The receptor for autocrine motility factor (AMFR) is known to be involved in the process of AMF-mediated cell migration and metastasis. This paper describes the procedures of non-radioactive in situ hybridization (ISH...The receptor for autocrine motility factor (AMFR) is known to be involved in the process of AMF-mediated cell migration and metastasis. This paper describes the procedures of non-radioactive in situ hybridization (ISH) detection of AMFR mRNA in both paraffin-embedded surgical sections and cultured cells using either biotinylated oligonucleotide probes or digoxigenin-labeled RNA probes. The results showed that the AMFR mRNA was expressed at an enhanced level in hyperplaJstic and malignant tissues of breast and prostate cancer patient surgical specimens, indicating that the elevated AMFR expression was associated with the tissue malignancy Moreover, AMFR mRNA was detected in both normal and earcinoma cells when cultured at a subconfluent density. However, AMFR expression was inhibited in confluent normal (3T3-A31 murine fibroblast and FHs738BL human bladder) cells while it continued to express in carcinoma (J82 human bladder)and metastatic (3T3-M murine fibroblast) cells irrespective of cell density This suggested a cell-cell contact downregulation of AMFR mRNA expression in normal but not in cancer cells. The ISH data obtained in this study are closely consistent with the AMFR protein expression pattern previously reported, implying that the differential expression of AMFR gene may be regulated and controlled at the transcriptional level.展开更多
Background:During Enterovirus type 71(EV71)infection,the structural viral protein 1(VP1)activates endoplasmic reticulum(ER)stress associated with peripheral myelin protein 22(PMP22)accumulation and induces autophagy.H...Background:During Enterovirus type 71(EV71)infection,the structural viral protein 1(VP1)activates endoplasmic reticulum(ER)stress associated with peripheral myelin protein 22(PMP22)accumulation and induces autophagy.However,the specific mechanism behind this process remains elusive.Methods:In this research,we used the VP1-overexpressing mouse Schwann cells(SCs)models co-transfected with a PMP22 silencing or Autocrine motility factor receptor(AMFR/gp78)overexpressing vector to explore the regulation of gp78 on PMP22 and its relationship with autophagy and apoptosis.Results:The activity of gp78 could be influenced by EV71-VP1,leading to a decrease in the ubiquitination and degradation of PMP22,resulting in PMP22 accumulation in ER.In VP1-overexpressing mouse SCs,all three ER stress sensors,including pancreatic endoplasmic reticulum kinase(PERK),activating transcription factor 6(ATF6)and inositol-requiring enzyme 1(IRE1)and the related downstream signals(C/EBP-homologous protein(CHOP)and Caspase 12)were activated,as well as the ER-resident chaperone Glucose-regulated protein 78(GRP78).In addition,VP1 upregulated the autophagy marker Microtubule-associated protein 1 light chain 3 beta(LC3B),while PMP22 silencing or gp78 overexpression reversed the phenomenon.Meanwhile,PMP22 silencing or gp78 overexpression increased proliferation of EV71-VP1-transfected mouse SCs.Conclusion:Gp78 could regulate PMP22 accumulation through ubiquitination degradation and cause ER stress and autophagy in EV71-VP1-overexpressing mouse SCs.Therefore,the gp78/PMP22/ER stress axis might emerge as a promising therapeutic target for myelin and neuronal damage induced by EV71 infection.展开更多
MicroRNAs （miRNAs） that exert function by posttran- scriptional suppression have recently brought insight in our understanding of the role of non.protein-coding RNAs in carcinogenesis and metastasis. In this study, ...MicroRNAs （miRNAs） that exert function by posttran- scriptional suppression have recently brought insight in our understanding of the role of non.protein-coding RNAs in carcinogenesis and metastasis. In this study, we described the function and molecular mechanism of miR-139-5p in colorectal cancer （CRC） and its potential clinical application in CRC. We found that miR-139-5p was significantly downregulated in 73.8% CRC samples compared with adjacent noncancerous tissues （NCTs）, and decreased miR-139-5p was associated with poor prognosis. Functional analyses demonstrated that ectopic expression of miR-139-5p suppressed CRC cell migration and invasion in vitro and metastasis in vivo. Mechanistic investigations revealed that miR-139-5p suppress CRC cell invasion and metastasis by targeting AMFR and NOTCH1. Knockdown of the two genes phe- nocopied the inhibitory effect of miR-139-5p on CRC metastasis. Furthermore, the protein levels of the two genes were upregulated in CRC samples compared with NCTs, and inversely correlated with the miR-139-5p expression. Increased NOTCH1 protein expression was correlated with poor prognosis of CRC patients. Together, our data indicate that miR-139-5p is a potentialtumor suppressor and prognostic factor for CRC, and targeting miR-139-5p may repress the metastasis of CRC and improve survival.展开更多
Presently,we develop a simplified corticothalamic(SCT)model and propose a single-pulse alternately resetting stimulation(SARS)with sequentially applying anodic(A,“+”)or cathodic(C,“−”)phase pulses to the thalamic ...Presently,we develop a simplified corticothalamic(SCT)model and propose a single-pulse alternately resetting stimulation(SARS)with sequentially applying anodic(A,“+”)or cathodic(C,“−”)phase pulses to the thalamic reticular(RE)nuclei,thalamus-cortex(TC)relay nuclei,and cortical excitatory(EX)neurons,respectively.Abatement effects of ACC-SARS of RE,TC,and EX for the 2 Hz-4 Hz spike and wave discharges(SWD)of absence seizures are then concerned.The m∶n on-off ACC-SARS protocol is shown to effectively reduce the SWD with the least current consumption.In particular,when its frequency is out of the 2 Hz-4 Hz SWD dominant rhythm,the desired seizure abatements can be obtained,which can be further improved by our proposed directional steering(DS)stimulation.The dynamical explanations for the SARS induced seizure abatements are lastly given by calculating the averaged mean firing rate(AMFR)of neurons and triggering averaged mean firing rates(TAMFRs)of 2 Hz-4 Hz SWD.展开更多
The hepatic endoplasmic reticulum(ER)-anchored cytochromes P450(P450s)are mixedfunction oxidases engaged in the biotransformation of physiologically relevant endobiotics as well as of myriad xenobiotics of therapeutic...The hepatic endoplasmic reticulum(ER)-anchored cytochromes P450(P450s)are mixedfunction oxidases engaged in the biotransformation of physiologically relevant endobiotics as well as of myriad xenobiotics of therapeutic and environmental relevance.P450 ER-content and hence function is regulated by their coordinated hemoprotein syntheses and proteolytic turnover.Such P450 proteolytic turnover occurs through a process known as ER-associated degradation(ERAD)that involves ubiquitindependent proteasomal degradation(UPD)and/or autophagic-lysosomal degradation(ALD).Herein,on the basis of available literature reports and our own recent findings of in vitro as well as in vivo experimental studies,we discuss the therapeutic and pathophysiological implications of altered P450 ERAD and its plausible clinical relevance.We specifically(i)describe the P450 ERAD-machinery and how it may be repurposed for the generation of antigenic P450 peptides involved in P450 autoantibodypathogenesis in drug-induced acute hypersensitivity reactions and liver injury,or viral hepatitis;(ⅱ)discuss the relevance of accelerated or disrupted P450-ERAD to the pharmacological and/or toxicological effects of clinically relevant P450 drug substrates;and(ⅲ)detail the pathophysiological consequences of disrupted P450 ERAD,contributing to non-alcoholic fatty liver disease(NAFLD)/non-alcoholic steatohepatitis(NASH)under certain synergistic cellular conditions.展开更多
Enhanced endoplasmic reticulum （ER）-associated protein degradation （ERAD） activity by the unfolded protein response （UPR） represents one of the mechanisms for restoring ER homeostasis. In vitro evidence indicate...Enhanced endoplasmic reticulum （ER）-associated protein degradation （ERAD） activity by the unfolded protein response （UPR） represents one of the mechanisms for restoring ER homeostasis. In vitro evidence indicates that the mammalian gp78 protein is an E3 ubiquitin ligase that facilitates ERAD by polyubiquitinating and targeting proteins for proteasomal degradation under both physiologic and stress conditions. However, the in vivo function of gp78 in maintaining ER protein homeostasis remains untested. Here we show that like its mammalian counterpart, the zebrafish gp78 is also an E3 ubiquitin ligase as revealed by in vitro ubiquitination assays. Expression analysis uncovered that gp78 is highly expressed in several organs, including liver and brain, of both larval and adult fish. Treatment of larvae or adult fish with tunicamycin induces ER stress and upregulates the expression of several key components of the gp78 ERAD complex in the liver. Moreover, liver-specific overexpression of the dominant-negative form of gp78 （gp78-R2M） renders liver more sensitive to tunicamycin-induced ER stress and enhances the expression of sterol response element binding protein （Srebp）-target genes, which was largely suppressed in fish overexpressing wild-type gp78. Together, these data indicate that gp78 plays a critical role in protecting against ER stress in liver.展开更多
文摘The receptor for autocrine motility factor (AMFR) is known to be involved in the process of AMF-mediated cell migration and metastasis. This paper describes the procedures of non-radioactive in situ hybridization (ISH) detection of AMFR mRNA in both paraffin-embedded surgical sections and cultured cells using either biotinylated oligonucleotide probes or digoxigenin-labeled RNA probes. The results showed that the AMFR mRNA was expressed at an enhanced level in hyperplaJstic and malignant tissues of breast and prostate cancer patient surgical specimens, indicating that the elevated AMFR expression was associated with the tissue malignancy Moreover, AMFR mRNA was detected in both normal and earcinoma cells when cultured at a subconfluent density. However, AMFR expression was inhibited in confluent normal (3T3-A31 murine fibroblast and FHs738BL human bladder) cells while it continued to express in carcinoma (J82 human bladder)and metastatic (3T3-M murine fibroblast) cells irrespective of cell density This suggested a cell-cell contact downregulation of AMFR mRNA expression in normal but not in cancer cells. The ISH data obtained in this study are closely consistent with the AMFR protein expression pattern previously reported, implying that the differential expression of AMFR gene may be regulated and controlled at the transcriptional level.
基金The study was supported by Guangdong Natural Science Foundation(Grant Numbers 2020A1515010014,2022A1515012411)Science and Technology Key Project for People’s Livelihood of Guangzhou,China(Grant Number 202206010060)+1 种基金Guangzhou Science and Technology Bureau Basic Research Project(SL2024A03J01288)Innovative Project of Children’s Research Institute,Guangzhou Women and Children’s Medical Center,China(Grant Numbers Pre-NSFC-2019-002,NKE PRE-2019-015).
文摘Background:During Enterovirus type 71(EV71)infection,the structural viral protein 1(VP1)activates endoplasmic reticulum(ER)stress associated with peripheral myelin protein 22(PMP22)accumulation and induces autophagy.However,the specific mechanism behind this process remains elusive.Methods:In this research,we used the VP1-overexpressing mouse Schwann cells(SCs)models co-transfected with a PMP22 silencing or Autocrine motility factor receptor(AMFR/gp78)overexpressing vector to explore the regulation of gp78 on PMP22 and its relationship with autophagy and apoptosis.Results:The activity of gp78 could be influenced by EV71-VP1,leading to a decrease in the ubiquitination and degradation of PMP22,resulting in PMP22 accumulation in ER.In VP1-overexpressing mouse SCs,all three ER stress sensors,including pancreatic endoplasmic reticulum kinase(PERK),activating transcription factor 6(ATF6)and inositol-requiring enzyme 1(IRE1)and the related downstream signals(C/EBP-homologous protein(CHOP)and Caspase 12)were activated,as well as the ER-resident chaperone Glucose-regulated protein 78(GRP78).In addition,VP1 upregulated the autophagy marker Microtubule-associated protein 1 light chain 3 beta(LC3B),while PMP22 silencing or gp78 overexpression reversed the phenomenon.Meanwhile,PMP22 silencing or gp78 overexpression increased proliferation of EV71-VP1-transfected mouse SCs.Conclusion:Gp78 could regulate PMP22 accumulation through ubiquitination degradation and cause ER stress and autophagy in EV71-VP1-overexpressing mouse SCs.Therefore,the gp78/PMP22/ER stress axis might emerge as a promising therapeutic target for myelin and neuronal damage induced by EV71 infection.
文摘MicroRNAs （miRNAs） that exert function by posttran- scriptional suppression have recently brought insight in our understanding of the role of non.protein-coding RNAs in carcinogenesis and metastasis. In this study, we described the function and molecular mechanism of miR-139-5p in colorectal cancer （CRC） and its potential clinical application in CRC. We found that miR-139-5p was significantly downregulated in 73.8% CRC samples compared with adjacent noncancerous tissues （NCTs）, and decreased miR-139-5p was associated with poor prognosis. Functional analyses demonstrated that ectopic expression of miR-139-5p suppressed CRC cell migration and invasion in vitro and metastasis in vivo. Mechanistic investigations revealed that miR-139-5p suppress CRC cell invasion and metastasis by targeting AMFR and NOTCH1. Knockdown of the two genes phe- nocopied the inhibitory effect of miR-139-5p on CRC metastasis. Furthermore, the protein levels of the two genes were upregulated in CRC samples compared with NCTs, and inversely correlated with the miR-139-5p expression. Increased NOTCH1 protein expression was correlated with poor prognosis of CRC patients. Together, our data indicate that miR-139-5p is a potentialtumor suppressor and prognostic factor for CRC, and targeting miR-139-5p may repress the metastasis of CRC and improve survival.
基金Project supported by the National Natural Science Foundation of China(Nos.11702018,11932003,and 11672074)。
文摘Presently,we develop a simplified corticothalamic(SCT)model and propose a single-pulse alternately resetting stimulation(SARS)with sequentially applying anodic(A,“+”)or cathodic(C,“−”)phase pulses to the thalamic reticular(RE)nuclei,thalamus-cortex(TC)relay nuclei,and cortical excitatory(EX)neurons,respectively.Abatement effects of ACC-SARS of RE,TC,and EX for the 2 Hz-4 Hz spike and wave discharges(SWD)of absence seizures are then concerned.The m∶n on-off ACC-SARS protocol is shown to effectively reduce the SWD with the least current consumption.In particular,when its frequency is out of the 2 Hz-4 Hz SWD dominant rhythm,the desired seizure abatements can be obtained,which can be further improved by our proposed directional steering(DS)stimulation.The dynamical explanations for the SARS induced seizure abatements are lastly given by calculating the averaged mean firing rate(AMFR)of neurons and triggering averaged mean firing rates(TAMFRs)of 2 Hz-4 Hz SWD.
基金supported by NIDDK Center Grant DK26743supported by NIH Grants GM44037 and DK26506(USA)to Maria Almira Correia.
文摘The hepatic endoplasmic reticulum(ER)-anchored cytochromes P450(P450s)are mixedfunction oxidases engaged in the biotransformation of physiologically relevant endobiotics as well as of myriad xenobiotics of therapeutic and environmental relevance.P450 ER-content and hence function is regulated by their coordinated hemoprotein syntheses and proteolytic turnover.Such P450 proteolytic turnover occurs through a process known as ER-associated degradation(ERAD)that involves ubiquitindependent proteasomal degradation(UPD)and/or autophagic-lysosomal degradation(ALD).Herein,on the basis of available literature reports and our own recent findings of in vitro as well as in vivo experimental studies,we discuss the therapeutic and pathophysiological implications of altered P450 ERAD and its plausible clinical relevance.We specifically(i)describe the P450 ERAD-machinery and how it may be repurposed for the generation of antigenic P450 peptides involved in P450 autoantibodypathogenesis in drug-induced acute hypersensitivity reactions and liver injury,or viral hepatitis;(ⅱ)discuss the relevance of accelerated or disrupted P450-ERAD to the pharmacological and/or toxicological effects of clinically relevant P450 drug substrates;and(ⅲ)detail the pathophysiological consequences of disrupted P450 ERAD,contributing to non-alcoholic fatty liver disease(NAFLD)/non-alcoholic steatohepatitis(NASH)under certain synergistic cellular conditions.
基金supported by the National Science Foundation(No.1120833)to SFa research grant (MB-8716-08) from United States-Israel Binational Agriculture Research and Development Fund to SD
文摘Enhanced endoplasmic reticulum （ER）-associated protein degradation （ERAD） activity by the unfolded protein response （UPR） represents one of the mechanisms for restoring ER homeostasis. In vitro evidence indicates that the mammalian gp78 protein is an E3 ubiquitin ligase that facilitates ERAD by polyubiquitinating and targeting proteins for proteasomal degradation under both physiologic and stress conditions. However, the in vivo function of gp78 in maintaining ER protein homeostasis remains untested. Here we show that like its mammalian counterpart, the zebrafish gp78 is also an E3 ubiquitin ligase as revealed by in vitro ubiquitination assays. Expression analysis uncovered that gp78 is highly expressed in several organs, including liver and brain, of both larval and adult fish. Treatment of larvae or adult fish with tunicamycin induces ER stress and upregulates the expression of several key components of the gp78 ERAD complex in the liver. Moreover, liver-specific overexpression of the dominant-negative form of gp78 （gp78-R2M） renders liver more sensitive to tunicamycin-induced ER stress and enhances the expression of sterol response element binding protein （Srebp）-target genes, which was largely suppressed in fish overexpressing wild-type gp78. Together, these data indicate that gp78 plays a critical role in protecting against ER stress in liver.
