Purification of Myelin Basic Protein from Bovine Brain

Chevalier, Dominique; Allen, Bruce G.

doi:10.1006/prep.1999.1183

Cited by 12 publications

(5 citation statements)

References 28 publications

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“…Myelin basic protein was enriched with modifications to the protocol of Chevalier and Allen [ 7 ]. Briefly, the enriched myelin fraction was re-suspended in 50 mM Tris buffer (pH 7.4) and centrifuged (21,000 g , 20 min).…”

Section: Methodsmentioning

confidence: 99%

Isoaspartic acid is present at specific sites in myelin basic protein from multiple sclerosis patients: could this represent a trigger for disease onset?

Friedrich

Hancock

Raftery

et al. 2016

acta neuropathol commun

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Multiple sclerosis (MS) is associated with breakdown of the myelin sheath that coats neurons in the central nervous system. The cause of MS is not known, although the pathogenesis involves destruction of myelin by the immune system. It was the aim of this study to examine the abundant myelin protein, myelin basic protein (MBP), to determine if there are sites of modification that may be characteristic for MS. MBP from the cerebellum was examined from controls and MS patients across the age range using mass spectrometry and amino acid analysis. Amino acid racemization data indicated that myelin basic protein is long-lived and proteomic analysis of MBP showed it to be highly modified. A common modification of MBP was racemization of Asp and this was significantly greater in MS patients. In long-lived proteins, L-Asp and L-Asn can racemize to three other isomers, D-isoAsp, L-isoAsp and D-Asp and this is significant because isoAsp formation in peptides renders them immunogenic.Proteomic analysis revealed widespread modifications of MBP with two surface regions that are altered in MS. In particular, isoAsp was significantly elevated at these sites in MS patients. The generation of isoAsp could be responsible for eliciting an immune response to modified MBP and therefore be implicated in the etiology of MS.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0348-x) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Isoaspartic acid is present at specific sites in myelin basic protein from multiple sclerosis patients: could this represent a trigger for disease onset?

Friedrich

Hancock

Raftery

et al. 2016

acta neuropathol commun

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“…ERK immune complex assays were performed as described previously ( Chevalier and Allen, 2000a ) with modifications. ERK was precipitated from cell lysates (100 μg) using ERK‐CT antisera (0.5 μg).…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant canine hsp27, cloned into the pET24a expression vector ( Larsen et al , 1995 ), was a kind gift from Dr William Gerthoffer, Reno, NV. Myelin basic protein was purified from bovine brain as described previously ( Chevalier and Allen, 2000a ). Anti‐ERK1‐CT antisera, which recognizes both ERK1 and ERK2, was from Stressgen (Victoria, BC, Canada).…”

Section: Methodsmentioning

confidence: 99%

Activation of ET_B receptors regulates the abundance of ET‐1 mRNA in vascular endothelial cells

Farhat

Matouk

Mamarbachi

et al. 2008

British J Pharmacology

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Background and purpose: The factors that influence the cellular levels of endothelin-1 (ET-1) include transcription, mRNA localization, stability and translation, post-translational maturation of preproET-1 and degradation of ET-1. We investigated the regulation of ET-1 mRNA abundance by extracellular ET-1 in porcine aortic endothelial cells (PAECs). Experimental approach: Passsage one cultures of PAECs were incubated in starving medium in the presence or absence of ET-1 and antagonists or pharmacological inhibitors. PreproET-1 mRNA, endothelin-1 promoter activity, Erk and p38 MAPK activation were determined. Key results: Exogenous ET-1 reduced cellular ET-1 mRNA content: a reduction of 10 000-fold was observed after 4 h. ET-1 simultaneously reduced the stability of ET-1 mRNA and increased the loading of RNA Polymerase II at the endothelin-1 promoter. In the absence of exogenous ET-1, the ET B -selective antagonist, BQ788, increased ET-1 mRNA. An ET A -selective antagonist had no effect. ET-1 mRNA returned to control levels within 24 h. Whereas activation of p38 MAPK induced by ET-1 peaked at 30 min and returned to control levels within 90 min, Erk1/2 remained active after 4 h of stimulation. Inhibition of p38 MAPK prevented the ET-1-induced decrease in ET-1 mRNA. In contrast, Erk1/2 inhibition increased ET-1 mRNA. Similarly, inhibition of receptor internalization increased ET-1 mRNA in the presence or absence of exogenous ET-1. Conclusions and implications: These results suggest that extracellular ET-1 regulates the abundance of ET-1 mRNA in PAECs, in an ET B receptor-dependent manner, by modulating both mRNA stability and transcription via mechanisms involving receptor endocytosis and both ERK and p38 MAPK pathways.

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“…HRP-conjugated secondary antibodies were from Jackson Laboratories. Myelin basic protein (MBP) was purified as described previously [27]. Anti-humanMK2 phospho-threonine-222 (3044), plus phosphorylated, active, recombinant ERK1, ERK2, p38α, β 2 , δ, and γ were from Upstate Cell Signaling Solutions.…”

Section: Methodsmentioning

confidence: 99%

Characterization of a novel MK3 splice variant from murine ventricular myocardium

Moïse

Dingar

Mamarbachi

et al. 2010

Cellular Signalling

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p38 MAP kinase (MAPK) isoforms alpha, beta, and gamma, are expressed in the heart. p38alpha appears pro-apoptotic whereas p38beta is pro-hypertrophic. The mechanisms mediating these divergent effects are unknown; hence elucidating the downstream signaling of p38 should further our understanding. Downstream effectors include MAPK-activated protein kinase (MK)-3, which is expressed in many tissues including skeletal muscles and heart. We cloned full-length MK3 (MK3.1, 384 aa) and a novel splice variant (MK3.2, 266 aa) from murine heart. For MK3.2, skipping of exons 8 and 9 resulted in a frame-shift in translation of the first 85 base pairs of exon 10 followed by an in-frame stop codon. Of 3 putative phosphorylation sites for p38 MAPK, only Thr-203 remained functional in MK3.2. In addition, MK3.2 lacked nuclear localization and export signals. Quantitative real-time PCR confirmed the presence of these mRNA species in heart and skeletal muscle; however, the relative abundance of MK3.2 differed. Furthermore, whereas total MK3 mRNA was increased, the relative abundance of MK3.2 mRNA decreased in MK2(-/-) mice. Immunoblotting revealed 2 bands of MK3 immunoreactivity in ventricular lysates. Ectopically expressed MK3.1 localized to the nucleus whereas MK3.2 was distributed throughout the cell; however, whereas MK3.1 translocated to the cytoplasm in response to osmotic stress, MK3.2 was degraded. The p38alpha/beta inhibitor SB203580 prevented the degradation of MK3.2. Furthermore, replacing Thr-203 with alanine prevented the loss of MK3.2 following osmotic stress, as did pretreatment with the proteosome inhibitor MG132. In vitro, GST-MK3.1 was strongly phosphorylated by p38alpha and p38beta, but a poor substrate for p38delta and p38gamma. GST-MK3.2 was poorly phosphorylated by p38alpha and p38beta and not phosphorylated by p38delta and p38gamma. Hence, differential regulation of MKs may, in part, explain diverse downstream effects mediated by p38 signaling.

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Purification of Myelin Basic Protein from Bovine Brain

Cited by 12 publications

References 28 publications

Isoaspartic acid is present at specific sites in myelin basic protein from multiple sclerosis patients: could this represent a trigger for disease onset?

Isoaspartic acid is present at specific sites in myelin basic protein from multiple sclerosis patients: could this represent a trigger for disease onset?

Activation of ET_B receptors regulates the abundance of ET‐1 mRNA in vascular endothelial cells

Characterization of a novel MK3 splice variant from murine ventricular myocardium

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Purification of Myelin Basic Protein from Bovine Brain

Cited by 12 publications

References 28 publications

Isoaspartic acid is present at specific sites in myelin basic protein from multiple sclerosis patients: could this represent a trigger for disease onset?

Isoaspartic acid is present at specific sites in myelin basic protein from multiple sclerosis patients: could this represent a trigger for disease onset?

Activation of ETB receptors regulates the abundance of ET‐1 mRNA in vascular endothelial cells

Characterization of a novel MK3 splice variant from murine ventricular myocardium

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Activation of ET_B receptors regulates the abundance of ET‐1 mRNA in vascular endothelial cells