In the alternative splicing, intron retention, of histamine H3 receptors in rats and mice, the short transcript isoforms that are excised alternatively spliced introns are easily detected in a very low level in rats a...In the alternative splicing, intron retention, of histamine H3 receptors in rats and mice, the short transcript isoforms that are excised alternatively spliced introns are easily detected in a very low level in rats and are undetectable in mice using the regular PCR protocol. The retained introns have common 5＇ splice site and different 3＇ splice sites. The detailed mechanism for the special alternative splicing remains largely unclear. In this study, we developed a minigene splicing system to recapitulate natural alternative splicing of the receptors and investigated the effects of 5＇ and 3＇ splice sites on intron retention in HeLa cells. Mutating weak 5＇ and 3＇ splice sites of the alternatively spliced introns toward the canonical consensus sequences promoted the splicing of the corresponding introns in rat and mouse minigenes. The effect of splice site strength was context-dependent and much more sigiaificant for the 3＇ splice site of the longer alternative intron than for the 3＇ splice site of the shorter alternative intron and the common 5＇ splice sites; it was also more significant in the rat minigene than in the mouse minigene. Mutating the 3＇ splice site of the longer alternative intron resulted in almost complete splicing of the intron and made the corresponding isoform to become the nearly exclusive transcript in the rat minigene.展开更多
In the intrinsic pathway of apoptosis, stresses of mitochondrial reactive oxygen species （mitoROS） might be sensed as more effective signals than those in cytosol, as mitochondria are the major sources of reactive o...In the intrinsic pathway of apoptosis, stresses of mitochondrial reactive oxygen species （mitoROS） might be sensed as more effective signals than those in cytosol, as mitochondria are the major sources of reactive oxygen species （ROS） and pivotal components during cell apoptosis. Mitochondrial superoxide dismutase （SOD2） takes the leading role in eliminating mitoROS, and inhibition of SOD2 might induce severe disturbances overwhelming the mitochondrial oxidative equilibrium, which would elevate the intracellular oxidative stresses and drive cells to death. Herein, we report a general strategy to kill cancer cells by targeted inhibition of SOD2 using 2-methoxyestradiol （2-ME, an inhibitor for the SOD family） via a robust mitochondria-targeted mesoporous silica nanocarrier （mtMSN）, with the expected elevation of mitoROS and activation of apoptosis in HeLa cells. Fe304@MSN was employed in the mitochondria-targeted drug delivery and selective inhibition of mitochondrial enzymes, and was shown to be stable with good biocompatibility and high loading capacity. Due to the selective inhibition of SOD2 by 2-ME/mtMSN, enhanced elevation of mitoROS （132% of that with free 2-ME） was obtained, coupled with higher efficiency in initiating cell apoptosis （395% of that with free 2-ME in 4 h）. Finally, the 2-ME/mtMSN exhibited powerful efficacy in targeted killing of HeLa cells by taking advantage of both biological recognition and magnetic guiding, causing 97.0% cell death with only 2 Dg/mL 2-ME/mtMSN, hinting at its great potential in cancer therapy through manipulation of the delicate mitochondrial oxidative balance.展开更多
The human serum proteome is closely associated with the state of the body.Endogenous peptides derived from proteolytic enzymes cleaving on serum proteins are widely studied due to their potential application in diseas...The human serum proteome is closely associated with the state of the body.Endogenous peptides derived from proteolytic enzymes cleaving on serum proteins are widely studied due to their potential application in disease-specific marker discovery.However,the reproducibility of peptidome analysis of endogenous peptides is significantly influenced by the proteolytic enzymes within body fluids,thereby limiting the clinical use of the endogenous peptides.We comprehensively investigated the N and C terminus of endogenous peptides using peptidomics.The cleavage site patterns of the N and C terminus and adjacent sites from all the identified endogenous peptides were highly conserved under different sample preparation conditions,including long-term incubation at 37℃ and pretreatment with repeated freeze-thaw cycles.Furthermore,a distinguishable cleavage site pattern was obtained when a different disease serum was analyzed.The conserved cleavage site pattern derived from proteolytic enzymes holds potential in highly specific disease diagnosis.展开更多
基金supported by the National Basic Research Program of China (973 Program) (No.2006CB943601)
文摘In the alternative splicing, intron retention, of histamine H3 receptors in rats and mice, the short transcript isoforms that are excised alternatively spliced introns are easily detected in a very low level in rats and are undetectable in mice using the regular PCR protocol. The retained introns have common 5＇ splice site and different 3＇ splice sites. The detailed mechanism for the special alternative splicing remains largely unclear. In this study, we developed a minigene splicing system to recapitulate natural alternative splicing of the receptors and investigated the effects of 5＇ and 3＇ splice sites on intron retention in HeLa cells. Mutating weak 5＇ and 3＇ splice sites of the alternatively spliced introns toward the canonical consensus sequences promoted the splicing of the corresponding introns in rat and mouse minigenes. The effect of splice site strength was context-dependent and much more sigiaificant for the 3＇ splice site of the longer alternative intron than for the 3＇ splice site of the shorter alternative intron and the common 5＇ splice sites; it was also more significant in the rat minigene than in the mouse minigene. Mutating the 3＇ splice site of the longer alternative intron resulted in almost complete splicing of the intron and made the corresponding isoform to become the nearly exclusive transcript in the rat minigene.
文摘In the intrinsic pathway of apoptosis, stresses of mitochondrial reactive oxygen species （mitoROS） might be sensed as more effective signals than those in cytosol, as mitochondria are the major sources of reactive oxygen species （ROS） and pivotal components during cell apoptosis. Mitochondrial superoxide dismutase （SOD2） takes the leading role in eliminating mitoROS, and inhibition of SOD2 might induce severe disturbances overwhelming the mitochondrial oxidative equilibrium, which would elevate the intracellular oxidative stresses and drive cells to death. Herein, we report a general strategy to kill cancer cells by targeted inhibition of SOD2 using 2-methoxyestradiol （2-ME, an inhibitor for the SOD family） via a robust mitochondria-targeted mesoporous silica nanocarrier （mtMSN）, with the expected elevation of mitoROS and activation of apoptosis in HeLa cells. Fe304@MSN was employed in the mitochondria-targeted drug delivery and selective inhibition of mitochondrial enzymes, and was shown to be stable with good biocompatibility and high loading capacity. Due to the selective inhibition of SOD2 by 2-ME/mtMSN, enhanced elevation of mitoROS （132% of that with free 2-ME） was obtained, coupled with higher efficiency in initiating cell apoptosis （395% of that with free 2-ME in 4 h）. Finally, the 2-ME/mtMSN exhibited powerful efficacy in targeted killing of HeLa cells by taking advantage of both biological recognition and magnetic guiding, causing 97.0% cell death with only 2 Dg/mL 2-ME/mtMSN, hinting at its great potential in cancer therapy through manipulation of the delicate mitochondrial oxidative balance.
基金supported by the Creative Research Group Project of National Natural Science Foundation of China(Grant No.21021004)the National Basic Research Program(973 Program)(Nos.2012CB910601,2012CB910101)the Analytical Method Innovation Program of MOST(No.2010IM030500)(H.Z.)。
文摘The human serum proteome is closely associated with the state of the body.Endogenous peptides derived from proteolytic enzymes cleaving on serum proteins are widely studied due to their potential application in disease-specific marker discovery.However,the reproducibility of peptidome analysis of endogenous peptides is significantly influenced by the proteolytic enzymes within body fluids,thereby limiting the clinical use of the endogenous peptides.We comprehensively investigated the N and C terminus of endogenous peptides using peptidomics.The cleavage site patterns of the N and C terminus and adjacent sites from all the identified endogenous peptides were highly conserved under different sample preparation conditions,including long-term incubation at 37℃ and pretreatment with repeated freeze-thaw cycles.Furthermore,a distinguishable cleavage site pattern was obtained when a different disease serum was analyzed.The conserved cleavage site pattern derived from proteolytic enzymes holds potential in highly specific disease diagnosis.
