Solution structure of the SWIRM domain of human histone demethylase LSD1

SWIRM is an evolutionarily conserved domain involved in several chromatin-modifying complexes. Recently, the LSD1 protein, which bears a SWIRM domain, was found to be a demethylase for Lys4-methylated histone H3. Here, we report a solution structure of the SWIRM domain of human LSD1. It forms a compact fold composed of 6 alpha helices, in which a 20 amino acid long helix (alpha4) is surrounded by 5 other short helices. The SWIRM domain structure could be divided into the N-terminal part (alpha1-alpha3) and the C-terminal part (alpha4-alpha6), which are connected to each other by a salt bridge. While the N-terminal part forms a SWIRM-specific structure, the C-terminal part adopts a helix-turn-helix (HTH)-related fold. We discuss a model in which the SWIRM domain acts as an anchor site for a histone tail.     

Over-expression of Kv1.5 in rat cardiomyocytes extremely shortens the duration of the action potential and causes rapid excitation

BACKGROUND: Genetically abnormal action potential duration (APD) can be a cause of arrhythmias that include long and short QT interval syndrome. PURPOSE: The aim of this study was to evaluate the arrhythmogenic effect of short QT syndrome induced by the over-expression of Kv1.5 in rat. METHODS: From Sprague-Dawley rats on fetal days 18-19, cardiomyocytes were excised and cultured with and without transfection with the Kv-1.5 gene using an adenovirus vector. The expression of Kv1.5 was proven by immunohistochemistry and Western blot analysis. In the culture dish and in the whole cells, the electrical activities were recorded using the whole-cell patch-clamp technique and the effects of 4-AP and verapamil were tested. RESULTS: After transfection with Kv1.5 for 12h, immunohistochemical staining and Western blot analysis were positive for Kv1.5 while they were negative in the control transfected with only Lac-Z. In the culture dish, the myocytes showed spontaneous beating at 115beats/min (bpm) just prior to the transfection with Kv1.5 and increased to 367bpm at 24h. The control myocytes showed stable beating rates during culturing. 4-AP at 200microM slowed down the rate and verapamil abolished the beating. In the whole cells, the maximal resting membrane potential was slightly depolarized and APD was extremely abbreviated both at 50% and 90% of repolarization compared with those of the control. Rapid spontaneous activities were found in a single myocyte with Kv1.5 transfection and 4-AP slowed down the frequency of the activities with a reversal of the shortened APD. CONCLUSION: The over-expression of Kv1.5 induced short APD and triggered activities in rat cardiomyocytes. This model can be used to study the arrhythmogenic substrate of short QT syndrome.     

The trehalose transporter 1 gene sequence is conserved in insects and encodes proteins with different kinetic properties involved in trehalose import into peripheral tissues

We recently cloned a trehalose transporter gene (Tret1) that contributes to anhydrobiosis induction in the sleeping chironomid Polypedilum vanderplanki Hinton. Because trehalose is the main haemolymph sugar in most insects, they might possess Tret1 orthologs involved in maintaining haemolymph trehalose levels. We cloned Tret1 orthologs from four species in three insect orders. The similarities of the amino acid sequence to TRET1 in P. vanderplanki were 58.5–80.4%. Phylogenetic analysis suggested the Tret1 sequences were conserved in insects. The Xenopus oocyte expression system showed apparent differences in the K_m and V_max values for trehalose transport activity among the six proteins encoded by the corresponding orthologs. The TRET1 orthologs of Anopheles gambiae (K_m: 45.74 ± 3.58 mM) and Bombyx mori (71.58 ± 6.45 mM) showed low trehalose affinity, whereas those of Apis mellifera (9.42 ± 2.37 mM) and Drosophila melanogaster (10.94 ± 7.70 mM) showed high affinity. This difference in kinetics might be reflected in the haemolymph trehalose:glucose ratio of each species. Tret1 was expressed not only in the fat body but also in muscle and testis. These findings suggest that insect TRET1 is responsible for the release of trehalose from the fat body and the incorporation of trehalose into other tissues that require a carbon source, thereby regulating trehalose levels in the haemolymph.     

Monoclonal antibody specific for α2→8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing 28-linked polysialic acid (8Neu5Gc2) _n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (8KDN2) _n 6(KDN23Gal13GlNAc13) GalNAc1 residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues. The mAb.kdn8kdn was shown specifically to recognize the 28-linked oligo/polyKDN sequences, (8KDN2) _n , and to be able to distinguish specifically (8KDN2) _n chains from (8Neu5Ac2) _n and (8Neu5Gc2) _n chains. The antibody was used successfully for the immunohistochemical detection of reactive KDN epitopes in sections of paraffin embedded rat pancreas. Several controls verified the specificity of the immunohistochemical staining, thus providing the first demonstration of (8KDN2) _n sequences in a mammalian tissue. The mAb.kdn8kdn can now be used to search further for glycoconjugates containing (8KDN2) _n chains and will facilitate studies on their biosynthesis, intracellular localization and function.     

Glial uptake of neurotransmitter glutamate from the extracellular fluid studied in vivo by microdialysis and (13)C NMR

Glial uptake of neurotransmitter glutamate (GLU) from the extracellular fluid was studied in vivo in rat brain by (13)C NMR and microdialysis combined with gas-chromatography/mass-spectrometry. Brain GLU C5 was (13)C enriched by intravenous [2,5-(13)C]glucose infusion, followed by [(12)C]glucose infusion to chase (13)C from the small glial GLU pool. This leaves [5-(13)C]GLU mainly in the large neuronal metabolic pool and the vesicular neurotransmitter pool. During the chase, the (13)C enrichment of whole-brain GLU C5 was significantly lower than that of extracellular GLU (GLU(ECF)) derived from exocytosis of vesicular GLU. Glial uptake of neurotransmitter [5-(13)C]GLU(ECF) was monitored in vivo through the formation of [5-(13)C,(15)N]GLN during (15)NH(4)Ac infusion. From the rate of [5-(13)C,(15)N]GLN synthesis (1.7 +/- 0.03 micromol/g/h), the mean (13)C enrichment of extracellular GLU (0.304 +/- 0.011) and the (15)N enrichment of precursor NH(3) (0.87 +/- 0.014), the rate of synthesis of GLN (V'(GLN)), derived from neurotransmitter GLU(ECF), was determined to be 6.4 +/- 0.44 micromol/g/h. Comparison with V(GLN) measured previously by an independent method showed that the neurotransmitter provides 80-90% of the substrate GLU pool for GLN synthesis. Hence, under our experimental conditions, the rate of 6.4 +/- 0.44 micromol/g/h also represents a reasonable estimate for the rate of glial uptake of GLU(ECF), a process that is crucial for protecting the brain from GLU excitotoxicity.     

cDNA cloning of putative rat acetyl-CoA transporter and its expression pattern in brain

Rat acetyl-CoA transporter gene (Acatn) encodes a hydrophobic multi-transmembrane protein involved in the O-acetylation of gangliosides. O-acetylated gangliosides have been found to play important roles in the embryonic development of the nervous system. We have isolated rat Acatn cDNA by PCR cloning. The amino acid sequence of rat Acatn exhibited 92% and 96% homology with human and mouse sequences, respectively. The mRNA was expressed in brain at all developmental stages. Acatn expression was higher in embryonic and postnatal rats than in adult rats. Cellular localization of Acatn mRNA in adult rat brain was also analyzed by in situ hybridization. Acatn mRNA expression was detected in the neuronal cells of cerebellum, hippocampus, hypothalamus, cortex, olfactory bulb, and dorsal and ventral anterior olfactory nucleus in adult rat brain.     

The target of rapamycin complex 2 controls dendritic tiling of Drosophila sensory neurons through the Tricornered kinase signalling pathway

To cover the receptive field completely and non‐redundantly, neurons of certain functional groups arrange tiling of their dendrites. In Drosophila class IV dendrite arborization (da) neurons, the NDR family kinase Tricornered (Trc) is required for homotypic repulsion of dendrites that facilitates dendritic tiling. We here report that Sin1, Rictor, and target of rapamycin (TOR), components of the TOR complex 2 (TORC2), are required for dendritic tiling of class IV da neurons. Similar to trc mutants, dendrites of sin1 and rictor mutants show inappropriate overlap of the dendritic fields. TORC2 components physically and genetically interact with Trc, consistent with a shared role in regulating dendritic tiling. Moreover, TORC2 is essential for Trc phosphorylation on a residue that is critical for Trc activity in vivo and in vitro. Remarkably, neuronal expression of a dominant active form of Trc rescues the tiling defects in sin1 and rictor mutants. These findings suggest that TORC2 likely acts together with the Trc signalling pathway to regulate the dendritic tiling of class IV da neurons, and thus uncover the first neuronal function of TORC2 in vivo.     

Potentiation of nerve growth factor-induced neurite outgrowth by fluvoxamine: role of sigma-1 receptors, IP3 receptors and cellular signaling pathways

Background

Selective serotonin reuptake inhibitors (SSRIs) have been widely used and are a major therapeutic advance in psychopharmacology. However, their pharmacology is quite heterogeneous. The SSRI fluvoxamine, with sigma-1 receptor agonism, is shown to potentiate nerve-growth factor (NGF)-induced neurite outgrowth in PC 12 cells. However, the precise cellular and molecular mechanisms underlying potentiation by fluvoxamine are not fully understood. In this study, we examined the roles of cellular signaling pathways in the potentiation of NGF-induced neurite outgrowth by fluvoxamine and sigma-1 receptor agonists.

Methods and Findings

The effects of three SSRIs (fluvoxamine, sertraline, paroxetine) and three sigma-1 receptor agonists (SA4503, 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP), and dehydroepiandrosterone (DHEA)-sulfate) on NGF-induced neurite outgrowth in PC12 cells were examined. Also examined were the effects of the sigma-1 receptor antagonist NE-100, inositol 1,4,5-triphosphate (IP₃) receptor antagonist, and specific inhibitors of signaling pathways in the potentiation of NGF-induced neurite outgrowth by selective sigma-1 receptor agonist SA4503. Fluvoxamine (but not sertraline or paroxetine) and the sigma-1 receptor agonists SA4503, PPBP, and DHEA-sulfate significantly potentiated NGF-induced neurite outgrowth in PC12 cells in a concentration-dependent manner. The potentiation by fluvoxamine and the three sigma-1 receptor agonists was blocked by co-administration of the selective sigma-1 receptor antagonist NE-100, suggesting that sigma-1 receptors play a role in blocking the enhancement of NGF-induced neurite outgrowth. Moreover, the potentiation by SA4503 was blocked by co-administration of the IP₃ receptor antagonist xestospongin C. In addition, the specific inhibitors of phospholipase C (PLC-γ), phosphatidylinositol 3-kinase (PI3K), p38MAPK, c-Jun N-terminal kinase (JNK), and the Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways blocked the potentiation of NGF-induced neurite outgrowth by SA4503.

Conclusion

These findings suggest that stimulation of sigma-1 receptors and subsequent interaction with IP₃ receptors, PLC-γ, PI3K, p38MAPK, JNK, and the Ras/Raf/MAPK signaling pathways are involved in the mechanisms of action of sigma-1 receptor agonists such as fluvoxamine and SA4503.