Endosomal location of dopamine receptors in neuronal cell cytoplasm

Five subtypes of dopamine receptor exist in two subfamilies: two D₁-like (D₁ and D₅) and three D₂-like (D₂, D₃ and D₄). We produced novel monoclonal antibodies against all three D₂-like receptors and used them to localize receptors in Ntera-2 (NT-2) cells, the human neuronal precursor cell line. Most of the immunostaining for all three receptors colocalized with mannose-6-phosphate receptor, a marker for late endosomes formed by internalization of the plasma membrane. This result was obtained with antibodies against three different epitopes on the D₃ receptor, to rule out the possibility of cross-reaction with another protein, and controls without primary antibody or in the presence of competitor antigen were completely negative. In rat cerebral cortex and hippocampus, some of the dopamine receptor staining was found in similar structures in neuronal cell cytoplasm. Only some of the neurons were positive for dopamine receptors and the pattern was consistent with previously-reported patterns of innervation by dopamine-producing neurons. Endosomal dopamine receptors may provide a useful method for identifying cell bodies of dopamine-responsive neurons to complement methods that detect only active receptors in the neuronal cell membrane.     

Role of P2 purinergic receptors in synaptic transmission under normoxic and ischaemic conditions in the CA1 region of rat hippocampal slices

The role of ATP and its stable analogue ATPγS [adenosine-5′-o-(3-thio)triphosphate] was studied in rat hippocampal neurotransmission under normoxic conditions and during oxygen and glucose deprivation (OGD). Field excitatory postsynaptic potentials (fEPSPs) from the dendritic layer or population spikes (PSs) from the soma were extracellularly recorded in the CA1 area of the rat hippocampus. Exogenous application of ATP or ATPγS reduced fEPSP and PS amplitudes. In both cases the inhibitory effect was blocked by the selective A₁ adenosine receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and was potentiated by different ecto-ATPase inhibitors: ARL 67156 (6-N,N-diethyl-D-β,γ-dibromomethylene), BGO 136 (1-hydroxynaphthalene-3,6-disulfonate) and PV4 [hexapotassium dihydrogen monotitanoundecatungstocobaltate(II) tridecahydrate, K₆H₂[TiW₁₁CoO₄₀]·13H₂O]. ATPγS-mediated inhibition was reduced by the P2 antagonist suramin [8-(3-benzamido-4-methylbenzamido)naphthalene-1,3,5-trisulfonate] at the somatic level and by other P2 blockers, PPADS (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonate) and MRS 2179 (2′-deoxy-N ⁶-methyladenosine 3′,5′-bisphosphate), at the dendritic level. After removal of both P2 agonists, a persistent increase in evoked synaptic responses was recorded both at the dendritic and somatic levels. This effect was prevented in the presence of different P2 antagonists. A 7-min OGD induced tissue anoxic depolarization and was invariably followed by irreversible loss of fEPSP. PPADS, suramin, MRS2179 or BBG (brilliant blue G) significantly prevented the irreversible failure of neurotransmission induced by 7-min OGD. Furthermore, in the presence of these P2 antagonists, the development of anoxic depolarization was blocked or significantly delayed. Our results indicate that P2 receptors modulate CA1 synaptic transmission under normoxic conditions by eliciting both inhibitory and excitatory effects. In the same brain region, P2 receptor stimulation plays a deleterious role during a severe OGD insult.     

HUPO BPP pilot study: a proteomics analysis of the mouse brain of different developmental stages

This study is a part of the HUPO Brain Proteome Project (BPP) pilot study, which aims at obtaining a reliable database of mouse brain proteome, at the comparison of techniques, laboratories, and approaches as well as at preparing subsequent proteome studies of neurologic diseases. The C57/Bl6 mouse brains of three developmental stages at embryonic day 16 (E16), postnatal day 7 (P7), and 8 wk (P56) (n = 5 in each group) were provided by the HUPO BPP executive committee. The whole brain proteins of each animal were individually prepared using 2-DE coupled with PDQuest software analysis. The protein spots representing developmentally related or stably expressed proteins were then prepared with in-gel digestion followed with MALDI-TOF/TOF MS/MS and analyzed using the MASCOT search engines to search the Swiss-Prot or NCBInr database. The 2-DE gel maps of the mouse brains of all of the developmental stages were obtained and submitted to the Data Collection Centre (DCC). The proteins alpha-enolase, stathmin, actin, C14orf166 homolog, 28,000 kDa heat- and acid-stable phosphoprotein, 3-mercaptopyruvate sulfurtransferase and 40 S ribosomal protein S3a were successfully identified. A further Western blotting analysis demonstrated that enolase is a protein up-regulated in the mouse brain from embryonic stage to adult stage. These data are helpful for understanding the proteome changes in the development of the mouse brain.     

International Symposium on High Performance Proteomics 2007: a contribution to Proteomics May 14-16, 2007 Dortmund, Germany

The symposium High Performance Proteomics was held in Dortmund on May 14-16, 2007, to celebrate the opening of the Zentrum für Angewandte Proteomik as well as the 6(th) anniversary of the German Human Brain Proteome Project. It offered an outstanding opportunity to obtain a broad overview about all fields of proteomics and related fields, combining the expertise of biochemists, physicians, bioinformatics, mathematicians and other researchers in Life Sciences. The main topics were the presentation of state-of-the-art proteomics technologies as well as possible transfer models for industrial applications. An accompanying industrial exhibition, as well as a discussion panel, offered the possibility to get in contact with colleagues and potential industrial partners. A visit to the former colliery Zeche Zollern and the social event at the Harenberg City-Center with an excellent view around Dortmund also left time for further communication between the more than 200 attendees.     

情绪的脑机制研究在社会认知神经科学中的进展

社会认知神经科学是近几年国外新兴起的交叉学科,旨在阐述社会性、情绪性的体验与行为的心理和神经基础。它综合了认知神经科学与社会心理学研究的长处,对刻板印象、态度与态度改变、他人知觉、自我认知以及情绪与认知交互作用等方面进行了深入研究。主要范式是应用认知神经科学的方法来验证社会心理学在这些范畴上的各种不同理论观点,并在某些方面取得了突破性进展,但仍存在着广泛的发展空间。随着当前各种脑成像技术的革新,人们对情绪状态下大脑的神经活动的了解在原来认知的层面上有了进一步提升。本文主要阐述社会认知神经科学在情绪的脑机制研究上所取得的进展。     

Comments on the eyes of tardigrades

A survey is given on the scarce information on the visual organs (eyes or ocelli) of Tardigrada. Many Eutardigrada and some Arthrotardigrada, namely the Echiniscidae, possess inverse pigment-cup ocelli, which are located in the outer lobe of the brain, and probably are of cerebral origin. Occurrence of such organs in tardigrades, suggested as being eyeless, has never been checked. Depending on the species, response to light (photokinesis) is negative, positive or indifferent, and may change during the ontogeny. The tardigrade eyes of the two eutardigrades examined up to now comprise a single pigment cup cell, one or two microvillous (rhabdomeric) sensory cells and ciliary sensory cell(s). In the eyes of the eutardigrade Milnesium tardigradum the cilia are differentiated in an outer branching segment and an inner (dendritic) segment. Because of the scarcity of information on the tardigrade eyes, their homology with the visual organs of other bilaterians is currently difficult to establish and further comparative studies are needed. Thus, the significance of these eyes for the evolution of arthropod visual systems is unclear yet.     

肺癌脑转移预后的多因素分析

目的:探索肺癌患者脑转移的预后影响因素。方法:回顾性分析我院2001~2010年216例肺癌患者的临床资料。运用SPSS软件包,通过比例风险模型(Cox模型)进行单因素和多因素分析,采用Kaplan-Merer法进行生存分析并描绘生存曲线和进行Log-rank时序检验。结果:通过单因素分析显示PS评分(p=0.006)、脑转移病灶数目(P=0.038)、有无脑转移的症状(P=0.001)、有无颅外器官转移(P=0.037)和治疗方法(P=0.045)等对生存期有影响,而年龄(P=0.887)、性别(P=0.207)、病理类型(P=0.727)、肺癌的部位(P=0.104)、脑转移病灶部位(P=0.401)对生存期无影响。多因素分析显示影响肺癌脑转移预后的主要因素依次为PS评分、有无脑转移的症状和治疗方法,而有无颅外器官转移和脑转移灶的数目等因素被削弱。结论:PS评分、有无颅外器官转移和脑转移病症灶的多少是脑转移肺癌患者的独立预后因素。     

Genetic associations with brain cortical thickness in multiple sclerosis

Multiple sclerosis (MS) is characterized by temporal and spatial dissemination of demyelinating lesions in the central nervous system. Associated neurodegenerative changes contributing to disability have been recognized even at early disease stages. Recent studies show the importance of gray matter damage for the accrual of clinical disability rather than white matter where demyelination is easily visualized by magnetic resonance imaging (MRI). The susceptibility to MS is influenced by genetic risk, but genetic factors associated with the disability are not known. We used MRI data to determine cortical thickness in 557 MS cases and 75 controls and in another cohort of 219 cases. We identified nine areas showing different thickness between cases and controls (regions of interest, ROI) (eight of them were negatively correlated with Kurtzke's expanded disability status scale, EDSS) and conducted genome‐wide association studies (GWAS) in 464 and 211 cases available from the two data sets. No marker exceeded genome‐wide significance in the discovery cohort. We next combined nominal statistical evidence of association with physical evidence of interaction from a curated human protein interaction network, and searched for subnetworks enriched with nominally associated genes and for commonalities between the two data sets. This network‐based pathway analysis of GWAS detected gene sets involved in glutamate signaling, neural development and an adjustment of intracellular calcium concentration. We report here for the first time gene sets associated with cortical thinning of MS. These genes are potentially correlated with disability of MS.     

Identification of the Cysteine Residue Responsible for Disulfide Linkage of Na+ Channel α and β2 Subunits

Voltage-gated Na⁺ channels in the brain are composed of a single pore-forming α subunit, one non-covalently linked β subunit (β1 or β3), and one disulfide-linked β subunit (β2 or β4). The final step in Na⁺ channel biosynthesis in central neurons is concomitant α-β2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding β2) null mice have reduced Na⁺ channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant β2 cDNA constructs to investigate the cysteine residue(s) responsible for α-β2 subunit covalent linkage. We demonstrate that a single cysteine-to-alanine substitution at extracellular residue Cys-26, located within the immunoglobulin (Ig) domain, abolishes the covalent linkage between α and β2 subunits. Loss of α-β2 covalent complex formation disrupts the targeting of β2 to nodes of Ranvier in a myelinating co-culture system and to the axon initial segment in primary hippocampal neurons, suggesting that linkage with α is required for normal β2 subcellular localization in vivo. WT β2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant β2 subunits, which cannot form disulfide bonds with α, are removed by detergent. Taken together, our results demonstrate that α-β2 covalent association via a single, extracellular disulfide bond is required for β2 targeting to specialized neuronal subcellular domains and for β2 association with the neuronal cytoskeleton within those domains.     

Estrogen receptor β and its domains interact with casein kinase 2, phosphokinase C, and N-myristoylation sites of mitochondrial and nuclear proteins in mouse brain

The localization of estrogen receptor (ER)β in mitochondria suggests ERβ-dependent regulation of genes, which is poorly understood. Here, we analyzed the ERβ interacting mitochondrial as well as nuclear proteins in mouse brain using pull-down assay and matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS). In the case of mitochondria, ERβ interacted with six proteins of 35-152 kDa, its transactivation domain (TAD) interacted with four proteins of 37-172 kDa, and ligand binding domain (LBD) interacted with six proteins of 37-161 kDa. On the other hand, in nuclei, ERβ interacted with seven proteins of 30-203 kDa, TAD with ten proteins of 31-160 kDa, and LBD with fourteen proteins of 42-179 kDa. For further identification, these proteins were cleaved by trypsin into peptides and analyzed by MALDI-MS using mascot search engine, immunoprecipitation, immunoblotting, and far-Western blotting. To find the consensus binding motifs in interacting proteins, their unique tryptic peptides were analyzed by the motif scan software. All the interacting proteins were found to contain casein kinase (CK) 2, phosphokinase (PK)C phosphorylation, and N-myristoylation sites. These were further confirmed by peptide pull-down assays using specific mutations in the interacting sites. Thus, the present findings provide evidence for the interaction of ERβ with specific mitochondrial and nuclear proteins through consensus CK2, PKC phosphorylation, and N-myristoylation sites, and may represent an essential step toward designing selective ER modulators for regulating estrogen-mediated signaling.