to lipid in each hemisphere. Isolated animal and vegetal hemisphere cortices exhibit similar PIP2 concentrations, suggesting that the 2-fold higher total PIP2 in the vegetal half is not due to a gradient of PIP2 in the plasma membrane, but rather implies that cytoplasmic organelle membranes also contain PIP2.     

GAP在近世代数教学中的应用

针对近世代数研究内容较抽象的特点,介绍一款适合开展近世代数数学实验的软件GAP,借助实例说明如何在群论的教学和实验中运用GAP.     

Synaptic defects and compensatory regulation of inositol metabolism in inositol polyphosphate 1-phosphatase mutants

Phosphoinositides function as important second messengers in a wide range of cellular processes. Inositol polyphosphate 1-phosphatase (IPP) is an enzyme essential for the hydrolysis of the 1-phosphate from either Ins(1,4)P2 or Ins(1,3,4)P3. This enzyme is Li+ sensitive, and is one of the proposed targets of Li+ therapy in manic-depressive illness. Drosophila ipp mutants accumulate IP2 in their system and are incapable of metabolizing exogenous Ins(1,4)P2. Notably, ipp mutants demonstrate compensatory upregulation of an alternative branch in the inositol-phosphate metabolism tree, thus providing a means of ensuring continued availability of inositol. We demonstrate that ipp mutants have a defect in synaptic transmission resulting from a dramatic increase in the probability of vesicle release at larval neuromuscular junctions. We also show that Li+ phenocopies this effect in wild-type synapses. Together, these results support a role for phosphoinositides in synaptic vesicle function in vivo and mechanistically question the "lithium hypothesis."     

Bridging the gap with the five-factor model.

Comments on the original article Personality traits and the classification of mental Disorders: Toward a more complete integration in DSM–5 and an empirical model of psychopathology by Robert F. Krueger and Nicholas R. Eaton (see record 2010-13810-003). Some researchers had hoped the forthcoming Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) would ask psychiatrists (and the clinical psychologists and researchers who are also tied to the DSM) to leap the gap and embrace a trait-based taxonomy of personality pathology (Widiger & Trull, 2007). Krueger and Eaton (pp. 97–118, this issue) take a more pragmatic stance: They hope to coax psychiatrists across by introducing personality dimensions as an adjunct to familiar PD types; they envision that DSM-5 might serve “as a bridge” (p. 110, this issue) to a fully dimensional Diagnostic and Statistical Manual of Mental Disorders, Sixth Edition (DSM-6). We acknowledge the wisdom of this strategy and suggest ways to strengthen it. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Bridging the gap from bench to bedside in wound care

OBJECTIVES: Hepatitis B vaccination has been proven to be effective and well-tolerated. Certain neurological, ocular or systemic complications have, however, been reported to be induced by the vaccine. Clinicians should be aware of exceptional ocular complications. CLINICAL REPORT: Four patients under 50 years of age developed occlusion of the central vein of the retina after vaccination with recombinant hepatitis B vaccine. None of the classical causes of occlusion of the central vein of the retina could be evidenced. DISCUSSION: Several pathophysiological hypotheses have been proposed to explain these ocular manifestations after vaccination: role of immunocomplexes, antigenic cross-reactions, role of immediate hypersensitivity, simulation of a pathogenic lymphocyte repertoire. None of these hypotheses is entirely satisfactory. It is important however to emphasize the need for a complete general evaluation, including an ophthalmological examination in the presence of unexplained ocular manifestations following hepatitis B vaccination.     

Retinoid-X receptor signalling in the developing spinal cord

Retinoids regulate gene expression through the action of retinoic acid receptors (RARs) and retinoid-X receptors (RXRs), which both belong to the family of nuclear hormone receptors. Retinoids are of fundamental importance during development, but it has been difficult to assess the distribution of ligand-activated receptors in vivo. This is particularly the case for RXR, which is a critical unliganded auxiliary protein for several nuclear receptors, including RAR, but its ligand-activated role in vivo remains uncertain. Here we describe an assay in transgenic mice, based on the expression of an effector fusion protein linking the ligand-binding domain of either RXR or RAR to the yeast Gal4 DNA-binding domain, and the in situ detection of ligand-activated effector proteins by using an inducible transgenic lacZ reporter gene. We detect receptor activation in the spinal cord in a pattern that indicates that the receptor functions in the maturation of limb-innervating motor neurons. Our results reveal a specific activation pattern of Gal4-RXR which indicates that RXR is a critical bona fide receptor in the developing spinal cord.     

Calcium signalling in glial cells

Glial cells respond to a variety of external stimuli such as neurotransmitters, hormones or even mechanical stress by generating complex changes in the cytoplasmic Ca2+ concentration. This Ca2+ signal is controlled by an interplay of different mechanisms including plasmalemmal and intracellular Ca2+ channels, Ca2+ transporters and cytoplasmic Ca2+ buffers. In astrocytes, the Ca2+ signal can travel as waves within the syncytium spreading via gap junctions which might be regarded as a possible means for interglial communication. Ca2+ signalling is also an important medium for neurone-glia interaction: neuronal activity can trigger Ca2+ signals in glial cells and, in turn, there is evidence that glial Ca2+ signals can elicit responses in neurones. While glial cells are not equipped with the proper channels to generate action potentials, Ca2+ signalling could be the instrument by which these cells integrate and propagate signals in the CNS.     

Polyisoprenyl phosphates in intracellular signalling

In response to environmental stimuli, leukocyte membrane remodelling generates biologically active lipids that can serve as both intra- and extracellular mediators. There are several classes of lipids that can mediate inflammatory reactions. We report here on a new intracellular lipid signal that regulates oxygen-radical formation in neutrophils, a key response in microbial killing, inflammation and tissue injury. Screening of neutrophil-derived extracts rich in phosphorylated, non-saponifiable lipids revealed a potent inhibitor of superoxide anion (O2-) production. Structural analysis of biologically active fractions gave four major phosphorylated lipids: most abundant was presqualene diphosphate (PSDP). Upon activation of neutrophil receptors, PSDP and its monophosphate form, presqualene monophosphate (PSMP), undergo rapid remodelling. At submicromolar concentrations, PSDP but not PSMP inhibit O2- production by human neutrophil cell-free oxidase preparations. We prepared PSDP and PSMP by total organic synthesis and matched both the physical properties and biological activity of the neutrophil-derived compounds. Our results indicate that PSDP, a recognized intermediate of cholesterol biosynthesis, is present in immune effector cells and is a potent regulator of the cellular response in host defence.