Purification and recognition of recombinant mouse P2X(1) receptors expressed in a baculovirus system
Pharmacology & Pharmacy; protein; binding; gated ion channels; agonist; cation channels; activation; extracellular; site; Ion channels; endoplasmic-reticulum; affinity chromatography; ATP; nadph-p450 oxidoreductase; nucleotides; p-2x receptors; polyacrylamide-gel electrophoresis; structural motif
The hexahistidine-tagged mouse P2X(1) receptor (H-mP2X(1)R), an ATP-gated ion channel receptor, was expressed in a baculovirus system using the pAcHLT-B transfer vector containing a hexahistidine tag. Both widely used denaturing (8M urea) acid nondenaturing (such as 1% Triton X-100) solubilization conditions were compared, resulting in about 30% of the P2X(1) receptors being solubilized (S1). However, at pH 13 most of the n-mP2X(1)R from the initially insoluble pellet fraction was solubilized (S2) and remained in the soluble fraction (S3) after dialyzing against a nondenaturing buffer. H-mP2X(1)Rs were purified sequentially through cobalt and ATP affinity columns. Receptors purified from S3 had higher purity than those from S1 (i.e., similar to 90% vs. similar to 75%). Circular dichroism spectra indicated identical protein secondary structures of the receptors from both sources. Autoradiographic data showed that the purified receptors from S3 had higher affinity for 8-azido-ATP-gamma-P-32 than the receptors from S1. The binding of 8-azido-ATP-gamma-P-32 to H-mP2X(1)R was inhibited by ATP-gamma -S, alpha,beta -me-ATP, and PPADS, but not by a nucleoside analog (N-6-methyl-2'-deoxy-adenosine). In the presence of 2 mM Ca2+ or Mg2+ the binding was increased, but not when using a partially purified receptor fraction, in which unidentified proteins bound 8-azido ATP-gamma-P-32 or were phosphorylated at 4 degreesC in the presence of 2 mM Mg2+. These data suggest that the decrease in potency of ATP in the presence of Ca2+ and Mg2+, as observed in functional studies, is not due to a direct effect of the cations on the binding of ATP to the receptor. Both cyanogen bromide and hydroxylamine cleavage further confirmed the peptide structure of the purified H-mP2X1R. Autoradiographic analysis of the cleavage products showed that 8-azido-ATP-gamma-P-32 was crosslinked to the carboxyl side of the extracellular domain of the receptor. Drug Dev. Res. 51:7-19, 2000. Published 2000 Wiley-Liss, Inc.dagger
Chen L P; Hardwick J P; McPhie P; Sitkovsky M V; Jacobson K A
Drug Development Research
2000
2000-09
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1002/1098-2299(20000901)51:1%3C7::aid-ddr2%3E3.3.co;2-n" target="_blank" rel="noreferrer noopener">10.1002/1098-2299(20000901)51:1%3C7::aid-ddr2%3E3.3.co;2-n</a>
Cross-Talk Between Stem Cells and the Dysfunctional Brain is Facilitated by Manipulating the Niche: Evidence from an Adhesion Molecule
adhesion molecule; Aging; aging and age-related diseases; astrocytes; axonal regeneration; Biotechnology & Applied Microbiology; Cell Biology; central-nervous-system; chaperone effect; differentiation; dopamine; extracellular; functional recovery; Hematology; L1; matrix; migration; model; molecules; motor-neurons; mouse; MPTP; neural stem cells; neurodegeneration; neuroprotection; neurotrophic factors; niche; Oncology; oxidative stress; parkinsons-disease; parkinsons-disease; protect neurons; recognition; spinal-cord-injury; transplantation
In the injured brain, the behavior of neural stem/progenitor cells (NSCs) is regulated by multiple converging factors encountered in the niche, which is composed of several neural and non-neural cell types. Signals emanating from the host influence the migration, survival, distribution, and fate of transplanted NSCs, which in turn can create host microenvironments that favor a return to homeostasis. We tested the hypothesis that overexpression of key facilitatory molecules that de. ne the injury niche might enhance this bidirectional stem cell host interaction to therapeutic advantage. As proof of concept, we investigated whether conditioning the niche with the neural cell adhesion molecule L1 might enhance recovery in a prototypical neurodegenerative milieu-the MPTP-induced model of Parkinson's disease in aged mice-where cross-talk between NSCs and imperiled host dopaminergic neurons is known to be pivotal in rescuing the function and connectivity of the latter. In lesioned mice (and in unlesioned controls), we overexpressed L1 in the NSCs to be transplanted into the ventral mesencephalon. Several pairwise experimental combinations were tested based on variations of engrafting L1 overexpressing versus nonoverexpressing NSCs into wild-type (WT) versus L1-overexpressing transgenic mice (specifically L1 transcribed from the GFAP promoter and, hence, overexpressed in host astrocytes). Enrichment for L1-particularly when expressed simultaneously in both donor NSCs and host brain-led to rapid and extensive distribution of exogenous NSCs, which in turn rescued (with an efficacy greater than in nonengineered controls) dysfunctional host dopaminergic nigral neurons, even when grafting was delayed by a month. L1 overexpression by NSCs also enhanced their own differentiation into tyrosine hydroxylase-expressing neurons in both WT and transgenic hosts. Graft-host interactions were thus favored by progressively increasing levels of L1. More broadly, this study supports the view that manipulating components of the niche (such as an adhesion molecule) that facilitate cross-talk between stem cells and the dysfunctional brain may offer new strategies for more efficacious neurotransplantation, particularly when treatment is delayed as in chronic lesions or advanced stages of a neurodegenerative disease. STEM CELLS 2009; 27: 2846-2856
Ourednik V; Ourednik J; Xu Y F; Zhang Y; Lynch W P; Snyder E Y; Schachner M
Stem Cells
2009
2009-11
Journal Article
<a href="http://doi.org/10.1002/stem.227" target="_blank" rel="noreferrer noopener">10.1002/stem.227</a>