bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hypochlorite‐induced white matter damage
Authors: Konat G. W. , Tirumalai P. 
Published: July 2010

The extent and severity of inflammation‐mediated white matter destruction correlates with neutrophil infiltration. Although the mechanisms are not fully understood, the generation of vast quantities of hypochlorite (HC) by activated neutrophils may be the major damaging factor. This study was undertaken to determine the toxicity of HC to white matter using brain stem slices from 2 month‐old female rats. Because of its high volatility under the incubation conditions (half‐life of ∼ 7.5 min), HC was administered every 30 min as 1 mm boluses providing an average concentration of ∼ 0.27 mm. Exposure period (1–3 h) was followed by 1 h recovery in normal medium, and by 2 h labelling with 3H‐leucine. Subsequently, slices were homogenized, and myelin was isolated by sucrose gradient ultracentrifugation. HC exposure inhibited total homogenate protein synthesis in the concentration‐ and time‐dependent manner. Thus, following 1, 2 and 3 h of exposure, total homogenate protein synthesis was reduced to ∼ 65, 45 and 30% of control. HC exposure preferentially suppressed the assembly of major structural proteins into the myelin sheath. Thus, 1 h exposure reduced the assembly of proteolipid protein and basic protein to ∼ 30% of control value. A significant amount of 3H‐leucine was also incorporated into myelin lipids. Unexpectedly, HC exposure profoundly stimulated 3H‐labelling of myelin lipids. Thus, after 1 and 2 h of exposure the labelling of myelin lipids was increased by ∼ 40 and 90% over control, respectively. In contrast, the labelling of nonmyelin lipids decreased to ∼ 50% of control. These results demonstrate that HC drastically alters myelin metabolism, and thus, may underlie neutrophil‐mediated inflammatory demyelination in multiple sclerosis.Acknowledgements:  Supported by NMSS research grant 3067A1.
Poster Sessions BP05: Neuroimmunology. Inhibition of adoptive transfer of EAE by nemo‐binding domain (NBD) peptides
Authors: Dasgupta S. , Zhou Y. , Jana M. , Pahan K. 
Published: July 2010

Experimental allergic encephalomyelitis (EAE) is the animal model for Multiple Sclerosis (MS), the chronic autoimmune disease of the central nervous system (CNS). Activation of NF‐κB requires the activity of IkB kinase (IKK) complex containing (IKKa and IKKb) and the regulatory protein NEMO (NF‐κB essential modifier). Recently it has been shown that peptides corresponding to the NEMO‐binding domain (NBD) of IKKa or IKKb specifically inhibit the induction of NF‐κB activation without inhibiting the basal NF‐κB activity. The present study underlines the importance of cell‐permeable NBD peptides in inhibiting the disease process of adoptively‐transferred EAE in female SJL/J mice. Immunocytochemical analysis of spinal cords of EAE mice showed that there was marked induction of NF‐κB activation as evidenced by enhanced p65 (the RelA subunit of NF‐κB) expression compared to that of control mice. Double‐labelling analysis of p65 and cell‐specific markers showed that p65 was mainly expressed by astrocytes, microglia and infiltrating macrophages. Next we examined the effect of NBD peptides on the disease process of EAE. Interestingly, clinical symptoms of EAE were much lower in mice receiving wild type NBD peptides. In contrast, mutated NBD peptides had no effect on the clinical symptoms of EAE. Taken together, our results support the conclusion that activation of NF‐κB participates in the disease process of EAE and that inhibitors of NF‐κB activation may ameliorate the neuroinflammatory disease process in MS patients.Acknowledgements:  This study was supported by NIH grants (NS39940 and AG19487.
Induction of nitric oxide synthase in microglial cells by MBP‐primed T cells
Authors: Dasgupta S. , Jana M. , Liu X. , Pahan K. 
Published: July 2010

The present study was undertaken to investigate the role of MBP‐primed T cells in the expression of iNOS in microglial cells. MBP‐primed T cells alone markedly induced the production of NO and the expression of iNOS protein and mRNA in mouse BV‐2 microglial cells. This induction of NO production was primarily dependent on the contact between MBP‐primed T cells and microglia. Similarly, MBP‐primed T cells also induced the contact‐mediated production of NO in mouse primary microglia. To understand the mechanism of induction of iNOS, we investigated the role of NF‐κB, transcription factor responsible for the induction of iNOS. MBP‐primed T cells alone were able to induce the activation of NF‐κB. Consistently, Dp65, a dominant‐negative mutant of p65, and SN50, a specific inhibitor of p50 NF‐κB, but not SN50M, a nonfunctional peptide mutant, inhibited MBP‐primed T cell‐induced production of NO in BV‐2 microglial cells suggesting that MBP‐primed T cell‐microglia contact induces the expression of iNOS through the activation of NF‐κB. Since IFN‐β has been used to treat MS patients, we examined the effect of IFN‐β on T cell‐mediated production of NO and activation of NF‐κB. Surprisingly, IFN‐β alone induced the production of NO and the activation NF‐κB in microglial cells. However, the pretreatment of MBP‐primed T cells with IFN‐β markedly inhibited the ability of MBP‐primed T cells to induce the production of NO and the activation NF‐κB in microglial cells, mechanisms which can help account for its efficacy in MS.Acknowledgements:  This study was supported by NIH grants (NS39940 and AG19487).
In vitro specification of CNS stem cells into either oligodendrocytes or neurons: their fate in vivo
Authors: Espinosa‐Jeffrey A. , Zhao P. M. , Becker‐Catania S. G. , Cole R. , De Vellis J. 
Published: July 2010

Cell loss within the central nervous system due to disease, trauma, or aging results in severe disability. Recent research indicates that proliferating populations of neural precursors are a promising source of cells for neural grafting and CNS repair. In our study, neural stem cells (NSCs) isolated from embryonic day (ED) 16 Sprague–Dawley rats were cultured in a novel serum‐free stem cell medium that allowed their propagation for extended periods of time, retaining immunoreactivity for both nestin and PSA‐NCAM, two markers characteristic of the stem cell phenotype. Alternatively, oligospheres were obtained from NSCs neurospheres by using a combination of an oligodendrocyte (OL) specification medium and the NSC medium. When neurospheres or oligospheres were plated as single spheres on poly‐d‐lysine and fed with OL specification medium their progenies gave rise to only OL progenitors that matured into MBP+ cells. Preliminary data from transplantation studies showed that when either neurospheres or oligospheres were grafted into the md rat brain cells survived and migrated extensively. Some of the neurosphere derived cells differentiated into NeuN+ cells. In contrast, oligosphere grafted into the md brain gave rise to transferrin, but not to GFAP or NeuN expressing cells. These results indicate that oligosphere derived cells only gave rise to OL progenitors, but not to neurons or astrocytes.Acknowledgements:  NICHD #06576 and AT Foundation.
Poster Sessions CP07: Ions, Channels, Pumps and Transporters. Functional relationship between Na/K‐pump and glutamate receptors in cerebellum granule cells
Authors: Boldyrev A. A. , Schoner W. 
Published: July 2010

Stationary level of reactive oxygen species (ROS) in cerebellum granule cells of 12‐day‐old‐rats was measured using three fluorescent dyes characteristic of different location within the neuronal cell: BODIPY 581/591 (for LOO.radicals), DCF‐DA (for H202) and DHR123 (OH‐radicals in mitochondria). When the neurons were activated by N‐methyl‐d‐aspartate (NMDA) a dose‐ and time‐dependent rise of the fluorescent signal was registered with each of the three dyes; the former dye provided the smallest and the latter the largest response. 3‐HPG, a ligand for metabotropic receptors decreases ROS fluorescence and suppressed the NMDA‐induced effect. NMDA and kainic acid presented simultaneously cumulatively increased ROS levels. Ouabain, specific inhibitors of	Na/K‐pump induced a considerable increase in ROS fluorescence, which was decreased by 2.5–5 mm KCl, 50 mkM Vanadate or 10 mkM D‐AP5, an inhibitor of NMDA‐activated ionic channels. The K0.5 for activation of ROS generation by Ouabain was more than 250 mkM, which is much higher than that for inhibition of Na/K‐ATPase or its rubidium pumping activity. The data show that the Na/K‐pump protein regulates ROS production by NMDA‐receptors and that the E1(Na) conformation of the Na/K‐pump being less sensitive to ouabain may be responsible for the effects. The data illustrate functional interaction between ionotropic and metabotropic receptors and Na/K‐ATPase.Acknowledgements:  Supported by DAAD, Grant 325‐sm, Germany.
Identification of a GM1/Sodium–Calcium exchanger complex in the nuclear envelope of non‐neuronal cells
Authors: Xie X. , Wu G. , Lu Z‐H. , Fontainhas A. , Ledeen R. W. 
Published: July 2010

Our previous studies identified a Na–Ca exchanger (NCX) that is tightly associated with GM1 ganglioside and potentiated by it in the nuclear envelope (NE) of NG108‐15 cells and primary neurons. The purpose of the present study was to explore whether this is a general phenomena or limited to neurons. Non‐neuronal C6 (glioma), HeLa (Epithelial carcinoma) and NCTC (connective tissue) cell lines were used. Immunocytochemical staining with anti‐NCX antibody and cholera toxin B subunit revealed that NCX and GM1 coexist in the nuclei from all 3 cell lines; in relation to plasma membrane, only HeLa cells showed staining for both NCX and GM1. Purified NE and non‐nuclear membrane mixture (mainly plasma membrane) from the 3 cell lines were immunoprecipitated with a mouse monoclonal anti‐NCX antibody and the precipitated proteins separated on SDS–PAGE. Analysis by immunoblot, showed that NCX is tightly associated with GM1 in the NE of all 3 cell lines. In contrast, NCX and the more loosely associated GM1 from plasma membrane of HeLa cells were separated by SDS–PAGE. Isolated nuclei from C6 cells were used for 45Ca2+ uptake experiments, which provided functional evidence that this exchanger protein is strongly potentiated by GM1. In similar experiments with Jurkat cells (T lymphocyte), no NCX was found. These results suggest a possible new and widely distributed mechanism for regulation of nuclear calcium by NCX in association with GM1.Acknowledgements:  supported by NIH grant NS 33912.
Prolactin‐dependent activation of ERK and TYR‐phosphorylation of cortactin in postmitotic neurons
Authors: Mangoura D. , Singh D. , Johnson S. , Sakellaridis N. 
Published: July 2010

Prolactin (PRL), originally identified as a hormone involved in reproduction, is now investigated as a growth factor. We have previously demonstrated that PRL is a mitogen in PC12 cells and astrocytes, acting independently of ERK activation. To investigate the potential role of prolactin as a neurotrophic factor, we used cultured postmitotic cortical neurons derived from embryonic day 6 chick. Western blot analysis with antiphosphotyrosine antibodies established functional expression of PRL receptors by showing that several proteins were modified with time of PRL exposure, including the PRL receptor and JAK2 kinase. Using both kinase in‐gel assays and Western blots with antiactive ERK antibodies we found that PRL stimulated activation of ERK1/2 in a sustained fashion, typical of differentiation. While active ERK was detected in the nucleus, some was associated with the cytoskeleton. We then investigated in nuclei‐free fractions the identity of proteins that coimmunoprecipitated with ERK and were tyrosine‐phosphorylated in response to prolactin. The most prominent protein was identified as the F‐actin‐binding cortactin. Upon stimulation with PRL, an activated ERK coimmunoprecipitated cortactin as early as 2′ and as long as 15′ after exposure. Tyr‐phosphorylation of cortactin and translocation to the Triton X‐100 fraction was also established. In the long term PRL induced a small but significant increase of cortactin expression. In lieu of our recent data that the developmentally regulated cortactin may supress several parameters of the GABAergic program, these results suggest that prolactin may have a role in neuronal differentiation and that signal specificity may be a result of protein interactions between cytoskeleton and signaling molecules.Acknowledgements:  Support: HD‐09402 to DM.
Poster Sessions CP09: Kinases and Phosphatases. Protein kinase C inhibitors counteract ethanol effects on myelin basic protein expression in CG‐4 oligodendrocytes
Authors: Bichenkov E. , Ellingson J. S. 
Published: July 2010

Abnormal myelin formation appears to be one defect contributing to the neuropathology associated with the fetal alcohol syndrome, the major cause of noncongenital mental retardation. Using the CG‐4 cell line we previously showed that 25–75 mm ethanol (EtOH) down‐regulates myelin basic protein (MBP) expression in differentiating oligodendrocytes (OLGs) without affecting the 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP) expression or morphological development (Bichenkov and Ellingson 2001). Here we observed that a relatively low concentration of 12‐phorbol‐13‐myristate acetate (PMA) mimicked the EtOH‐caused inhibition of MBP expression without affecting CNP expression or morphology. The inhibition of MBP expression by 100 mm EtOH or 1 nm PMA was completely counteracted by three inhibitors of protein kinase C (PKC); bisinodoylmaleimide I, chelerythrine chloride, and calphostin C, indicating that EtOH down‐regulated MBP expression by activating PKC. We investigated whether the EtOH‐caused activation resulted in part from up‐regulation of the expression of PKC isozymes. Of 11 PKC isozymes examined, CG‐4 OLGs expressed nine; PKC α, β1, β2, δ, ε, λ, μ, nu and zeta; while PKC isozymes γ and theta were not detected. Only five PKC isozymes, α, β1, β2, μ, and nu, displayed developmental changes in expression. However, EtOH did not up‐regulate the early expression of any PKC isozyme during the first two days of differentiation, the developmental stage when it down‐regulates the MBP expression in CG‐4 cells. The results indicate that EtOH delays MBP expression by activating at least one phorbol ester‐sensitive PKC isozyme in differentiating oligodendrocytes without up‐regulating its expression.Acknowledgements:  Support: NIAAA Grant AA072185.
Aromatic hydrocarbon 1,2‐diacetylbenzene cross‐links neurofilament and other proteins
Authors: Sabri M. I. , Hashemi S. , Kim M. S. , Spencer P. S. 
Published: July 2010

Rats treated with 1,2‐diacetylbenzene (1,2‐DAB) develop giant proximal axonal swellings packed with 10‐nm neurofilaments in lumbar spinal motor neurons (Kim et al. 2001; Toxicol. Appl. Pharmacol. 177, 121–131), an early pathological feature in amyotrophic lateral sclerosis. This study examines the reactivity of 1,2‐DAB with a number of proteins. Incubation of purified bovine serum albumin or ribonuclease with DAB isomers (1–5 mm) revealed the formation of high molecular weight polymers with 1,2‐DAB, but not with the non‐neurotoxic analog 1,3‐DAB. Western blots of rat spinal cord revealed that 1,2‐DAB (1–10 mm), but not 1,3‐DAB, produces a concentration‐dependent decrease in neurofilament proteins with increased high‐molecular‐weight protein species. Native beta‐tubulin was least affected by 1,2‐DAB. Western blots of rat sciatic nerves exposed in situ to 1,2‐DAB (5–50 mm) showed a decrease of the NF‐M band with corresponding increased staining in the high‐molecular‐weight region. Protein polymerization was not detected with monoclonal antibodies to either NF‐L or beta‐tubulin. Preliminary results show that 1,2‐DAB, but not 1,3‐DAB, significantly reduces the levels of other axonal proteins (e.g. microtubule‐associated protein, tau and motor proteins kinesin and dynein). In summary, 1,2‐DAB reacts differentially with a number of axonal proteins; which reactions underlie the genesis of axonal pathology is unknown.Acknowledgements:  Supported by NIEHS grants ES10338 and ES11384, and the State of Oregon's Worker's Benefit Fund.
Acidification‐induced changes in Cx43 protein–protein interactions
Authors: Duffy H. S. , Sorgen P. , Coombs W. , Taffet S. , Girvin M. , Delmar M. , Spray D. C. 
Published: July 2010

Astrocytic gap junctions close in response to low intracellular pH, a process that may occur during ischemia. We propose that pH gating of Connexin43 (Cx43) involves binding of the carboxyl terminal domain (Cx43CT) to a pore‐affiliated ‘receptor’ and, possibly, interaction of Cx43 with other molecular partners. We studied the role of intra– and intermolecular interactions in Cx43 pH gating. Peptides were made corresponding to intracellular regions of Cx43. Using Surface Plasmon resonance (SPR) we found pH dependent binding of the second half of the cytoplasmic loop to Cx43CT (Kd = 80 and 225 mm, pH 6.5 and 7.4, respectively). Binding was inhibited by preincubation with a synthetic peptide from region 346–362 of Cx43CT and by an antibody to this epitope, suggesting the binding site lies partially within this region. Nuclear magnetic resonance (NMR) studies showed formation of alpha helices in response to low pH in amino acid sequences VEMHL (aa 123–127) and IEEHGK (aa 139–143) in the loop, and a helical region within the suggested Cx43CT binding site. In addition, we found that acidification of astrocytes in vitro led to dissociation of Cx43 from its scaffold protein ZO‐1. SPR showed that this was not due to pH dependence of Cx43‐ZO‐1 interaction (Kd = 2.23 mm at pH 7.4, 1.71 mm at pH 6.5) but to a strong pH–dependent interaction of Cx43 with c‐Src (Kd = 0.63 mm at pH 7.4, 0.04 mm at pH 6.5) which caused ZO‐1 to dissociate from Cx43CT. Separate experiments showed activation of astrocytic src at low pH. These changes in the composition of the Cx43 Nexus may play a major role in limiting tissue damage during brain ischemia.Acknowledgements:  Supported by NIH grants NS 07098 (HSD) NS34931 & NS41282 (DCS) and GM57691 (MD).
Aggregation of peripheral myelin protein 22 in Trembler J neuropathy nerves
Authors: Notterpek L. , Fortun J. , Ferguson T. A. 
Published: July 2010

Mutations in the peripheral myelin protein 22 (PMP22) gene are associated with demyelinating neuropathies leading to muscle atrophy. Altered subcellular trafficking and aggregation of mutant PMP22s have been proposed as mechanisms for the neuropathy. Indeed, wild type PMP22 accumulates in aggresomes when the ubiquitin‐proteasome pathway is inhibited or PMP22 is overexpressed. Therefore, we investigated the presence of PMP22 aggregates in sciatic nerves of adult heterozygote Trembler J (TrJ) mice. Our studies show the accumulation of PMP22 in perinuclear, as well as peripheral aggregates in affected samples. PMP22 aggregates are surrounded by a vimentin cage and often colocalize with ubiquitin. Other myelin proteins, including myelin basic protein, are diverted to PMP22 aggregates in TrJ nerves. In addition, connexin 32 and protein 0 aggregates are detected in affected Schwann cells. Biochemical analyses of nerve lysates confirm the up‐regulation of the ubiquitin‐proteasomal and lysosomal protein degradation pathways. In addition, missfolding of the mutant TrJ PMP22 induces a stress response in the Schwann cells, as revealed by the elevated levels of molecules involved in protein folding, such as Hsp60, Hsp70 and alpha β crystalline. Our results support a possible mechanism by which the L16P mutation in PMP22 increases the tendency of the protein to aggregate and sequester other proteins, such as chaperones and myelin proteins, interfering with normal processes in Schwann cells.Acknowledgements:  These studies are supported by funds from the NIH and the Muscular Dystrophy Association.
Hierarchical interactions among cytokines establish the functional state of microglia
Authors: Levison S. W. 
Published: July 2010

Microglia rapidly respond to CNS injury yet the mechanisms leading to their activation and inactivation remain poorly defined. In particular, few studies have established how interactions among inflammatory mediators affect the innate immune response of microglia. To begin to understand the hierarchy of cytokine signalling we examined the effects of several cytokines on purified newborn and adult rat microglia in vitro, and we have examined the microglial response to injury in mice deficient in the IL‐1 type 1 receptor (IL‐1R1). Using several indices of activation, we find that IL‐1β, TNF‐α, and IL‐6 are potent microglial activators. By contrast, TGF‐β1 did not activate the cells and when TGFβ1 was administered prior to IL‐1β, it blocked the effects of IL‐1β. However, TGFβ1 was ineffective in antagonizing IL‐6. In null mice lacking the IL‐1R1, microglia inefficiently responded to injury, and IL‐6 induction was severely curtailed. These data establish a model of hierarchical signalling, whereby constitutive expression of TGF‐β1 in the CNS maintains microglia in a resting state. IL‐1, while an important microglial activator, is modifiable, whereas, the downstream cytokine, IL‐6, is a strong stimulus that is unaffected by other modifiers of the innate immune response.Acknowledgements:  Supported by NMSS award #RG 3837.
Desensitization, phosphorylation and sequestration of the human P2Y2 nucleotide receptor
Authors: Gonzalez F. A. , Aquino E. , Flores R. V. , Garrad R. C. , Weisman G. A. 
Published: July 2010

Human 1321 N1 astrocytoma cells transfected with HA‐tagged P2Y2 receptors cDNA were used to investigate the molecular mechanisms involved in P2Y2 receptor signalling. Immunoprecipitation of wild‐type P2Y2 receptor revealed a heterogeneous distribution of the receptor protein ranging in Mr from 57 to 76 kDa which is typical of membrane glycoproteins with heterogeneous complex glycosylation. The human HA‐tagged P2Y2 receptor stably transfected in 1321 N1 cells exhibited an EC50 value of 0.25 ± 0.03 that is similar to that of the P2Y2 receptor endogenously expressed in human monocytic U‐937 cells. The HA‐P2Y2 receptor is rapidly desensitized with UTP showing an IC50 of 0.43 ± 0.1 μm. The protein phosphatase inhibitor, okadaic acid, inhibited the recovery of receptor activity from the desensitized state, suggesting a role for P2Y2 receptor phosphorylation in desensitization. Indeed, a 3.8 ± 0.2‐fold increase in [32P]‐PO43– radioactivity content in the immunoprecipitated	HA‐P2Y2 receptor was detected in cells after a 15‐min treatment with 100 μm UTP when compared receptor from to control untreated cells. Receptor sequestration studies revealed that greater than 80% of the surface HA‐P2Y2 receptors were internalized after a 15 min stimulation of 100 μm UTP. Our results support the hypothesis that receptor desensitization implicates an increase in receptor phosphorylation of sequestered P2Y2 nucleotide receptors. These results lay the grounds for further studies to identify the specific amino acid residues that are phosphorylated and the protein kinases involved in homologous desensitization of the P2Y2 nucleotide receptor.