Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports....Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e.,≥95°C), forming a large molecular weight band when analyzed by immuno-blotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immuno-blot analysis. These findings provide very useful information for efficient mammalian expression and immuno-blotting of membrane receptors.展开更多
Sulfur dioxide（SO_2） pollution in the atmospheric environment causes brain inflammatory insult and inflammatory-related microvasculature dysfunction.However,there are currently no effective medications targeting the...Sulfur dioxide（SO_2） pollution in the atmospheric environment causes brain inflammatory insult and inflammatory-related microvasculature dysfunction.However,there are currently no effective medications targeting the harmful outcomes from chemical inhalation.Endocannabinoids（eCBs） are involved in neuronal protection against inflammation-induced neuronal injury.The 2-arachidonoylglycerol（2-AG）,the most abundant eCBs and a full agonist for cannabinoid receptors（CB1 and CB2）,is also capable of suppressing proinflammatory stimuli and improving microvasculature dysfunction.Here,we indicated that endogenous 2-AG protected against neuroinflammation in response to SO_2 inhalation by inhibiting the activation of microglia and astrocytes and attenuating the overexpression of inflammatory cytokines,including tumor necrosis factor alpha（TNF-a）,interleukin（IL）-1β,and inducible nitric oxide synthase（iNOS）.In addition,endogenous 2-AG prevented cerebral vasculature dysfunction following SO_2 inhalation by inhibiting endothelin 1（ET-1）,vascular cell adhesion molecule-1（VCAM-1） and intercellular adhesion molecule 1（ICAM-1） expression,elevating endothelial nitric oxide synthase（eNOS） level,and restoring the imbalance between thromboxane A2（TXA2） and prostaglandin 12（PGI2）.In addition,the action of endogenous 2-AG on the suppression of inflammatory insult and inflammatory-related microvasculature dysfunction appeared to be mainly mediated by CB1 and CB2 receptors.Our results provided a mechanistic basis for the development of new therapeutic approaches for protecting brain injuries from SO_2 inhalation.展开更多
基金supported by a grant from Army Medical Research Program of China(No.08G168)
文摘Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e.,≥95°C), forming a large molecular weight band when analyzed by immuno-blotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immuno-blot analysis. These findings provide very useful information for efficient mammalian expression and immuno-blotting of membrane receptors.
基金supported by the National Science Foundation of China(Nos.91543203,21477070,21377076,21307079)the Research Project Supported by Shanxi Scholarship Council of China(No.2015-006)
文摘Sulfur dioxide（SO_2） pollution in the atmospheric environment causes brain inflammatory insult and inflammatory-related microvasculature dysfunction.However,there are currently no effective medications targeting the harmful outcomes from chemical inhalation.Endocannabinoids（eCBs） are involved in neuronal protection against inflammation-induced neuronal injury.The 2-arachidonoylglycerol（2-AG）,the most abundant eCBs and a full agonist for cannabinoid receptors（CB1 and CB2）,is also capable of suppressing proinflammatory stimuli and improving microvasculature dysfunction.Here,we indicated that endogenous 2-AG protected against neuroinflammation in response to SO_2 inhalation by inhibiting the activation of microglia and astrocytes and attenuating the overexpression of inflammatory cytokines,including tumor necrosis factor alpha（TNF-a）,interleukin（IL）-1β,and inducible nitric oxide synthase（iNOS）.In addition,endogenous 2-AG prevented cerebral vasculature dysfunction following SO_2 inhalation by inhibiting endothelin 1（ET-1）,vascular cell adhesion molecule-1（VCAM-1） and intercellular adhesion molecule 1（ICAM-1） expression,elevating endothelial nitric oxide synthase（eNOS） level,and restoring the imbalance between thromboxane A2（TXA2） and prostaglandin 12（PGI2）.In addition,the action of endogenous 2-AG on the suppression of inflammatory insult and inflammatory-related microvasculature dysfunction appeared to be mainly mediated by CB1 and CB2 receptors.Our results provided a mechanistic basis for the development of new therapeutic approaches for protecting brain injuries from SO_2 inhalation.
