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MBP
Terms modified by MBP Selected AbstractsEffects of early weaning on anxiety and prefrontal cortical and hippocampal myelination in male and female wistar ratsDEVELOPMENTAL PSYCHOBIOLOGY, Issue 4 2008Yuka Kodama Abstract We investigated developmental changes in myelin formation in the prefrontal cortex and the hippocampus, and behavioral effects of early weaning in Wistar rats. Early-weaned rats showed decreased numbers of open-arm entries in an elevated plus-maze in both sexes at 4 weeks old; this effect persisted in males, but ceased in females after this age. Expression of myelin basic protein (MBP) showed both age-dependent increases and sex differences; 4-week-old males exhibited higher MBP levels in the hippocampus, whereas 7-week-old males showed lower MBP levels in the prefrontal cortex compared to females of the same age. There was a tendency for group differences from weaning for the 21.5-kDa isoform in the prefrontal cortex. Although these results suggest that male rats are more vulnerable than females to early-weaning effects on anxiety-related behaviors, further detailed analysis is needed to clarify the functional relationship between myelination and anxiety-related behaviors. © 2008 Wiley Periodicals, Inc. Dev Psychobiol 50: 332,342, 2008. [source] Effect of detergent on electromigration of proteins: CE of very low density lipoprotein receptor modules and viral proteinsELECTROPHORESIS, Issue 20 2007Leopold Kremser Dr. Abstract The different electrophoretic behavior of the members of two groups of proteins with respect to the absence or presence of detergent additives in the BGE was explored. Recombinant soluble concatemers of repeat 3 of the very low density lipoprotein (VLDL)-receptor fused at their N -terminus to maltose-binding protein (MBP) exhibited different electrophoretic mobilities in borate buffer (pH,8.3) in the absence and in the presence of dodecyl-PEG ether (D-PEG). This enabled the separation of the receptor fragments from MBP after enzymatic cleavage. In the presence of SDS, the mobilities of all proteins approached the same values with increase in detergent concentrations. In contrast, viral capsid proteins of a human rhinovirus (HRV) exhibited different migration in the presence of the additive. For the receptor proteins, extreme apparent high plate numbers were observed when the SDS concentration in the sample and the separation buffer differed. This effect might be erroneously interpreted as a high efficiency. However, it is due to the conductivity boundaries caused by the sample and leads to a total loss of separation. [source] Disruption effects of monophthalate exposures on inter-Sertoli tight junction in a two-compartment culture modelENVIRONMENTAL TOXICOLOGY, Issue 3 2008Yun-Hui Zhang Abstract Phthalates are suspect environmental endocrine disruptors that may affect male reproduction and development by disturbing androgen synthesis and cell,cell interactions in the seminiferous epithelium. The in vivo metabolites, monophthalates, are thought to be the active agents, and toxicant effects including testicular damage and decreased sperm motility have been described previously. In this study, the aim was to investigate the effect of monophthalates on Sertoli cells using a two-compartment cell culture model, asking whether tight junction protein structures are affected, compromising the blood-testis barrier and contributing to male-mediated toxicity. Sertoli cells were isolated from Sprague Dawley rat testes and seeded onto the filters of two-compartment wells. A Sertoli cell monolayer was allowed to form, whereupon the cultures were treated with 0, 10, 30, 150, and 600 ,mol/L monobutyl phthalate (MBP) or mono-2-ethylhexyl phthalate (MEHP) for 24 h. Effects on the tight junctions between adjacent Sertoli cells were studied by light and transmission electron microscopy, the transepithelial electrical resistance (TEER) assay, and immunofluorescence localization. Results showed that exposures to monophthalates destroyed tight junctional structure in Sertoli cell monolayers in a dose-depended manner, as evidenced by a loss of single-cell layer organization in the cultures, decline of TEER value, and decreased expression of proteins associated with tight junctions such as zonula occludens-1 (ZO-1), F-actin, and Occludin. The changes were observed at doses of 150 and 600 ,mol/L, which is 10,100 times higher relative to estimated human exposures from the environment. These results are consistent with monophthalate-induced damage to tight junctions between adjacent Sertoli cells, suggesting that damage to Sertoli cell tight junctions induced by monophthalates may be an underlying mechanism of their male-mediated reproductive toxicity. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008. [source] L-Selectin-deficient SJL and C57BL/6 mice are not resistant to experimental autoimmune encephalomyelitisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2008Chiara Uboldi Abstract L-selectin has been suggested to play a role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Here we demonstrate that L-selectin,/, SJL mice are susceptible to proteolipid protein (PLP)-induced EAE because the compromised antigen-specific T cell proliferation in peripheral lymph nodes is fully compensated by the T cell response raised in their spleen. Transfer of PLP-specific T cells into syngeneic recipients induced EAE independent of the presence or absence of L-selectin on PLP-specific T cells or in the recipient. Leukocyte infiltration into the central nervous system parenchyma was detectable independent of the mode of disease induction and the presence or absence of L-selectin. In addition, we found L-selectin,/, C57BL/6 mice to be susceptible to myelin oligodendrocyte glycoprotein-induced EAE. Taken together, we demonstrate that in SJL and C57BL/6 mice L-selectin is not required for EAE pathogenesis. The apparent discrepancy of our present observation to previous findings, demonstrating a role of L-selectin in EAE pathogenesis in C57BL/6 mice or myelin-basic protein (MBP)-specific TCR-transgenic B10.PL mice, may be attributed to background genes rather than L-selectin and to a unique role of L-selectin in EAE pathogenesis in MBP-TCR-transgenic mice. [source] Copolymer effects on microglia and T,cells in the central nervous system of humanized miceEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2005Zsolt Illes The random amino acid copolymers FYAK and VWAK ameliorate EAE in a humanized mouse model expressing both a human transgenic myelin basic protein (MBP)85,99-specific T,cell receptor and HLA-DR2. Here we show that microglia isolated from the central nervous system (CNS) of humanized mice with EAE induced by MBP85,99 and treated with these copolymers had reduced expression of HLA-DR, and thus reduced capacity to present MBP85,99 and activate transgenic T,cells. In vitro microglia up-regulated empty HLA-DR2 upon activation with GM-CSF with or without LPS or IFN-,, but not with IL-4 or IL-10. Correspondingly, gene chip arrays showed that the CNS of untreated and YFAK-treated mice differentially expressed pro- and anti-inflammatory molecules during MBP85,99-induced EAE. Interestingly, microglia expressed the full-length ,,,and ,,,subunits of the tetrameric adaptor protein complexes AP-1 and AP-2 respectively, but after treatment with GM-CSF these complexes were cleaved, as had been found in immature dendritic cells derived from bone marrow. Strikingly, in vivo the perivascular lymphocyte infiltration seen in untreated mice immunized with MBP85,99 was composed of equal numbers of hV,2+ MPB85,99-specific transgenic and hV,2, endogenous T,cells, while the much smaller infiltration seen after treatment with YFAK was composed predominantly of hV,2, endogenous T,cells. [source] Delay of myelin formation in arylsulphatase A-deficient miceEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2005Afshin Yaghootfam Abstract Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulphatase A (ASA). This leads to the accumulation of the sphingolipid 3-O-sulphogalactosylceramide (sulphatide) and progressive demyelination in the nervous system of MLD patients. The mechanisms and development of pathology in the disease are still largely unknown. In this study we investigate how the inability to degrade sulphatide affects the formation of myelin in ASA-deficient (ASA,/,) mice. In mice at 2 weeks of age there was a substantial reduction in myelin basic protein (MBP) mRNA and protein. This was confirmed by an immunohistochemical analysis. MBP mRNA and protein, however, reach normal levels at 3 weeks of age. Proteolipid protein (PLP) and MAL mRNA were also reduced in ASA,/, mice at 2 weeks of age; whereas the level of PLP mRNA was normal at 26 weeks of age, MAL mRNA expression remained reduced up to this age. In situ hybridization revealed no significant changes in the number of myelinating oligodendrocytes or oligodendrocyte precursor cells in ASA,/, mice. These results suggest that oligodendrocyte differentiation was normal in ASA,/, mice. No differences were found in the expression of the sulphatide synthesizing enzymes cerebroside sulphotransferase and UDP-galactose : ceramide galactosyltransferase. Our data demonstrate a delay in myelin formation in ASA,/, mice. This raises the possibility that similar alterations in MLD patients may contribute to the pathology of the disease. [source] Cardiovascular Response to Graded Lower Body Negative Pressure in Young and Elderly ManEXPERIMENTAL PHYSIOLOGY, Issue 3 2001R. van Hoeyweghen Lower body negative pressure (LBNP) reduces central venous pressure (CVP) and cardiac output. The elderly are reported to have a limited capacity to increase cardiac output by increasing heart rate (HR), are especially dependent on end diastolic volume to maintain stroke volume and therefore should be especially vulnerable to LBNP. The present study compared the effects of LBNP in the young and old. Stroke volume was assessed non-invasively as stroke distance (SD) by aortovelography. Two groups of healthy male volunteers were studied: eight young (29.7 ± 2.0 years, mean ± S.E.M.) and nine old (70.1 ± 0.9 years). LBNP was applied progressively at 17.5, 35 and 50 mmHg in 20 min steps, with measurements taken during each steady state. There were similar, significant, falls in CVP in both groups. SD fell significantly in both groups from respective control values of 24.8 ± 1.6 and 16.6 ± 0.9 cm to 12.5 ± 1.3 and 8.9 ± 0.4 cm at a LBNP of 50 mmHg. Although SD in the elderly was significantly lower than in the young, the LBNP-induced changes were not different between groups. Both groups produced similar significant increases in vascular resistance, HR, plasma vasopressin (AVP) and noradrenaline. Mean arterial blood pressure (MBP) and plasma adrenaline did not change significantly. Therefore healthy old men respond to LBNP in a similar manner to the young, although MBP and SD are regulated around different baselines in the two groups. [source] Specific Ser-Pro phosphorylation by the RNA-recognition motif containing kinase KISFEBS JOURNAL, Issue 14 2000Alexandre Maucuer We present here a first appraisal of the phosphorylation site specificity of KIS (for ,kinase interacting with stathmin'), a novel mammalian kinase that has the unique feature among kinases to possess an RNP type RNA-recognition motif (RRM). In vitro kinase assays using various standard substrates revealed that KIS has a narrow specificity, with myelin basic protein (MBP) and synapsin I being the best in vitro substrates among those tested. Mass spectrometry and peptide sequencing allowed us to identify serine 164 of MBP as the unique site phosphorylated by KIS. Phosphorylation of synthetic peptides indicated the importance of the proline residue at position +1. We also identified a tryptic peptide of synapsin I phosphorylated by KIS and containing a phosphorylatable Ser-Pro motif. Altogether, our results suggest that KIS preferentially phosphorylates proline directed residues but has a specificity different from that of MAP kinases and cdks. [source] Cloning and characterization of CmGPD1, the Candida magnoliae homologue of glycerol-3-phosphate dehydrogenaseFEMS YEAST RESEARCH, Issue 8 2008Dae-Hee Lee Abstract Glycerol-3-phosphate dehydrogenase (GPDH) plays a central role in glycerol metabolism. A genomic CmGPD1 gene encoding NADH-dependent GPDH was isolated from Candida magnoliae producing a significant amount of glycerol. The gene encodes a polypeptide of 360 amino acids, which shows high homology with known NADH-dependent GPDHs of other species. The CmGPD1 gene was expressed in recombinant Escherichia coli with the maltose-binding protein (MBP) fusion system and purified to homogeneity using simple affinity chromatography. The purified CmGpd1p without the MBP fusion displayed an apparent molecular mass of 40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The CmGpd1p enzyme exhibited a Kcat/Km value of 195 min,1 mM,1 for dihydroxyacetone phosphate whereas Kcat/Km for glycerol-3-phosphate is 0.385 min,1 mM,1. In a complementation study, CmGpd1p rescued the ability of glycerol synthesis and salt tolerance in a Saccharomyces cerevisiae GPD1,GPD2, mutant strain. The overall results indicated that CmGPD1 encodes a functional homologue of S. cerevisiae GPDH. [source] Human remyelination promoting antibody inhibits apoptotic signaling and differentiation through Lyn kinase in primary rat oligodendrocytesGLIA, Issue 15 2010J. Watzlawik Abstract Purpose: Human remyelination promoting IgM mAbs target oligodendrocytes (OLs) and function in animal models of multiple sclerosis (MS). However, their mechanism of action is unknown. This study seeks to identify the cellular mechanism of action of a recombinant human IgM on OL survival. Methods: Binding of rHIgM22 to the surface of rat OLs was studied by co-localization with various markers. RHIgM22-mediated effects on apoptotic signaling in OLs, differentiation markers, and signaling molecules were detected by Western blotting and immunoprecipitation. Results: RHIgM22 co-localized with integrin ,3 but not other integrin ,-chains in OLs. Downstream of integrin ,3 we identified Src family kinase (SFK) Lyn as a key player of rHIgM22-mediated actions in OLs. Lyn immunoprecipitated in a complex together with integrin ,v,3 and PDGF,R. Lyn expression was 9-fold up-regulated and Lyn activation was 3-fold higher inrHIgM22-treated OL cultures compared with controls. RHIgM22 inhibited apoptotic signaling by greater than 10-fold reduction of caspase-3 and capsase-9 cleavage and reduced by 4-fold expression of differentiation markers MBP and MOG in OLs. SFK inhibitors PP2 and SU6656 inhibited Lyn activity and restored caspase-cleavage in OLs. A human IgM that did not promote remyelination and medium wereused as controls. Conclusions: rHIgM22 prevented apoptotic signaling andinhibited OL differentiation by Lyn implying thatIgM-mediated remyelination is due toprotection of OPC and OLs rather than promotion of OPC differentiation. © 2010 Wiley-Liss, Inc. [source] Identification of Tmem10/Opalin as an oligodendrocyte enriched gene using expression profiling combined with genetic cell ablationGLIA, Issue 11 2008Neev Golan Abstract Oligodendrocytes form an insulating multilamellar structure of compact myelin around axons, which allows efficient and rapid propagation of action potentials. However, little is known about the molecular mechanisms operating at the onset of myelination and during maintenance of the myelin sheath in the adult. Here we use a genetic cell ablation approach combined with Affymetrix GeneChip microarrays to identify a number of oligodendrocyte-enriched genes that may play a key role in myelination. One of the "oligogenes" we cloned using this approach is Tmem10/Opalin, which encodes for a novel transmembrane glycoprotein. In situ hybridization and RT-PCR analysis revealed that Tmem10 is selectively expressed by oligodendrocytes and that its expression is induced during their differentiation. Developmental immunofluorescence analysis demonstrated that Tmem10 starts to be expressed in the white matter tracks of the cerebellum and the corpus callosum at the onset of myelination after the appearance of other myelin genes such as MBP. In contrast to the spinal cord and brain, Tmem10 was not detected in myelinating Schwann cells, indicating that it is a CNS-specific myelin protein. In mature oligodendrocytes, Tmem10 was present at the cell soma and processes, as well as along myelinated internodes, where it was occasionally concentrated at the paranodes. In myelinating spinal cord cultures, Tmem10 was detected in MBP-positive cellular processes that were aligned with underlying axons before myelination commenced. These results suggest a possible role of Tmem10 in oligodendrocyte differentiation and CNS myelination. © 2008 Wiley-Liss, Inc. [source] Seasonal changes in herbage production and soil phosphorus contents in Japanese lawngrass (Zoysia japonica Steud.) and tall fescue (Festuca arundinacea Schreb.) pasturesGRASSLAND SCIENCE, Issue 1 2008Makoto Kaneko Abstract Seasonal changes in the above-ground phosphorus (P), soil total P (TP), soil Olsen P (OP) and soil microbial biomass P (MBP) were investigated for 2 years in Japanese lawngrass (Zy) and tall fescue (Tf) pastures on Japanese Andosol, with the goal of clarifying P characteristics in the Zy pasture in comparison with the Tf pasture. The soil P attributes were measured in two soil layers (root mat layer, 0,2.5 cm depth; under layer, 5,10 cm depth). The P concentration of the above-ground herbage in the Zy pasture, which was higher than the standard value and similar to those in the Tf pasture, might have contributed to the large amounts of the above-ground P mass. The lack of plowing management and the coverage with Japanese lawngrass might have changed soil TP. The TP, the OP and the OP/TP in the Zy pasture were higher than those in the Tf pasture, and the TP, the OP and the OP/TP at the root mat layer were higher than those at the under layer. A large amount of the TP and high P availability in the soil caused the large amounts of OP. Soil pH, soil microorganisms and MBP might have affected soil P availability in the Zy pasture. Plant litter in the root mat layer of the Zy pasture may have increased soil P accumulation and its availability, which might be reasons for the high P uptake in the present study. Japanese lawngrass pasture may be a system with improved soil P utilization efficiency based on P cycling. [source] Hypermethylation of gene promoters in hematological neoplasiaHEMATOLOGICAL ONCOLOGY, Issue 4 2002C. S. Chim Abstract Cancer cells are associated with global hypomethylation but with focal hypermethylation of specific gene promoters organized as CpG island. DNA methyltransferases, DNMT1 and 3 (3a and 3b), have been implicated in mediating maintenance and de novo methylation. Hypermethylation of gene promoters results in the inactivation of the corresponding genes, by preclusion of the formation of the transcription complex, due to the recruitment of MBP, MeCPs and histone deacetylase. This results in the deacetylation of histone and thus a compact chromatin complex unfavourable for the initiation of transcription. This methylation-associated gene silencing has been demonstrated in various genes including tumour suppressor genes (p15, p16, p73, VHL). Therefore, gene promoter hypermethylation collaborates with other mechanisms of gene inactivation such as deletion and intragenic mutations to fulfil Knudson's hypothesis. Hypermethylation may serve as a molecular disease marker for the detection of minimal residual disease. Emerging evidence suggests a possible prognostic value of gene promoter hypermethylation. Moreover, gene hypermethylation may also serve as a target for therapeutic invention by hypomethylating agents. Copyright © 2002 John Wiley & Sons, Ltd. [source] T-cell seeding: neonatal transfer of anti-myelin basic protein T-cell lines renders Fischer rats susceptible later in life to the active induction of experimental autoimmune encephalitisIMMUNOLOGY, Issue 1 2009Ilan Volovitz Summary Fischer strain rats resist active induction of experimental autoimmune encephalomyelitis (EAE) following immunization with guinea-pig myelin basic protein (MBP) in complete Freund's adjuvant (CFA). Nevertheless, we now report that an encephalitogenic CD4+ anti-MBP T-cell line could be developed from actively immunized Fischer rats. Adoptive transfer of the activated line mediated acute EAE in adult Fischer rats, but not in 1-day-old rats. Moreover, we found that both resting and activated anti-MBP T cells injected 1 day post-natally rendered these rats susceptible later in life to the active induction of EAE by immunization with MBP/CFA. The actively induced EAE manifested the accelerated onset of a secondary, memory-type response. Resting anti-MBP T cells injected even up to 2 weeks post-natally produced no clinical signs but seeded 50,100% of the recipients for an active encephalitogenic immune response to MBP. An earlier T-cell injection (1,2 days) produced a higher incidence and stronger response. The transferred resting T cells entered the neonatal spleen and thymus and proliferated there but did not change the total anti-MBP precursor number in adults. Splenocytes harvested from rats that were injected neonatally but not exposed to MBP in vivo proliferated strongly and produced significant amounts of interferon-, to MBP in vitro. Similar results were observed in rats injected with resting T-cell lines reactive to ovalbumin, suggesting that the neonatal injection of resting T cells specific for a self or for a foreign antigen can seed the immune system with the potential for an enhanced effector response to that antigen later in life. [source] T helper cell type 1 (Th1), Th2 and Th17 responses to myelin basic protein and disease activity in multiple sclerosisIMMUNOLOGY, Issue 2 2008Chris J. Hedegaard Summary Autoreactive T cells are thought to play an essential role in the pathogenesis of multiple sclerosis (MS). We examined the stimulatory effect of human myelin basic protein (MBP) on mononuclear cell (MNC) cultures from 22 patients with MS and 22 sex-matched and age-matched healthy individuals, and related the patient responses to disease activity, as indicated by magnetic resonance imaging. The MBP induced a dose-dependent release of interferon-, (IFN-,), tumour necrosis factor-, (TNF-,) and interleukin-10 (IL-10) by patient-derived MNCs. The patients' cells produced higher amounts of IFN-, and TNF-,, and lower amounts of IL-10, than cells from healthy controls (P < 0·03 to P < 0·04). Five patients with MS and no controls, displayed MBP-induced CD4+ T-cell proliferation. These high-responders exhibited enhanced production of IL-17, IFN-,, IL-5 and IL-4 upon challenge with MBP, as compared with the remaining patients and the healthy controls (P < 0·002 to P < 0·01). A strong correlation was found between the MBP-induced CD4+ T-cell proliferation and production of IL-17, IFN-,, IL-5 and IL-4 (P < 0·0001 to P < 0·01) within the patient group, and the production of IL-17 and IL-5 correlated with the number of active plaques on magnetic resonance images (P = 0·04 and P = 0·007). These data suggest that autoantigen-driven CD4+ T-cell proliferation and release of IL-17 and IL-5 may be associated with disease activity. Larger studies are needed to confirm this. [source] Effects of sevoflurane on collagen production and growth factor expression in rats with an excision woundACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 7 2010H.-J. LEE Background: Sevoflurane is a widely used inhalation anesthetic, but there are no studies on its effect on the wound-healing process. This study was undertaken to evaluate the effect of exposure time to sevoflurane on wound healing. Method: Male Sprague,Dawley rats were used. Two circular full-thickness skin defects 8 mm in diameter were made on the dorsum of the rats. The animals were divided into six groups according to exposed gas type and time: S1 (sevoflurane, 1 h), S4 (sevoflurane, 4 h), S8 (sevoflurane, 8 h), O1 (oxygen, 1 h), O4 (oxygen, 4 h), and O8 (oxygen, 8 h). The surface area of the wounds was measured 0, 1, 3, and 7 days after surgery. Separately, the mean blood pressures (MBP) and arterial oxygen pressures (PaO2) were monitored during the sevoflurane exposure. Collagen type I production and transforming growth factor-,1 (TGF-,1) and basic fibroblast growth factor (bFGF) expression on the wound surface were analyzed. Routine histological analysis was also performed. Result: Exposure duration to sevoflurane had no influence on MBP and PaO2. The reduction in wound size and collagen type I production was delayed in S8. The expression of TGF-,1 and bFGF on the wound surface in S8 was significantly attenuated in S8. The histology of the S8 demonstrated a delayed healing status. Conclusions: Prolonged exposure to sevoflurane might alter the inflammatory phase of the wound-healing process by attenuation of growth factor expression such as TGF-,1 and bFGF and subsequently by reduced collagen production. [source] Agonists specific for the transcription factor PPARdelta accelerate differentiation of oligodendrocytesJOURNAL OF NEUROCHEMISTRY, Issue 2002R. P. Skoff Peroxisome proliferator activated receptors (PPARs) are transcription factors belonging to the nuclear hormone receptor superfamily that regulate key genes involved in lipid metabolism. PPAR, is ubiquitously expressed at low levels in many tissues and its function has remained elusive. However, we have shown that PPAR, is abundantly expressed in oligodendrocytes (Ols), suggesting this receptor plays a critical role in oligodendrocyte differentiation (Granneman et al. 1998 J. Neurosci. Res51, 563). We first investigated the effects of PPAR agonists on proliferation and differentiation of Ols in tissue culture. Primary glial and enriched Ol cultures were treated with ligands that specifically activate PPAR, and PPAR, (Berger et al. 1999 J. Biol. Chem. 274, 6717). PPAR, but not PPAR, agonists increased the size of OL membrane sheets within 24 h of application. The increase in membrane sheet size was mirrored by increases in MBP and PLP mRNA's. In enriched Ol cultures, the number of Ols was increased 70% with the PPAR, agonist but not the PPAR, agonist (Saluja et al. 2001 Glia33, 191). In vivo injections of PPAR, agonist into P2 and P3 mice show an increase of total macroglia in the ventral and dorsal funiculi of the spinal cord of 20,40% compared to controls. Preliminary observations suggest the Ols in agonist treated cultures are larger and more densely stained than controls. Our results show for the first time that a specific ligand for a transcription factor is capable of activating the program of Ol differentiation. Acknowledgements: Supported by NMSS. [source] Signal transduction pathways involved in interaction of galactosylceramide/sulfatide-containing liposomes with cultured oligodendrocytes and requirement for myelin basic protein and glycosphingolipidsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 7 2008Joan M. Boggs Abstract We showed previously that the addition to cultured oligodendrocytes (OLs) of multivalent carbohydrate in the form of liposomes containing the two major glycosphingolipids (GSLs) of myelin, galactosylceramide (GalC) and cerebroside sulfate (Sulf), or galactose conjugated to bovine serum albumin caused clustering of GalC on the extracellular surface and myelin basic protein (MBP) on the cytosolic surface. Multivalent carbohydrate also caused depolymerization of actin microfilaments and microtubules, indicating that interaction of the carbohydrate with the OL surface transmits a transmembrane signal to the cytoskeleton. In the present study we show that inhibition of GSL synthesis with fumonisin B1 prevents clustering of MBP in GalC/Sulf-negative oligodendrocytes, suggesting that GSLs are required for the effect. Because the effects of multivalent carbohydrate resemble those caused by the addition of anti-GalC/Sulf antibodies to OLs and because GalC and Sulf can interact with each other by trans carbohydrate,carbohydrate interactions across apposed membranes, these results support the conclusion that the OL receptor for GalC/Sulf in liposomes is GalC/Sulf in the OL membrane. Inhibition of MBP expression using MBP siRNA inhibited GalC clustering, suggesting that MBP is required for the effect. We also investigate the signal transduction pathways involved using a number of enzyme inhibitors. These indicated that the Akt and p42/p44 MAPK pathways, Rho GTPases, and GSK-3, are involved, consistent with their known involvement in regulation of the cytoskeleton. These interactions between GalC/Sulf-containing liposomes and the OL membrane may mimic interactions between GalC/Sulf-enriched signaling domains when OL cell membranes or the extracellular surfaces of compact myelin come into contact. © 2008 Wiley-Liss, Inc. [source] Insulin-like growth factor-I ameliorates demyelination induced by tumor necrosis factor-, in transgenic miceJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2007Ping Ye Abstract Our groups have reported that tumor necrosis factor-, (TNF-,) causes myelin damage and apoptosis of oligodendrocytes and their precursors in vitro and in vivo. We also have reported that insulin-like growth factor-I (IGF-I) can protect cultured oligodendrocytes and their precursors from TNF-,-induced damage. In this study, we investigated whether IGF-I can protect oligodendrocytes and myelination from TNF-,-induced damage in vivo by cross-breeding TNF-, transgenic (Tg) mice with IGF-I Tg mice that overexpress IGF-I exclusively in brain. At 8 weeks of age, compared with those of wild-type (WT) mice, the brain weights of TNF-, Tg mice were decreased by ,20%, and those of IGF-I Tg mice were increased by ,20%. The brain weights of mice that carry both TNF-, and IGF-I transgenes (TNF-,/IGF-I Tg mice) did not differ from those of WT mice. As judged by histochemical staining and immunostaining, myelin content in the cerebellum of TNF-,/IGF-I Tg mice was similar to that in WT mice and much more than that in TNF-, Tg mice. Consistently, Western immunoblot analysis showed that myelin basic protein (MBP) abundance in the cerebellum of TNF-,/IGF-I Tg mice was double that in TNF-, Tg mice. In comparison with WT mice, the number of oligodendrocytes was decreased by ,36% in TNF-, Tg mice, whereas it was increased in IGF-I Tg mice by ,40%. Oligodendrocyte number in TNF-,/IGF-I Tg mice was almost twice that in TNF-, Tg mice. Furthermore, IGF-I overexpression significantly reduced TNF-,-induced increases in apoptotic cell number, active caspase-3 abundance, and degradaion of MBP. Our results indicate that IGF-I is capable of protecting myelin and oligodendrocytes from TNF-,-induced damage in vivo. © 2007 Wiley-Liss, Inc. [source] Characterization of thromboxane A2 receptor signaling in developing rat oligodendrocytes: Nuclear receptor localization and stimulation of myelin basic protein expressionJOURNAL OF NEUROSCIENCE RESEARCH, Issue 7 2006Santosh Ramamurthy Abstract The present work investigates the role of thromboxane A2 (TXA2) receptors in the development of oligodendrocytes (OLGs). The results demonstrate that the proteins of the TXA2 signaling pathway, i.e., cyclooxygenase (COX-1), TXA2 synthase (TS), and TXA2 receptor (TPR) are expressed in the developing rat brain during myelination. Furthermore, culture of OLG progenitor cells (OPCs) revealed that the expression levels of these proteins as well as TXA2 synthesis increase during OLG maturation. Separate studies established that activation of TPRs by the agonist U46619 increases intracellular calcium in both OPCs and OLGs as visualized by digital fluorescence imaging. Immunocytochemical staining demonstrated that TPRs are localized in the plasma membrane and perinuclear compartments in OPCs. However, during OLG differentiation, TPRs shift their localization pattern and also become associated with the nuclear compartment. This shift to nuclear localization was confirmed by biochemical analysis in cultured cells and by immunocytochemical analysis in developing rat brain. Finally, it was found that U46619 activation of TPRs in maturing OLGs resulted in enhanced myelin basic protein (MBP) expression. Alternatively, inhibition of endogenous TPR signaling led to reduced MBP expression. Furthermore, TPR-mediated MBP expression was found to be associated with increased transcription from the MBP promoter using a MBP-luciferase reporter. Collectively, these findings suggest a novel TPR signaling pathway in OLGs and a potential role for this signaling during OLG maturation and myelin production. © 2006 Wiley-Liss, Inc. [source] Comparative analysis of neuroectodermal differentiation capacity of human bone marrow stromal cells using various conversion protocolsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2006Andreas Hermann Abstract Human adult bone marrow-derived mesodermal stromal cells (hMSCs) are able to differentiate into multiple mesodermal tissues, including bone and cartilage. There is evidence that these cells are able to break germ layer commitment and differentiate into cells expressing neuroectodermal properties. There is still debate about whether this results from cell fusion, aberrant marker gene expression or real neuroectodermal differentiation. Here we extend our work on neuroectodermal conversion of adult hMSCs in vitro by evaluating various epigenetic conversion protocols using quantitative RT-PCR and immunocytochemistry. Undifferentiated hMSCs expressed high levels of fibronectin as well as several neuroectodermal genes commonly used to characterize neural cell types, such as nestin, ,-tubulin III, and GFAP, suggesting that hMSCs retain the ability to differentiate into neuroectodermal cell types. Protocols using a direct differentiation of hMSCs into a neural phenotype failed to induce significant changes in morphology and/or expression of markers of early and mature glial/neuronal cells types. In contrast, a multistep protocol with conversion of hMSCs into a neural stem cell-like population and subsequent terminal differentiation in mature glia and neurons generated relevant morphological changes as well as significant increase of expression levels of marker genes for early and late neural cell types, such as nestin, neurogenin2, MBP, and MAP2ab, accompanied by a loss of their mesenchymal properties. Our data provide an impetus for differentiating hMSCs in vitro into mature neuroectodermal cells. Neuroectodermally converted hMSCs may therefore ultimately help in treating acute and chronic neurodegenerative diseases. Analysis of marker gene expression for characterization of neural cells derived from MSCs has to take into account that several early and late neuroectodermal genes are already expressed in undifferentiated MSCs. © 2006 Wiley-Liss, Inc. [source] Myelin proteolipid protein, basic protein, the small isoform of myelin-associated glycoprotein, and p42MAPK are associated in the Triton X-100 extract of central nervous system myelinJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2002Dina N. Arvanitis Abstract To further our understanding of the functions of the major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), and other myelin proteins, such as 2,3,-cyclic nucleotide 3,-phosphodiesterase (CNP) and myelin-associated glycoprotein (MAG), bovine brain myelin was extracted with Triton X-100, and protein complexes in the detergent-soluble fraction were isolated by coimmunoprecipitation and sucrose density gradient sedimentation. MBP, PLP, and the small isoform of MAG (S-MAG) were coimmunoprecipitated from the detergent-soluble fraction by anti-PLP, anti-MBP or anti-MAG monoclonal antibodies. Additionally, a 30 kDa phosphoserine-containing protein and two phosphotyrosine-containing proteins (Mr 30 and 42 kDa) were found in the coimmunoprecipitates. The 42 kDa protein is probably p42MAPK, in that MAPK was shown also to be present in the immunoprecipitated complex. CNP, the small PLP isoform DM20, the large MAG isoform L-MAG, MOG, CD44, MEK, p44MAPK, and actin were not present in the immunoprecipitates, although they were present in the detergent-soluble fraction. Lipid analysis revealed that the PLP,MBP,S-MAG coimmunoprecipitated with some phospholipids and sulfatide but not cholesterol or galactosylceramide. However, the complex had a high density, indicating that the lipid/protein ratio is low, and it was retained on a Sepharose CL6B column, indicating that it is not a large membrane fragment. Given that MAG is localized mainly in the periaxonal region of myelin, where it interacts with axonal ligands, the PLP,MBP,S-MAG complex may come from these regions, where it could participate in dynamic functions in the myelin sheath and myelin,axonal interactions. © 2002 Wiley-Liss, Inc. [source] Milk basic protein increases alveolar bone formation in rat experimental periodontitisJOURNAL OF PERIODONTAL RESEARCH, Issue 1 2007H. Seto Background and Objective:, It is conceivable that the active components extracted from milk whey protein (i.e. milk basic protein, MBP) stimulate bone formation and suppress bone resorption. Periodontitis is characterized by excessive alveolar bone resorption. We examined whether milk basic protein could recover alveolar bone loss in rat experimental periodontitis. Material and Methods:, A nylon ligature was placed around the cervix of molars in 8-wk-old male Fischer rats for 20 d. Then, the ligature was removed and a powder diet containing 0.2 or 1.0% milk basic protein was provided daily for another 45,90 d. On days 45 and 90, the maxillae were extracted and analyzed using microcomputerized tomography (micro-CT), followed by histological analysis. Results:, Micro-CT images showed that alveolar bone resorption was severely induced around the molar by the 20-d ligature procedure. Treatment with high-dose milk basic protein (1.0%) clearly recovered ligature-induced alveolar bone resorption on days 45 and 90, whereas low-dose milk basic protein (0.2%) did not show such a clear effect. Histological examination clarified that the osteoid thickness of alveolar bone was dose dependently increased by milk basic protein treatment for 90 d. Conclusion:, These findings suggest that a systemic administration of milk basic protein may be effective for the recovery of alveolar bone loss in periodontitis. [source] Electroencephalographic arousals during sleep do not alter the pressor response to Cheyne,Stokes respiration in subjects with chronic heart failureJOURNAL OF SLEEP RESEARCH, Issue 4 2007GRANT N. WILLSON Summary This study examined the influence of electroencephalographic (EEG) arousal on the magnitude and morphology of the pressor response to Cheyne,Stokes respiration (CSR) in subjects with congestive heart failure (CHF). Thirteen subjects with stable CHF (left ventricular ejection fraction, 26 ± 7%) and CSR (apnea,hypopnea index 52 ± 15 h,1) underwent overnight polysomnography with beat-to-beat measurement of systemic arterial blood pressure (BP). CSR events were divided into those with or without an EEG arousal defined according to the criteria of the American Sleep Disorders Association. The pressor response was quantified in terms of the delta BP change (difference between the minimum BP during apnea and maximum BP during hyperpnea). Changes in the morphology of the pressor response were assessed by subdividing individual respiratory events into six periods (three during apnea: A1, A2, A3; and three during hyperpnea: H1, H2, H3). Considerable fluctuations in BP and heart rate (HR) were observed across the CSR cycle (delta mean BP 20.2 ± 6.5 mmHg). The presence of an EEG arousal did not alter the amplitude of fluctuations in BP. Mean blood pressure (MBP) increased 21.0 ± 7.5 mmHg with arousal versus 19.3 ± 5.8 mmHg without arousal (NS). A repeated measures ANOVA showed no significant interaction between the presence of arousal and the proportional change in mean BP across the six periods, indicating that an EEG arousal had no effect on the morphology of MBP change during CSR [F(5,60) = 1.44, P = 0.22]. This study showed that EEG-defined arousal does not amplify the pressor response to CSR in CHF. [source] The role of eosinophil major basic protein in angiogenesisALLERGY, Issue 3 2009I. Puxeddu Background:, Eosinophil-derived major basic protein (MBP) plays an active role in allergic inflammation and tissue remodelling. However, its role in angiogenesis has not been established as yet. Therefore our objective was to investigate whether MBP exhibits any direct pro-angiogenic effects. Methods:, Rat aortic endothelial cells and human umbilical vascular endothelial cells were cultured with different concentrations of MBP and their viability (Trypan blue exclusion test), proliferation (thymidine incorporation) and capillary-like structure formation (matrigel assay) were investigated in vitro. The angiogenic activity of MBP was then tested in vivo using the chick chorio allantoic membrane (CAM) assay. Results:, Subcytotoxic concentrations of MBP induce endothelial cell proliferation and enhance the pro-mitogenic effect of vascular endothelial growth factor (VEGF), but do not affect their VEGF release. MBP promotes capillarogenesis by endothelial cells seeded on matrigel and sprouting formation in the CAM assay. Furthermore, we have shown that the pro-angiogenic effect of MBP is not due to its cationic charge since stimulation of the CAMs with the synthetic polycation, poly- l -arginine does not induce any angiogenic effects. Conclusions:, These data demonstrate that MBP has pro-angiogenic effects in vitro and in vivo, providing a novel mechanism whereby MBP can participate in tissue inflammation and remodelling in atopic diseases. [source] Exploration of twin-arginine translocation for expression and purification of correctly folded proteins in Escherichia coliMICROBIAL BIOTECHNOLOGY, Issue 5 2008Adam C. Fisher Summary Historically, the general secretory (Sec) pathway of Gram-negative bacteria has served as the primary route by which heterologous proteins are delivered to the periplasm in numerous expression and engineering applications. Here we have systematically examined the twin-arginine translocation (Tat) pathway as an alternative, and possibly advantageous, secretion pathway for heterologous proteins. Overall, we found that: (i) export efficiency and periplasmic yield of a model substrate were affected by the composition of the Tat signal peptide, (ii) Tat substrates were correctly processed at their N-termini upon reaching the periplasm and (iii) proteins fused to maltose-binding protein (MBP) were reliably exported by the Tat system, but only when correctly folded; aberrantly folded MBP fusions were excluded by the Tat pathway's folding quality control feature. We also observed that Tat export yield was comparable to Sec for relatively small, well-folded proteins, higher relative to Sec for proteins that required cytoplasmic folding, and lower relative to Sec for larger, soluble fusion proteins. Interestingly, the specific activity of material purified from the periplasm was higher for certain Tat substrates relative to their Sec counterparts, suggesting that Tat expression can give rise to relatively pure and highly active proteins in one step. [source] Topical glucocorticoids downregulate COX-1 positive cells in nasal polypsALLERGY, Issue 1 2009F. A. Ebbens Background: Influx of inflammatory cells is one of the hallmarks of nasal polyposis. As glucocorticoids (GC) are known to exhibit strong anti-inflammatory effects, these drugs are frequently used in the treatment of the disease. Part of the anti-inflammatory effects of GC is attributed to their interference with prostanoid synthesis. As cyclooxygenases (COX) are key enzymes in the synthesis of both pro- (COX-1, COX-2) and anti-inflammatory prostanoids (COX-2), we investigated the role of topical GC on COX-1, COX-2 and inflammatory markers in nasal polyps (NP). Methods: Immunohistochemical analysis of inflammatory markers (CD68, CD117, MBP, elastase, IgE, BB-1, IL-4, IL-5 and IL-6), COX-1 and COX-2 was performed on normal nasal mucosa (NM) (n = 18), non-GC treated NP (n = 27) and topical GC treated NP (n = 12). NP groups were matched for allergy, asthma and ASA intolerance. Results: Increased numbers of eosinophils, IL-5+ cells and IgE+ cells and decreased numbers of mastcells are striking features of NP inflammation (P < 0.05). In addition, increased numbers of COX-1+ cells are observed in NP epithelium compared to NM (P < 0.05). Conclusion: Topical GC significantly reduce the number of COX-1+ NP cells (P < 0.05), but have no significant effect on COX-2+ NP cells. No significant reduction in the number of eosinophils is observed for GC treated NP. The number of IL-5+ cells is however increased significantly upon GC treatment (P < 0.05). [source] Tethering of CpxP to the inner membrane prevents spheroplast induction of the Cpx envelope stress responseMOLECULAR MICROBIOLOGY, Issue 5 2000Tracy L. Raivio The Cpx envelope stress response of Escherichia coli is controlled by a two-component regulatory system that senses misfolded proteins in extracytoplasmic compartments and responds by inducing the expression of envelope protein folding and degrading factors. We have proposed that in the absence of envelope stress the pathway is maintained in a downregulated state, in part through interactions between the periplasmic inhibitor molecule CpxP and the sensing domain of the histidine kinase CpxA. In this study, we show that depletion of the periplasmic contents of the cell by spheroplast formation does indeed lead to induction of the Cpx envelope stress response. Further, removal of CpxP is an important component of this induction because tethering an MBP,CpxP fusion protein to the spheroplast inner membranes prevents full activation by this treatment. Spheroplast formation has previously been demonstrated to induce the expression of a periplasmic protein of unknown function, Spy. Analysis of spy expression in response to spheroplast formation by Western blot analysis and by lacZ operon fusion in various cpx mutant backgrounds demonstrated that spy is a member of the Cpx regulon. Interestingly, although the only known spy homologue is cpxP, Spy does not appear to perform the same function as CpxP as it is not involved in inhibiting the Cpx envelope stress response. Rather, deletion of spy leads to activation of the ,E stress response. Because the ,E response is specifically affected by alterations in outer membrane protein biogenesis, we think it possible that Spy may be involved in this process. [source] Germinal vesicle materials are not required for the activation of MAP kinase in porcine oocyte maturationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2001K. Sugiura Abstract The requirement of the germinal vesicle (GV) for the normal kinetics of mitogen-activated protein (MAP) kinase activity during porcine oocyte maturation was investigated. Porcine follicular oocytes were enucleated, and the locations of their extracellular signal-regulated kinases 1 and 2 (ERK1/2), major MAP kinases in maturating porcine oocytes, were detected by indirect immunofluorescent microscopy. The MAP kinase activity was assayed as myelin basic protein (MBP) kinase activity, and the phosphorylation states of ERK1/2 were detected by immunoblotting analyses. Translocation of MAP kinase into the GV and association with the spindle were observed in intact oocytes, while MAP kinase in enucleated oocytes was distributed almost uniformly in cytoplasm throughout the culturing period. The phosphorylation and the activation of MAP kinase were induced, and the activity was comparable with that of control denuded oocytes. The high level of activity was maintained through maturation, even in the absence of spindle formation. These results indicate that the presence of nuclear material and translocation into the GV are dispensable for the activation of MAP kinase and that associating with the spindle is not required for maintenance of its activity though porcine oocyte maturation. Mol. Reprod. Dev. 59:215,220, 2001. © 2001 Wiley-Liss, Inc. [source] Sequential myelin protein expression during remyelination reveals fast and efficient repair after central nervous system demyelinationNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 1 2008M. Lindner To understand the mechanisms of remyelination and the reasons for regeneration failure is one of the major challenges in multiple sclerosis research. This requires a good knowledge and reliable analysis of experimental models. This work was undertaken to characterize the pattern of myelin protein expression during experimental remyelination. Acute demyelination of the corpus callosum was induced by feeding of 0.3% cuprizone for 6 weeks, followed by a 10-week remyelination period. We used a combination of Luxol fast blue (LFB) myelin staining, electron microscopy (EM) and immunohistochemistry for the myelin proteins 2,,3,-cyclic nucleotide 3, phosphodiesterase (CNPase), myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG). Early remyelination was detected by the re-expression of CNPase, MBP and PLP as early as 4 days. MOG, as a marker for late differentiation of oligodendrocytes, was not detectable until 2 weeks of remyelination. EM data correlated well with the LFB myelin staining and myelin protein expression, with 50% of the axons being rapidly remyelinated within 2 weeks. While particularly MBP but also PLP and CNPase are re-expressed very early before significant remyelination is observed by EM, the late marker MOG shows a lag behind the remyelination detected by EM. The presented data indicate that immunohistochemistry for various myelin proteins expressed early and late during myelin formation is a suitable and reliable method to follow remyelination in the cuprizone model. Furthermore, investigation of early remyelination confirms that the intrinsic repair programme is very fast and switched on within days. [source] | ||||||||||||||||||||||||||||||||||