bluuebunny/crossref_metadata_2025_split huggingface dataset:
The mediterranean fever gene modifies the progression of disability in non‐Ashkenazi Jewish multiple sclerosis patients
Authors: Shinar Y. , Livneh A. , Vila Y. , Pinhasov A. , Zeitoun I. , Achiron A. 
Published: July 2010

MS is an autoimmune, CNS demyelinating disease manifested in most patients with progressive disability. The progression rate varies between patients and may depend on modifier, immune related genes. The Mediterranean fever gene, expressed in peripheral blood leukocytes, is responsible for familial Mediterranean fever (FMF), a recessive, periodic autoinflammatory disease prevalent in Semitic populations, and less penetrant in Ashkenazi Jews. We related common, FMF associated MEFV mutations to the progression of disability in Jewish, relapsing remitting (RR) MS patients. The mutations 148Q, 694V, 695R and 726A were identified by enzymatic restriction of PCR‐amplified MEFV DNA. The progression to statuses 3 and 6 of the expanded disability status scale (EDSS) was analyzed on survival plots. 35% of 48 non‐Ashkenazi patients had one MEFV mutation. Compared to non‐carriers (n = 31) the heterozygous cohort (n = 17) represented with an increased fraction reaching both EDSS statuses (p < 0.05), and with a shorter median time to reach both EDSS = 3 (2 years in carriers vs. 10 years in non‐carriers, p < 0.01) and EDSS = 6 (6 vs. 23 years, respectively, p < 0.005). 17% of 71 Ashkenazi patients had one MEFV mutation. There was no significant difference in the fraction of disabled or in the progression of disability between Ashkenazi carrier patients and non‐carriers. The susceptibility of the non‐Ashkenazi group attributed, in part, to the detrimental non‐Ashkenazi 694V mutation. The results suggest phenotypic expression of one mutated MEFV gene in non‐ashkenazi patients, pertinent to the pathogenesis of disability.Acknowledgements:  Granted by the Israeli Ministry of Science (#6279).
Structure and regional distribution of low molecular weight myelin proteolipids
Authors: Bizzozero O. A. , Malkoski S. P. , Mobarak C. D. , Evans J. E. , Dejesus G. 
Published: July 2010

We have investigated the structure of the native PLP, DM‐20 and several low molecular weight proteolipids (LMWPs) by mass spectrometry (MS). The various proteolipid species were isolated from bovine spinal cord by size‐exclusion and ion‐exchange chromatography. Matrix‐assisted laser desorption ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) of PLP and DM‐20 revealed molecular masses of 31.6 and 27.2 kDa, respectively, which is consistent with the presence of six and four molecules of thioester‐bound fatty acids. Electrospray ionization‐MS analysis of the deacylated proteins produced the predicted molecular masses of the apoproteins, demonstrating that palmitoylation is the major modification of PLP, and that the majority of PLP and DM‐20 molecules in the CNS are fully acylated. A series of proteolipids with molecular masses from 12 to 18 kDa were also isolated and subjected to amino acid analysis, fatty acid analysis, N‐ and C‐terminal sequencing, tryptic digestion and peptide mapping by MALDI‐TOF‐MS. The results showed that these polypeptides correspond to the N‐terminal region (residues 1‐105/112) and C‐terminal region (residues 113/131‐276) of PLP, and they appear to be generated by natural proteolytic cleavage within the 50 amino acid‐long cytoplasmic domain. These LMWPs are derived from myelin, and are not generated during their isolation or during the postmortem period. Moreover, they are present in other species and are unevenly distributed across the CNS. It is tempting to speculate that these minor proteolipids may play a role in myelin formation.Acknowledgements:  Supported by PHHS grant NS 38325.
Adenovirus infection induces microglial activation: involvement of mitogen‐activated protein kinase pathways
Authors: Bhat N. R. , Fan F. 
Published: July 2010

Non‐replicating adenovirus (Ad) vectors represent effective tools for long‐term gene expression in the CNS, but they also elicit inflammation. The cellular and molecular mechanisms of such a response are not well‐understood. We show here that infection with Ad causes an activation of microglial cells, the key cells involved in inflammatory and immune‐regulatory functions in the brain. Exposure of cultured rat brain microglia to Ad resulted in an induced production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) and the pro‐inflammatory cytokine, TNFα. The roles of signal transduction pathways believed to be involved in microglial activation, in particular, mitogen‐activated protein kinases (MAPKs) and nuclear factor κB (NFκB) were explored by determining their activation in response to Ad infection and by testing the effects of specific pharmacological inhibitors. It was found that Ad strongly activates extracellular signal‐regulated kinase (ERK) and to a lesser extent, p38 MAPK but not NFκB. The kinase inhibitor i.e. PD98059, specific for the ERK pathway, potently inhibits and, in combination with the p38 MAPK inhibitor, SB203580, almost completely abolishes Ad‐induced expression of iNOS and TNFα. The results suggest that Ad uses cellular signal transduction machinery, in particular, the ERK pathway to elicit microglial activation and that increased production by these cells of inflammatory mediators may primarily contribute to CNS inflammatory responses commonly seen in models of gene therapy using Ad vectors.Acknowledgements:  Supported by NINDS grant # NS41035‐01.
Inhibition of interferon‐gamma‐induced nitric oxide production in BV‐2 microglial cells by polyphenolic compounds
Authors: Zheng Q. , Binek J. , Sun G. Y. 
Published: July 2010

Microglial cells are known to play an important role in mediating the host defense functions in the brain. These cells respond readily to pro‐inflammatory cytokines, which stimulate signalling pathways and induction of many genes, including the inducible nitric oxide synthase (iNOS). In the immortalized BV‐2 microglial cells, our studies have demonstrated that interferon gamma (IFNγ) alone could stimulate the induction of iNOS through activation of the nuclear factor‐kappa B (NF‐κB) pathway. Increased NO production in the brain has been implicated in enhanced oxidative stress associated with many neurodegenerative diseases. Many polyphenolic compounds in vegetables and fruits, including resveratrol, catechin, quercetin, genistein are regarded as phytoestrogens and are known to possess potent antioxidant properties. However, whether these compounds also offer anti‐inflammatory effects and inhibit cytokine‐induced NO production in microglial cells have not been examined in detail. The major goal for this project is to use BV‐2 microglial cells as cell model to investigate the effects of these polyphenolic compounds on IFNγ induction of NO. Results show that all the polyphenolic compounds tested could inhibit NO production albeit to different extents, with quercetin having the lowest IC50 and catechin the highest IC50. Surprising, daidzin, a non‐tyrosine kinase analog of genistein, also inhibited IFNγ‐induced NO production, suggesting that the inhibitory effects may not be due entirely to their action on the tyrosine kinase pathway. These results lead to the conclusion that polyphenolic compounds tested not only possess antioxidative but also anti‐inflammatory properties.Acknowledgements:  Supported by 5 P01 ES10535 from NIH.
Double‐stranded RNA induces nitric oxide synthase in human astrocytes
Authors: Auch C. J. , Dasgupta S. , Jana M. , Liu X. , Pahan K. 
Published: July 2010

Environmental factor(s), such as viral infection, has been implicated as one of the potential triggering events leading to demyelination in MS. This study underlines the importance of double‐stranded RNA (dsRNA), the active component of a viral infection that stimulates antiviral responses in infected cells, in inducing the expression of inducible nitric oxide synthase (iNOS) in human astroglia. Treatment of human U373MG astroglial cells with dsRNA in the form of synthetic polyinosinic‐polycytidylic acid (poly IC) induces the production of NO and the expression of iNOS in a dose‐dependent manner. Similar to human U373MG astroglial cells, poly IC also induces the expression of iNOS in human primary astroglia. Induction of human iNOS promoter‐derived luciferase activity by poly IC suggests that poly IC induces the transcription of iNOS. As activation of both NF‐κB and C/EBPb is necessary for the transcription of iNOS in human astroglia, we investigated the effect of poly IC on the activation of both NF‐κB and C/EBPb. Poly IC induced the activation of both NF‐κB and C/EBPb. Furthermore, inhibition of poly IC‐induced production of NO by Dp65, a dominant‐negative mutant of p65, and DC/EBPb, a dominant‐negative mutant of C/EBPb, suggests that poly IC induces the expression of iNOS in human astroglial cells through the activation of NF‐κB and C/EBPb. This study delineates a novel role of dsRNA in inducing the expression of iNOS in human astroglia which may participate in virus‐induced neurological abnormalities.Acknowledgements:  This study was supported by NIH grants (NS39940 and AG19487).
Effects of calpain inhibition on IkBα degradation following PBMC activation: identification of calpain cleavage sites
Authors: Rocchini A. , Schaecher K. , Goust J. M. , Banik N. L. 
Published: July 2010

We previously demonstrated calpain‐mediated activation of PBMCs. In order to examine whether calpain plays a role in the degradation of IkBα and NFkβ translocation in the mechanism of T cell activation, human PBMCs were activated using anti‐CD3/CD28 (HIT3A/CD28.2). Degradation of IkBα was monitored by Western blot, degradation products were sequenced, and calpain‐cleaved sites were determined. The activated cells degraded IkBα, the inhibitor of NFkβ, relative to non‐activated cells. IkBα degradation was progressive and inhibited by calpain inhibitor calpeptin in a dose and time dependent manner. Further, calpain inhibition increased IkBα levels above those observed in non‐activated negative controls. Recombinant IkBα was incubated with purified porcine mcalpain to initiate the degradation of IkBα. The degradation products were separated via SDS–PAGE and visualized using Coomassie Stain. The stained degradation products were isolated and subjected to sequencing. Calpain cleaved the recombinant IkBα most effectively when incubated with IkBα at a ratio of 1 : 1 for 20 min in the presence of 0.1% Triton‐X100. After incubation, several degradation products were detected, including a peptide corresponding to a cleavage of IkBα between amino acids 50 and 51 (glutamine and glutamic acid). The liberated fragment included the entire Signal Response Domain (SRD), a region containing key serine and threonine residues necessary for phosphorylation by the IKKinase complex and sites required for ubiquitination. These results show that calpain plays an important role in IkBα degradation, a crucial event in T cell activation and is involved in the normal turnover of IkBα.Acknowledgements:  Supported by grants from NIH‐NINDS.
Poster Sessions BP06: Neurogenesis, Stem Cells and Apoptosis ‘DECOY’ oligonucleotides' effects on NF‐κB regulation of bcl‐x gene expression in rat hippocampus
Authors: Qiu J. , Grafe M. R. , Martin S. S. , Kent T. A. , Rassin D. K. , Perez‐Polo J. R. 
Published: July 2010

The NF‐κB transcription factor regulates bcl‐x gene expression, which may determine hypoxia‐induced neuronal apoptosis. We examined hypoxia‐induced NF‐κB and Bcl‐xL changes in rat hippocampus. We showed differential hypoxia‐induced NF‐κB binding to the bcl‐x promoter CS4 and IgG–κB enhancer sequences by rat hippocampal nuclear extracts. The differential NF‐κB binding to these two promoter sequences was also determined in a contused spinal cord injury model and in vitro studies with LPS‐treated Hela cells. There was tissue‐, gene promoter‐specific and time‐dependent regulation of bcl‐x gene expression by NF‐κB in support of the hypothesis that NF‐κB has tissue‐ and gene‐specific regulatory effects and that these effects may account for both the pro‐ and anti‐apoptotic roles assigned to NF‐κB in different apoptotic processes. We applied ‘decoy’ oligonucleotides with sequences specific to different promoters to the rat hippocampus and measured ‘decoy’ inhibitory effects on nuclear NF‐κB binding. The IgG‐κB enhancer sequence ‘decoy’ showed stronger inhibition on nuclear NF‐κB c‐Rel/p50 binding to the bcl‐x gene promoter CS4 sequence when compared to NF‐κB p50/p50 binding to the same sequence. This result suggests that the ‘decoy’ approach has the potential to selectively manipulate NF‐κB regulation of gene expression in response to hypoxia.Acknowledgements:  Supported by NINDS NS‐39161, Shriner Grant 8710 and a Grant from the Sealy Center on Aging to J. Qiu.
Cyclic GMP is more effective than cyclic AMP or GDNF in protecting mature oligodendrocytes from cell death
Authors: Benjamins J. A. , Nedelkoska L. 
Published: July 2010

During immune‐mediated or ischemic attack, oligodendrocytes (OLs) receive a barrage of signals, both protective and injurious. The specific ligands at the site of damage in vivo vary widely in type and concentration. Using cultured mature mouse OLs, we have focused on four models of cell death which represent common pathways initiated by a number of ligands. These include (1) staurosporine, which induces peroxynitrite formation and subsequent release of cytochrome c; (2) thapsigargin, which disrupts Ca++ homeostasis in the endoplasmic reticulum; (3) kainate, which activates non‐NMDA receptors; and (4) SNAP and NOC‐18, which release NO. GDNF protects only against cell death induced by staurosporine, and acts via PI‐3‐kinase but not MAP kinase to protect. Elevation of cyclic AMP by addition of 8Br‐cyclic AMP or forskolin protects against staurosporine, but does not increase the protective effect of GDNF when added in combination. In addition, elevation of cyclic AMP protects against cell death initiated by thapsigargin, but not kainate or SNAP. Inhibition of protein kinase C with calphostin protects only against kainate. Of the possible protective agents tested, 8Br‐cyclic GMP is the only one which protects OLs in all four models of cell death. These results highlight the importance of signaling via cyclic GMP in survival pathways in mature OLs.Acknowledgements:  Supported by grant NS13143 from NIH and grant RG 3030A/6 from the National Multiple Sclerosis Society.
Prevention of apoptosis and preservation of function by protease inhibitors in cortical neurons exposed to glutamate
Authors: Ray S. K. , Nowak M. W. , Li L. , Wilford G. G. , Banik N. L. 
Published: July 2010

Traumatic injury and diseases of the central nervous system are often associated with release of L‐glutamate that causes neuronal death and dysfunction. Rat primary cortical neurons exposed to 0.5 μmL‐glutamate for 24 h died mostly by apoptosis (Wright staining, ApopTag assay). L‐glutamate exposure also caused up‐regulation of calpain and caspase‐3 compared with β‐actin, an internal control (Western blot). We tested calpain and caspase‐3 inhibitors to prevent death and dysfunction in following L‐glutamate exposure. Pre‐treatment of cells with 0.2 μm calpeptin (calpain‐specific inhibitor) or/and 100 μm z‐DEVD‐fmk (caspase 3‐specific inhibitor) prevented apoptosis. Electrophysiological properties and whole‐cell currents associated with voltage‐gated Na+ channels, NMDARs, and kainate/AMPARs were measured. There were no significant differences in VREST, membrane capacitance and IBASAL at −70 mV for inhibitor only or inhibitor plus L‐glutamate treated neurons relative to control neurons. The lack of a change in capacitance indicated that neurons treated with inhibitor plus L‐glutamate did not undergo apoptotic shrinkage and maintained the same size as the control neurons. Currents associated with Na+ channels, NMDARs, and kainate/AMPARs were similar in amplitude and activation/inactivation kinetics for control and treatments with inhibitor plus L‐glutamate. Spontaneous synaptic activity (as observed by miniature end‐plate currents) was also similar in control, inhibitor only, and inhibitor plus L‐glutamate treated cultures. Prevention of L‐glutamate‐induced apoptosis by protease inhibitors preserved normal electrophysiological properties and ion channel activity in cortical neurons.Acknowledgements:  Supported by the grants from NIH‐NINDS (NS31622, NS38146, NS41088).
A new method for measuring fibrillar amyloid β‐protein using diphenylhexatriene, a fluorescent probe
Authors: Chauhan A. , Chauhan V. 
Published: July 2010

The two current methods for the measurement of fibrillar (f) amyloid beta‐protein (Aβ), namely Thioflavin T (ThT) fluorescence spectroscopy and Congo red (CR) absorbance spectroscopy, are based on the interaction of fAβ with cationic ThT and anionic CR molecules. The presence of cations and colored factors in the reaction often interferes with ThT and CR assays. Recently, we reported that fibrillar Aβ forms a membrane‐like hydrophobic domain (Chauhan et al. 2001; Neuroreport12, 587–590). We report here that diphenylhexatriene (DPH), a neutral fluorescent probe, interacts with fibrillar Aβ 1‐40 and fibrillar Aβ 1‐42, but not with soluble Aβ. As a result, DPH fluorescence increases several fold in the presence of fibrillar Aβ at the excitation and emission wavelength of 360 and 430 nm, respectively. The blank sample (DPH in buffer) had minimal fluorescence, and soluble Aβ had no effect on DPH fluorescence. The interaction of DPH with fibrillar Aβ was dependent on the incubation time of these two constituents. At fixed Aβ concentration and varying DPH concentration, there was a lag phase of DPH fluorescence from 1 to 4 μm DPH, that was followed by a linear increase in fluorescence from 4 to 8 μm DPH which then reached a plateau. Studies at varying pH showed that emission spectra of fAβ/DPH can be studied at a wide range of pH from 4 to 8. Aβ concentration studies showed that the fluorescence of DPH increases linearly with increase in the concentration of fibrillar Aβ 1‐40/Aβ 1‐42. Kinetic studies showed that one mole of fAβ binds to 15.5 mol of DPH. These results suggest that DPH fluorescence can be used as a standard assay method for the measurement of fibrillar Aβ.
A role for the transcription factor ets‐2 in Down's syndrome neuropathology
Authors: Pelsman A. , Busciglio J. 
Published: July 2010

Down's Syndrome (DS) is the most frequent genetic cause of mental retardation. Most DS patients develop Alzheimer's disease by middle age. A transgenic model of ets‐2 that presents pathological features similar to DS suggests a role for ets‐2 in DS (Sumarsono et al. 1996; Nature379, 534). We analyzed the expression and activity of ets‐2 in normal and DS brain tissue and human cortical neurons (HCN) in culture. Western blot and immunocytochemistry revealed that ets‐2 is expressed in the human brain and in HCN during development. The level of ets‐2 was significantly increased in DS brain homogenates and DS HCN cultures. Interestingly, DS HCN cultures exhibited reduced bcl‐2 and increased bax and p53 protein levels consistent with reduced DS neuronal viability in culture. To determine the role of ets‐2 in neuronal degeneration, normal neurons were transfected with an ets‐2 expression vector. After 24 h, a significant increase in cell death was observed in ets‐2‐transfected neurons but not in neurons cotransfected with a dominant‐negative ets‐2 expression vector (ets‐2dn). Neuronal degeneration was dependent on p53 activity since a dominant‐negative mutant form of p53 significantly abolished ets‐2‐induced neuronal degeneration. Moreover, ets‐2 overexpression did not increase neuronal death in neuronal cultures derived from p53KO mice. Finally, the expression of ets‐2dn significantly reduced degeneration of DS cortical neurons in culture. In DS, ets‐2 overexpression may compromise neuronal viability directly by inducing neuronal apoptosis or indirectly by rendering neurons more sensitive to pro‐apoptotic stimuli such as Aβ neurotoxicity and oxidative stress.
Retinoic acid's role in sexually dimorphic neural homeosis
Authors: Rosa‐Molinar Eduardo 
Published: July 2010

The sexually dimorphic developmental program of the western mosquitofish, Gambusia affinis affinis radically reorganizes the typical two compartment teleost body plan and nervous system (e.g. pre‐caudal: vertebral bodies with ribs; and caudal: vertebral bodies without ribs and no ano‐urogenital plexus) into a three compartment body plan [e.g. pre‐caudal: vertebral bodies with ribs; and caudal: vertebral bodies without ribs and an ano‐urogenital region (a region composed of pre‐caudal and caudal vertebrae combined)] and a ano‐urogenital plexus and is reminiscent of homeotic transformations achieved by disturbing developmental programs using exogenous retinoic acid (RA). RA has not been thought to play a role in the patterning, organization and regulation of neural sexual dimorphism, a role considered to be the domain of steroids, specifically, testosterone and estrogen. Data discussed in this presentation will show the cell types responsible for synthesizing RA within the spinal cord of G. a. affinis. We will present data that the endogenous RA synthesized within the spinal cord is an instructive morphogen that (1) imparts identity and position of sex‐specific vertebral and motor neurons and (2) regulates the number of motor neurons within the spinal cord region associated with the ano‐urogenital region. These findings may represent the first described example of ‘sexually dimorphic neural homeosis’.Acknowledgements:  Research support through a grant from the National Science Foundation (IBN‐0091120).
Poster Sessions CP08: Signal Transduction. Synergistic modification of inducible and neuronal NOS expression by NGF and TNFα: relevance to CNS aging
Authors: Taglialatela G. , Thomas M. S. , Zhang W. R. , Macdonald N. J. , Andorn A. C. 
Published: July 2010

Inducible nitric oxide synthase (iNOS) and high levels of nitric oxide (NO) are present in the CNS of patients with Alzheimer's disease (AD), resulting in both DNA and protein oxidative damage. While iNOS can result in damaging levels of NO, the neuronal constitutive form of NOS (nNOS) has a role in cell signalling and can prevent neuronal apoptosis. iNOS can be induced by inflammatory cytokines such as tumor necrosis alpha (TNFα). TNFα is found in the CNS of AD, where neurons dependent on neurotrophins such as nerve growth factor (NGF) are particularly affected. Here we determined the effect of TNFα on the three NOS isoforms (endothelial, neuronal and inducible) in NGF‐responsive PC12 cells. We found that while TNFα and NGF alone were uneffective, their simultaneous addition resulted in iNOS induction and the release of NO. In addition TNFα and NGF synergistically reduced nNOS, independently of the presence of high NO levels promoted by iNOS, while no effect was observed on eNOS. A similar pattern was observed in the brain of aged human subjects as compared to young individuals. Our results suggest that synergistic iNOS induction by TNFα and NGF may occur in selective populations of NGF‐responsive neurons. Oxidative damage in such neurons could then occur in the presence of elevated levels of TNFα, that potentially occur in the brain of AD patients. This damaging scenario may further be aggravated by a concomitant reduction of nNOS, brought about by similar synergistic effects between TNFα and NGF.Acknowledgements:  Supported by NIA (AG13945) and Sealy Res. Dev. grants to GT.