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1.
Neurol India ; 69(4): 861-866, 2021.
Article in English | MEDLINE | ID: mdl-34507402

ABSTRACT

BACKGROUND: Spinal cord injury (SCI) and its negative impact on the quality of life (QOL) is a significant public health concern in India. People with SCI suffer from serious health, economic, and social consequences in their lives. Often, care for SCI survivors is left to their immediate family members in India. Appropriate planning is needed for prevention, rehabilitation, health, and psychological care for SCI in the country. PURPOSE: This study assessed the overall QOL of SCI survivors and their satisfaction levels with specific domains and their importance of QOL. MATERIALS AND METHODS: In this observational study, two instruments, Farrens and Power for QOL and Barthel Index for functional abilities, were administered to a convenience sample of participants drawn from Narayana Medical College, Nellore, in South India. RESULTS: Statistically, SCI survivors were found moderately and very satisfied with their QOL. Their perception about importance of health, functioning, social, and economic subscale also did not differ statistically.


Subject(s)
Quality of Life , Spinal Cord Injuries , Activities of Daily Living , Family , Humans , Personal Satisfaction
2.
PLoS One ; 14(12): e0220679, 2019.
Article in English | MEDLINE | ID: mdl-31877136

ABSTRACT

Kuwait is a semi-arid region with soils that are relatively nitrogen-poor. Thus, biological nitrogen fixation is an important natural process in which N2-fixing bacteria (diazotrophs) convert atmospheric nitrogen into plant-usable forms such as ammonium and nitrate. Currently, there is limited information on free-living and root-associated nitrogen-fixing bacteria and their potential to fix nitrogen and aid natural plant communities in the Kuwait desert. In this study, free living N2-fixing diazotrophs were enriched and isolated from the rhizosphere soil associated with three native keystone plant species; Rhanterium epapposum, Farsetia aegyptia, and Haloxylon salicornicum. Root-associated bacteria were isolated from the root nodules of Vachellia pachyceras. The result showed that the strains were clustered in five groups represented by class: γ-proteobacteria, and α-proteobacteria; phyla: Actinobacteria being the most dominant, followed by phyla: Firmicutes, and class: ß-proteobacteria. This study initially identified 50 nitrogen-fixers by16S rRNA gene sequencing, of which 78% were confirmed to be nitrogen-fixers using the acetylene reduction assay. Among the nitrogen fixers identified, the genus Rhizobium was predominant in the rhizosphere soil of R. epapposum and H. salicornicum, whereas Pseudomonas was predominant in the rhizosphere soil of F. aegyptia, The species Agrobacterium tumefaciens was mainly found to be dominant among the root nodules of V. pachyceras and followed by Cellulomonas, Bacillus, and Pseudomonas genera as root-associated bacteria. The variety of diazotrophs revealed in this study, signifying the enormous importance of free-living and root-associated bacteria in extreme conditions and suggesting potential ecological importance of diazotrophs in arid ecosystem. To our knowledge, this study is the first to use culture-based isolation, molecular identification, and evaluation of N2-fixing ability to detail diazotroph diversity in Kuwaiti desert soils.


Subject(s)
Microbiota/genetics , Nitrogen Fixation/physiology , Nitrogen/metabolism , Actinobacteria/genetics , Bacteria/genetics , DNA, Ribosomal/genetics , Desert Climate , Ecosystem , Kuwait , Phylogeny , RNA, Ribosomal, 16S/genetics , Rhizosphere , Soil/chemistry , Soil Microbiology
3.
Neuroreport ; 10(13): 2869-73, 1999 Sep 09.
Article in English | MEDLINE | ID: mdl-10511455

ABSTRACT

Activated microglia regulate immune and inflammatory responses in the CNS under a variety of stresses due to infection, injury and disease. In this study, we show that a stress-inducible small heat shock protein, alpha-crystallin, induces in vitro activation of microglia cultured from newborn rat brain. Exposure of microglia to alpha-crystallin resulted in an increased production of nitric oxide (NO) and the expression of the inducible NO synthase (iNOS) as determined by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. Alpha-crystallin also stimulated the synthesis of the pro-inflammatory cytokine, TNF alpha. The results presented showing microglial induction of the two key immune regulatory and inflammatory molecules, i.e., NO and TNF alpha, in response to a stress-inducible protein, suggest a link between environmental stress and the CNS immune response.


Subject(s)
Crystallins/pharmacology , Microglia/drug effects , Microglia/physiology , Animals , Animals, Newborn , Blotting, Western , Brain/cytology , Chromatography, High Pressure Liquid , Enzyme Inhibitors/pharmacology , Microglia/metabolism , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide/biosynthesis , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Nitrites/antagonists & inhibitors , Protein Isoforms/pharmacology , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Tumor Necrosis Factor-alpha/biosynthesis
4.
J Neurochem ; 72(2): 472-8, 1999 Feb.
Article in English | MEDLINE | ID: mdl-9930718

ABSTRACT

The induction of inducible nitric oxide synthase (iNOS) by proinflammatory cytokines was studied in an oligodendrocyte progenitor cell line in relation to mitogen-activated protein kinase (MAPK) activation and cytokine-mediated cytotoxicity. When introduced individually to cultures of CG4 cells, the cytokines, i.e., tumor necrosis factor-alpha (TNF alpha), interleukin-1 (IL-1), and interferon-gamma (IFN gamma), had either minimal (TNF alpha) or no (IL-1 and IFN gamma) detectable stimulatory effect on the production of nitric oxide. However, combinations of these factors, in particular, TNF alpha plus IFN gamma, elicited a strong enhancement of nitric oxide synthesis and, as revealed by western blot and RT-PCR analysis, the expression of iNOS. TNF alpha and IL-1 were able to activate p38 MAPK in a time- and dose-dependent manner and together showed a combinatorial effect. In contrast, IFN gamma neither activated on its own nor enhanced the activation of p38 MAPK in response to TNF alpha and IL-1. However, a specific inhibitor of p38 MAPK, i.e., SB203580, inhibited the induction of iNOS in cytokine combination-treated cells in a dose-dependent manner, thereby suggesting a role for the MAPK cascade in regulating the induction of iNOS gene expression in cytokine-treated cells. Blocking of nitric oxide production by an inhibitor of iNOS, i.e., nitro-L-arginine methyl ester, had a minimal protective effect against cytokine-mediated cytotoxicity that occurred before the elevation of nitric oxide levels, thereby indicating temporal and functional dissociation of nitric oxide production from cell killing.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cytokines/pharmacology , Mitogen-Activated Protein Kinases , Nitric Oxide Synthase/genetics , Oligodendroglia/enzymology , Animals , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cell Line , Culture Media/pharmacology , Cytotoxins/pharmacology , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Imidazoles/pharmacology , Interferon-gamma/pharmacology , Interleukin-1/pharmacology , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Oligodendroglia/cytology , Oligodendroglia/drug effects , Phosphorylation , Pyridines/pharmacology , RNA, Messenger/analysis , Tumor Necrosis Factor-alpha/pharmacology , p38 Mitogen-Activated Protein Kinases
5.
J Neurochem ; 72(1): 112-9, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9886061

ABSTRACT

Oxidative stress is known to induce cell death in a wide variety of cell types, apparently by modulating intracellular signaling pathways. In this study, we have examined the activation of mitogen-activated protein kinase (MAPK) cascades in relation to oxidant-induced cell death in an oligodendrocyte cell line, central glia-4 (CG4). Exposure of CG4 cells to hydrogen peroxide (H2O2) resulted in an increased tyrosine phosphorylation of several protein species, including the abundantly expressed platelet-derived growth factor (PDGF) receptor and the activation of the three MAPK subgroups, i.e., extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK). Dose-response studies showed differential sensitivities of PDGF receptor phosphorylation (>1 mM) and ERK/p38 MAPK (>0.5 mM) and JNK (>0.1 mM) activation by H2O2. The activation of ERK was inhibited by PD98059, a specific inhibitor of the upstream kinase, MAPK or ERK kinase (MEK). H2O2 also activated MAPK-activated protein kinase-2, and this activation was blocked by SB203580, a specific inhibitor of p38 MAPK. The oxidant-induced cell death was indicated by morphological changes, decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, and DNA fragmentation. These effects were suppressed dose-dependently by the MEK inhibitor PD98059. The results demonstrate that H2O2 induces the activation of multiple MAPKs in oligodendrocyte progenitors and that the activation of ERK is associated with oxidant-mediated cytotoxicity.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Hydrogen Peroxide/pharmacology , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinase Kinases , Mitogen-Activated Protein Kinases , Oligodendroglia/enzymology , Oxidants/pharmacology , Signal Transduction/physiology , Animals , Calcium-Calmodulin-Dependent Protein Kinases/analysis , Cell Death/drug effects , Cell Line , DNA Fragmentation , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Imidazoles/pharmacology , Immunoblotting , MAP Kinase Kinase 4 , Mitogen-Activated Protein Kinase 3 , Oligodendroglia/cytology , Oligodendroglia/drug effects , Oxidative Stress/physiology , Protein Kinases/analysis , Protein Kinases/metabolism , Pyridines/pharmacology , Signal Transduction/drug effects
6.
J Neurosci Res ; 54(5): 574-83, 1998 Dec 01.
Article in English | MEDLINE | ID: mdl-9843148

ABSTRACT

Oligodendrocytes in multiple sclerosis brain may be under a direct attack by proinflammatory cytokines, particularly tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma). In this study, we have examined the in vitro cytotoxic effects of the two cytokines, individually and in combination, on oligodendrocyte lineage cells using morphological criteria, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction assay (MTT), terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL), and agarose-gel electrophoretic analysis of fragmented DNA. IFNgamma exerted a dose-dependent cytotoxic effect on cultured CG4 cells, an oligodendrocyte progenitor cell line, and in primary cultures of purified oligodendrocyte progenitors. TNFalpha, while by itself being only mildly toxic, greatly potentiated the cytotoxicity of IFNgamma. The cytokine effects were developmentally modified in that their cytotoxic and cooperative effects became less evident in more differentiated cells. A cell-permeable peptide inhibitor (i.e., z-VAD.fmk) of caspases partially suppressed apoptotic changes elicited by the cytokine combination in CG4 cells but not in primary oligodendrocytes. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of mRNA prepared from cytokine-treated cultures revealed an increased expression of the death receptor, Fas. The results suggest particular vulnerability of oligodendrocyte progenitors to a combination of TNFalpha and IFNgamma involving an activation of the cell death program.


Subject(s)
Apoptosis/drug effects , Interferon-gamma/pharmacology , Oligodendroglia/drug effects , Stem Cells/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Caspase Inhibitors , Caspases/physiology , Cells, Cultured , Drug Synergism , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/physiology , Oligodendroglia/cytology , Protease Inhibitors/pharmacology , RNA, Messenger/biosynthesis , Rats , Recombinant Proteins/pharmacology , Stem Cells/cytology , fas Receptor/biosynthesis , fas Receptor/genetics
7.
J Neurosci ; 18(5): 1633-41, 1998 Mar 01.
Article in English | MEDLINE | ID: mdl-9464988

ABSTRACT

Tumor necrosis factor-alpha (TNFalpha) and nitric oxide (NO), the product of inducible NO synthase (iNOS), mediate inflammatory and immune responses in the CNS under a variety of neuropathological situations. They are produced mainly by "activated" astrocytes and microglia, the two immune regulatory cells of the CNS. In this study we have examined the regulation of TNFalpha and iNOS gene expression in endotoxin-stimulated primary glial cultures, focusing on the role of mitogen-activated protein (MAP) kinase cascades. The bacterial lipopolysaccharide (LPS) was able to activate extracellular signal-regulated kinase (ERK) and p38 kinase subgroups of MAP kinases in microglia and astrocytes. ERK activation was sensitive to PD98059, the kinase inhibitor that is specific for ERK kinase. The activity of p38 kinase was inhibited by SB203580, a member of the novel class of cytokine suppressive anti-inflammatory drugs (CSAIDs), as revealed by blocked activation of the downstream kinase, MAP kinase-activated protein kinase-2. The treatment of glial cells with either LPS alone (microglia) or a combination of LPS and interferon-gamma (astrocytes) resulted in an induced production of NO and TNFalpha. The two kinase inhibitors, at micromolar concentrations, individually suppressed and, in combination, almost completely blocked glial production of NO and the expression of iNOS and TNFalpha, as determined by Western blot analysis. Reverse transcriptase-PCR analysis showed changes in iNOS mRNA levels that paralleled iNOS protein and NO while indicating a lack of effect of either of the kinase inhibitors on TNFalpha mRNA expression. The results demonstrate key roles for ERK and p38 MAP kinase cascades in the transcriptional and post-transcriptional regulation of iNOS and TNFalpha gene expression in endotoxin-activated glial cells.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Gene Expression Regulation, Enzymologic , Lipopolysaccharides/pharmacology , Microglia/metabolism , Mitogen-Activated Protein Kinases , Nitric Oxide Synthase/biosynthesis , Tumor Necrosis Factor-alpha/biosynthesis , Animals , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cells, Cultured , Enzyme Activation , Enzyme Induction , Enzyme Inhibitors/pharmacology , Female , Microglia/enzymology , Mitogen-Activated Protein Kinase 1 , Nitric Oxide/antagonists & inhibitors , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type II , Polymerase Chain Reaction , Rats , Rats, Sprague-Dawley , Tumor Necrosis Factor-alpha/genetics , p38 Mitogen-Activated Protein Kinases
8.
Neurochem Res ; 23(2): 219-25, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9475517

ABSTRACT

Recently, we reported on the activation of c-Jun N-terminal kinase (JNK) in primary glial cells noting certain differences in the patterns of kinase activation in astrocytes and oligodendrocytes (Zhang et al., J Neurosci Res 46:114-121;1996). In this extended study, we have examined the activation and expression levels of JNK1 and JNK2 isoforms in different glial cell types including the two in vitro-defined astroglial subtypes (type-1 and type-2), oligodendrocytes and microglia. An in-gel kinase assay of cell extracts and JNK-immunoprecipitates revealed the activation of both JNK1 and JNK2 in type-1 astrocytes in response to TNFalpha, and in microglia, in response to TNFalpha and bacterial lipopolysaccharide. The strong activation of the two JNK isoforms in type-1 astrocytes and microglia contrasted with a predominant activation of JNK1 over JNK2 in type-2 astrocytes and oligodendrocytes, the two glial subtypes sharing a common lineage. Immunoblot and immunocytochemical analyses using isoform-specific antibodies showed a differential expression of the two isoforms in different glial cells thereby accounting for their observed differential activation.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/biosynthesis , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Isoenzymes/biosynthesis , Isoenzymes/metabolism , Mitogen-Activated Protein Kinases , Neuroglia/enzymology , Protein Kinases/biosynthesis , Protein Kinases/metabolism , Animals , Animals, Newborn , Cells, Cultured , Enzyme Activation/drug effects , JNK Mitogen-Activated Protein Kinases , Lipopolysaccharides/pharmacology , Mitogen-Activated Protein Kinase 9 , Neuroglia/drug effects , Rats , Tumor Necrosis Factor-alpha/pharmacology
9.
Dev Neurosci ; 19(2): 152-61, 1997.
Article in English | MEDLINE | ID: mdl-9097030

ABSTRACT

Developmental expression of neutral monoglycosylceramides (MGCs) has been examined in rat brain from embryonic day 15 (E15) to postnatal day 30 (P30) and adulthood. To this point, glucosylceramide (GlcCer) is the only MGC that has been characterized in embryonic brain. Galactosylceramide (GalCer) appears at P1, increases with age until P25 and remains constant thereafter. The developmental occurrence of GlcCer and GalCer agrees well with their respective glucosyl- and galactosyltransferase activities. Cerebroside fatty-acid and base compositions, examined by gas chromatography, also change during development. Several alkali-labile fast-migrating cerebrosides (FMCs) with a higher thin-layer chromatography RF than GalCer/ GlcCer are expressed early at P10, increase in concentration with age (P25-P30) and are unchanged until maturity. They are derivatives of GalCer. By employing a newly developed neutral methylation procedure, we have confirmed the structure of one of the FMCs as 6-acylGalCer. A reduction in brain FMC concentrations along with GalCer in murine genetic dysmyelinating disorders (jimpy and quaking) further supports the conclusion that they are myelin constituents.


Subject(s)
Brain Chemistry/physiology , Ceramides/metabolism , Animals , Brain/growth & development , Brain Chemistry/genetics , Ceramides/biosynthesis , Ceramides/chemistry , Cerebrosides/metabolism , Chromatography, Gas , Chromatography, Thin Layer , Demyelinating Diseases/metabolism , Immunochemistry , Mice , Mice, Jimpy , Mice, Quaking , Myelin Sheath/metabolism , Myelin Sheath/physiology , Rats
10.
J Neurosci Res ; 46(1): 114-21, 1996 Oct 01.
Article in English | MEDLINE | ID: mdl-8892112

ABSTRACT

Glial cells in the mammalian CNS are subject to environmental stress resulting from a variety of neuro-pathological conditions. In this study, we have examined the activation of a stress signal responsive kinase, i.e., stress-activated protein kinase (SAPK) or c-Jun N-terminal kinase (JNK), in primary cultures of rat brain glial cells (i.e., astrocytes and oligodendrocytes) and an oligodendrocyte progenitor cell line, CG4, in response to cytokines and other stress inducers. JNK/SAPK activity was measured by an immune complex kinase assay using polyclonal anti-JNK antibodies along with GST c-Jun (1-79) as the substrate. Among the cytokines tested, TNF-alpha had the strongest effect on JNK activation followed by TNF-beta in both the glial cell types while a substantial level of kinase activation was observed in response to IL-1 in astrocytes. JNK activation by TNF-alpha in astrocytes, but not in oligodendrocytes, showed a biphasic response. An in-gel kinase assay of cell extracts and immunoprecipitated JNK confirmed the activation of JNK1 in cells treated with TNF-alpha. JNK was also activated by several other stress-inducing factors including. UV light, heat shock, inhibitors of protein synthesis, and mechanical injury. Incubation of cells with bacterial sphingomyelinase and a cell-permeable ceramide stimulated JNK activity, suggesting that the ceramide pathway may play a role in JNK activation, although the time course of activation did not correspond to that of TNF-alpha. The results are discussed in terms of possible roles of JNK activation in signaling for gliosis in astrocytes and as a protective/toxic response in oligodendrocytes.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Mitogen-Activated Protein Kinases , Neuroglia/enzymology , Animals , Astrocytes/enzymology , Cells, Cultured , Cytokines/pharmacology , Electrophoresis, Polyacrylamide Gel , Enzyme Activation/physiology , Fibroblasts/metabolism , JNK Mitogen-Activated Protein Kinases , Oligodendroglia/enzymology , Rats , Sphingomyelin Phosphodiesterase , Stem Cells/metabolism , Tumor Necrosis Factor-alpha/pharmacology
11.
J Neurochem ; 66(5): 1986-94, 1996 May.
Article in English | MEDLINE | ID: mdl-8780027

ABSTRACT

The proliferation and differentiation of oligodendrocyte progenitors are stringently controlled by an interacting network of growth and differentiation factors. Not much is known, however, about the intracellular signaling pathways activated in oligodendrocytes. In this study, we have examined the activation of mitogen-activated protein (MAP) kinase [also called extracellular signal-regulated protein kinases (ERKs)] in primary cultures of developing oligodendrocytes and in a primary oligodendrocyte cell line, CG4, in response to platelet-derived growth factor (PDGF) and basic fibroblast growth factor. MAP kinase activation was determined by an ingel protein kinase renaturation assay using myelin basic protein (MBP) as the substrate. The specificity of MAP kinase activation was further confirmed by an immune complex kinase assay using anti-MAP kinase antibodies. Stimulation of oligodendrocyte progenitors with the growth factors PDGF and basic fibroblast growth factor and a protein kinase C-activating tumor promoter, phorbol 12-myristate 13-acetate, resulted in a rapid activation of p42mapk (ERK2) and, to a lesser extent, p44mapk (ERK1). Immunoblot analysis with anti-phosphotyrosine antibodies revealed an increased Tyr phosphorylation of a 42-kDa phosphoprotein band cross-reacting with anti-MAP kinase antibodies. The phosphorylation of p42mapk in PDGF-treated oligodendrocyte progenitors was preceded by a robust autophosphorylation of the growth factor receptor. Immunoblot analysis with anti-pan-ERK antibodies indicated the presence of ERK-immunoreactive species other than p42mapk and p44mapk in oligodendrocytes. The presence of some of the same pan-ERK-immunoreactive species and certain renaturable MBP kinase activities was also demonstrable in myelin preparations from rat brain, suggesting that MAP kinases (and other MBP kinases) may function not only during oligodendrogenesis but also in myelinogenesis.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Oligodendroglia/enzymology , Protein-Tyrosine Kinases/metabolism , Animals , Cells, Cultured , Cyclic AMP-Dependent Protein Kinases/physiology , Enzyme Activation , Glycogen Synthase Kinase 3 , Growth Substances/pharmacology , Immunoblotting , Mitogen-Activated Protein Kinase 1 , Mitogens/pharmacology , Myelin Sheath/enzymology , Rats , Rats, Sprague-Dawley , Signal Transduction , Stem Cells/enzymology , Tetradecanoylphorbol Acetate/pharmacology
12.
J Cell Physiol ; 165(2): 417-24, 1995 Nov.
Article in English | MEDLINE | ID: mdl-7593220

ABSTRACT

Thrombin is known to evoke numerous inflammatory and proliferative responses in a wide variety of its target cells. Recent studies have demonstrated morphoregulatory and mitogenic effects of thrombin on astroglial cells (astrocytes). The present study deals with thrombin-induced activation of mitogen-activated protein (MAP) kinase in primary cultures of rat astrocytes. Treatment of serum-starved astrocytes with thrombin resulted in a rapid activation of tyrosine (Tyr) phosphorylation of a set of proteins including a prominent one with a molecular mass of 42 kDa (p42). The identity of p42 with MAP kinase was confirmed by MAP kinase-immunoreactivity of isolated [i.e., immunoprecipitated with anti-phosphotyrosine (PY) antibodies] p42 and by increased myelin basic protein (MBP) kinase activity present in MAP kinase immunoprecipitates of thrombin-treated cultures. Pertussis toxin (PTX) pretreatment failed to inhibit thrombin stimulation of p42 phosphorylation, indicating the lack of involvement of PTX sensitive G proteins in the mechanism of activation of MAP kinase by thrombin. Chronic exposure of cultures to phorbol 12-myristate 13-acetate to down-regulate PKC resulted in an attenuation of thrombin-induced p42 Tyr phosphorylation, although H-7, a known PKC inhibitor, failed to block thrombin effect. However, staurosporine, a nonspecific protein kinase inhibitor, prevented the activation of p42 phosphorylation. It is concluded that thrombin induces MAP kinase activation in astrocytes by a mechanism involving a staurosporine-sensitive pathway.


Subject(s)
Astrocytes/drug effects , Astrocytes/enzymology , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Thrombin/pharmacology , Animals , Cells, Cultured , Enzyme Activation/drug effects , Mitogen-Activated Protein Kinase 1 , Pertussis Toxin , Phosphorylation , Precipitin Tests , Rats , Tetradecanoylphorbol Acetate/pharmacology , Tyrosine/metabolism , Virulence Factors, Bordetella/pharmacology
13.
Neuroreport ; 6(15): 2037-40, 1995 Oct 23.
Article in English | MEDLINE | ID: mdl-8580435

ABSTRACT

The interaction of hyaluronectin (HN), a hyaluronic acid-binding extracellular matrix (ECM) glycoprotein with two other ECM-associated molecules, laminin and fibronectin, was studied by ligand blot and solid phase ligand binding assays. Ligand blot analysis with biotin-labeled HN revealed a strong binding of HN to immobilized laminin and a weaker binding to fibronectin. Ligand binding studies indicated a concentration dependent, Ca(2+)-independent binding of HN to laminin. Binding of HN to laminin but not to fibronectin was resistant to increased salt concentrations indicating a non-electrostatic, protein-protein interaction of HN with laminin. The functional relevance of HN-laminin interaction was demonstrated by an inhibition of laminin-supported astroglial process formation by HN.


Subject(s)
Astrocytes/metabolism , Hyaluronic Acid/metabolism , Laminin/metabolism , Animals , Cells, Cultured/metabolism , Humans , Radioligand Assay , Rats
14.
Dev Neurosci ; 17(4): 256-63, 1995.
Article in English | MEDLINE | ID: mdl-8575345

ABSTRACT

Myristoylated alanine-rich C-kinase substrate (MARCKS), a major substrate of activated protein kinase C (PKC), is thought to be involved in PKC-mediated signal transduction events. In the present study, we have examined the expression of MARCKS in primary cultures of rat glial cells. Western blot analysis of different glial cell types (i.e., astrocytes, oligodendrocytes and microglia) revealed a relatively high level of MARCKS protein in oligodendrocytes. MARCKS protein and MARCKS mRNA levels in oligodendrocytes increased with time in culture, indicating a developmental regulation in MARCKS gene expression in differentiating oligodendrocytes. Immunocytochemical examination of developing oligodendrocytes indicated a strong labeling of MARCKS distributed both in the cell body and in the lacy network of processes. These findings, in concert with our previous observations on the role of PKC in oligodendrogenesis, strongly implicate a PKC-signaling system in oligodendrocyte development.


Subject(s)
Brain/growth & development , Intracellular Signaling Peptides and Proteins , Membrane Proteins , Oligodendroglia/metabolism , Protein Biosynthesis , Animals , Blotting, Western , Brain/cytology , Cell Differentiation , Cells, Cultured , Immunohistochemistry , Microscopy, Fluorescence , Myelin Sheath/metabolism , Myristoylated Alanine-Rich C Kinase Substrate , Oligodendroglia/cytology , Phosphorylation , Protein Kinase C/biosynthesis , RNA, Messenger/biosynthesis , Rats , Signal Transduction , Stem Cells/metabolism
15.
Dev Neurosci ; 17(5-6): 267-84, 1995.
Article in English | MEDLINE | ID: mdl-8829916

ABSTRACT

Growth factor-activated and cell adhesion-mediated signaling plays a crucial role in the development and differentiation of oligodendrocytes and astrocytes, the two macroglial cells of the CNS. Guided by the recent advances in the elucidation of intracellular signaling pathways, researchers have begun to investigate the mechanisms that regulate gliogenesis, glial cell differentiation and myelinogenesis. The progress made so far strongly implicates protein kinase C (PKC)- and protein kinase A (PKA)-mediated signaling in glial cell proliferation and differentiation. These pathways are also involved in the morphogenesis of astrocytes and in the myelin gene expression of oligodendrocytes. The cellular responses elicited by both PKC and PKA pathways in oligodendrocytes are developmentally controlled. The PKC pathway also seems to play a key role in phenotypic plasticity and regeneration of oligodendrocytes. Initial events of myelination requiring cell surface interactions may involve signaling via Fyn kinase which functionally associates with myelin-associated glycoprotein, an adhesion molecule implicated in myelinogenesis.


Subject(s)
Neuroglia/physiology , Signal Transduction/physiology , Animals , Humans
16.
J Neurosci Res ; 39(1): 1-10, 1994 Sep 01.
Article in English | MEDLINE | ID: mdl-7528816

ABSTRACT

Previous studies have demonstrated that inhibitors of glycoprotein processing glucosidases interfere with the development of oligodendrocyte properties in primary cultures of embryonic rat brain cells (Bhat, J Neurosci Res 20:158-164, 1988). The present study examines the effect of castanospermine, an inhibitor of the processing glucosidases, on the development and differentiation of isolated oligodendrocyte progenitor cells. Treatment of oligodendrocyte progenitors with castanospermine did not affect the developmental progression of the precursors to become committed oligodendrocytes as revealed by comparable increases in the percentages of cells positive for galactocerebroside (a surface marker for terminally differentiated oligodendrocytes) in control and drug-treated cultures. On the other hand, there was an impairment of the expression of differentiated properties of oligodendrocytes [i.e., sulfolipid synthesis, myelin basic protein (MBP)] and 2'3'-cyclic nucleotide 3'-phosphohydrolase in the drug-treated cultures. Immunocytochemical analysis with anti-MBP antibodies revealed a reduced number of MBP-positive cells in inhibitor-treated cultures. Furthermore, a majority of MBP-positive cells in such cultures displayed immunoreactive MBP in their cell body and not the processes, unlike in control cultures where both cell body and the processes of oligodendrocytes stained intensely for MBP. The strong inhibitory effect of castanospermine on the expression of oligodendrocyte-specific activities was contrasted with a relatively smaller effect of swainsonine, a mannosidase inhibitor on oligodendrocyte differentiation. Both castanospermine and swainsonine, however, effectively blocked the formation of complex-type oligosaccharides, suggesting thereby a lack of correlation between the inhibition of the formation of complex-type oligosaccharides and oligodendrocyte differentiation. It is suggested, therefore, that early trimming reactions involving the removal of glucose residues from the high mannose oligosaccharides in the endoplasmic reticulum may be essential for the cell surface localization and function of glycoproteins critically involved in surface interactions of oligodendrocytes with each other and/or with the substratum.


Subject(s)
Glucosidases/antagonists & inhibitors , Indolizines/pharmacology , Nerve Tissue Proteins/physiology , Oligodendroglia/drug effects , Oligosaccharides/metabolism , Phosphoric Diester Hydrolases , Protein Processing, Post-Translational/drug effects , Swainsonine/pharmacology , 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase , 2',3'-Cyclic-Nucleotide Phosphodiesterases/biosynthesis , 2',3'-Cyclic-Nucleotide Phosphodiesterases/genetics , Animals , Animals, Newborn , Antigens, Differentiation/biosynthesis , Antigens, Differentiation/genetics , Biomarkers , Cell Differentiation/drug effects , Cell Division/drug effects , Cells, Cultured , Endoplasmic Reticulum/metabolism , Gene Expression Regulation/drug effects , Glucose/metabolism , Glucosidases/physiology , Glycoside Hydrolase Inhibitors , Glycosylation/drug effects , Mannose/metabolism , Mannosidases/antagonists & inhibitors , Myelin Basic Protein/biosynthesis , Myelin Basic Protein/genetics , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/biosynthesis , Oligodendroglia/cytology , Oligodendroglia/enzymology , Rats , Stem Cells/cytology , Stem Cells/drug effects , Stem Cells/metabolism , alpha-Glucosidases , alpha-Mannosidase
17.
Glia ; 9(2): 157-62, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8244536

ABSTRACT

Endogenous opioids inhibit nervous system development by inhibiting the proliferation of certain neuronal and glial progenitors. To determine whether opioids affect the growth of preoligodendrocytes, the effects of the endogenous opioid [Met5]-enkephalin were examined in preoligodendrocytes in primary mixed-glial and preoligodendrocyte-enriched (> 98% pure) cultures. Proliferating preoligodendrocytes in mixed-glial or preoligodendrocyte-enriched cultures were continuously treated for a total of 40 h with either basal growth media (controls), 1 microM [Met5]-enkephalin, 1 microM [Met5]-enkephalin plus the opioid antagonist naloxone (3 microM), or naloxone alone (3 microM), and incubated in [3H]-thymidine (0.2 microCi/ml/4-6 h) after 34-36 h of opioid exposure. Opioid-dependent changes in DNA synthesis were assessed autoradiographically in O4-immunoreactive oligodendrocyte progenitors. Naloxone alone significantly decreased the rate of DNA synthesis and number of O4-immunoreactive preoligodendrocytes in mixed-glial cultures. However, naloxone and/or [Met5]-enkephalin did not affect DNA synthesis or the number of O4-immunoreactive preoligodendrocytes in cultures enriched in preoligodendrocytes. The results suggest that astrocytes, or perhaps another cell type, play a permissive role in opioid-dependent alterations in preoligodendrocyte proliferation. Endogenous opioids affect the genesis of neural cells by both direct and indirect mechanisms.


Subject(s)
Enkephalin, Methionine/physiology , Oligodendroglia/cytology , Stem Cells/cytology , Animals , Cell Division , DNA/biosynthesis , Naloxone/administration & dosage , Rats , Rats, Sprague-Dawley
18.
Anal Biochem ; 207(2): 304-10, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1481985

ABSTRACT

This paper describes a simple and direct procedure for assaying Ca(2+)-dependent protein kinase C (PKC) activity in membrane fractions isolated from purified murine B lymphocytes (B cells) treated with phorbol 12-myristate 13-acetate (PMA). The results indicate that membrane-bound PKC in B cells, treated with PMA, can be measured directly in the presence of 0.5% Brij 58 by assaying the transfer of 32P from [gamma-32P]ATP to histone type III-S. This method obviates the need for partial purification of the protein kinase by ion-exchange chromatography prior to assaying PKC activity. The properties of membrane-associated PKC activity in B cells have been characterized, and the kinetics of PMA-induced translocation of PKC in cultured murine B cells, the rat glial tumor clone C6, and primary neonatal osteoblastic cells have been defined by this direct assay. The results obtained with B cells and the other cell lines indicate that this direct assay procedure could be useful for studies on the factors controlling PKC translocation in a variety of cultured mammalian cells.


Subject(s)
B-Lymphocytes/enzymology , Cetomacrogol/pharmacology , Protein Kinase C/metabolism , Adenosine Triphosphate/metabolism , Animals , B-Lymphocytes/drug effects , Cell Fractionation , Cell Membrane/enzymology , Cells, Cultured , Cytosol/enzymology , Detergents/pharmacology , Female , Glioma , Kinetics , Mice , Mice, Inbred DBA , Osteoblasts/enzymology , Phosphorus Radioisotopes , Protein Kinase C/analysis , Radioisotope Dilution Technique , Rats , Tetradecanoylphorbol Acetate/pharmacology , Tumor Cells, Cultured
19.
J Neurosci Res ; 32(3): 340-9, 1992 Jul.
Article in English | MEDLINE | ID: mdl-1433384

ABSTRACT

Cell proliferation and the expression of the protooncogenes c-fos and c-jun have been examined in the primary cultures of oligodendroglial (OL) progenitor cells in response to phorbol 12-myristate 13-acetate (PMA), serum, insulin, insulin-like growth factor-I (IGF-I), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF). Combined [3H]thymidine autoradiography and immunocytochemistry was used to assess the mitogenic response of O4 (an oligodendrocyte-specific marker)-positive OL progenitors. In addition, the rate of DNA synthesis was measured by the incorporation of [3H]thymidine into acid-precipitable material. It was found that all of the agents tested stimulated DNA synthesis in OL progenitors and induced a rapid increase in c-fos and c-jun protooncogene expression. The induction of c-fos gene expression and DNA synthesis in response to PMA was completely blocked by 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C (PKC), thereby suggesting a role for PKC in the control of c-fos expression and cell proliferation in OL progenitors.


Subject(s)
Oligodendroglia/cytology , Proto-Oncogenes , Stem Cells/drug effects , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine , Animals , Autoradiography , Blotting, Northern , Cell Division/drug effects , Cells, Cultured , Female , Genes, fos/drug effects , Genes, jun/drug effects , Immunohistochemistry , Isoquinolines/pharmacology , Oligodendroglia/drug effects , Oligodendroglia/metabolism , Piperazines/pharmacology , Pregnancy , Protein Kinase Inhibitors , Rats , Rats, Sprague-Dawley , Tetradecanoylphorbol Acetate/pharmacology
20.
Brain Res Mol Brain Res ; 13(3): 199-206, 1992 Apr.
Article in English | MEDLINE | ID: mdl-1317493

ABSTRACT

Astrocytic activation plays a major role in homeostatic maintenance of the central nervous system in response to neuronal damage. To assess the reactivity of astrocytes in transient cerebral ischemia of the gerbil, we studied the levels of glial fibrillary acidic protein (GFAP) and its mRNA. GFAP mRNA increased by 4 h after carotid artery occlusion, reached peak levels by 72 h with a 12-fold increase over control and then started declining as early as 96 h postischemia. An examination of the specific regions of the brain revealed an increase in GFAP mRNA associated with the forebrain, midbrain, hippocampus and striatum. GFAP mRNA in the non-ischemic cerebellum however, remained expressed at constitutively low levels. Immunoblot analysis with anti-GFAP antibodies demonstrated a 2- to 3-fold increase in the protein after 24 and 48 h of reperfusion. Pretreatment with pentobarbital and 1-(5'-oxohexyl)-3-methyl-7-propyl xanthine (HWA 285), the drugs that have been shown to protect against ischemic damage, prevented the increase in GFAP mRNA in the cortex following ischemic injury. Forebrain ischemia also induced vimentin mRNA and protein quantities by 12 h of reperfusion in the cortex. The levels of c-fos and preproenkephalin mRNA increased rapidly within 1 h after ischemic injury, demonstrating a temporal difference in mRNA changes following ischemia. These results indicate that an increase in GFAP and vimentin, the two glial intermediate filament proteins in the area of the ischemic lesion may be associated with a glial response to injury.


Subject(s)
Astrocytes/metabolism , Cerebral Cortex/metabolism , Corpus Striatum/metabolism , Glial Fibrillary Acidic Protein/biosynthesis , Hippocampus/metabolism , Ischemic Attack, Transient/metabolism , Vimentin/biosynthesis , Animals , Enkephalins/biosynthesis , Enkephalins/metabolism , Gene Expression , Gerbillinae/metabolism , Glial Fibrillary Acidic Protein/genetics , Male , Protein Precursors/biosynthesis , Protein Precursors/metabolism , Proto-Oncogene Proteins c-fos/biosynthesis , Proto-Oncogene Proteins c-fos/genetics , RNA, Messenger/analysis , Vimentin/genetics
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