1.The mechanism of Stat3 nuclear import.
Zhong-De YE ; Bei-Fen SHEN ; Lun SONG
Chinese Journal of Biotechnology 2004;20(2):299-301
In order to investigate the mechanism of stat3 nuclear import. positioned a characterized NLS of the SV40 large T antigen into Stat3-GFP, Dstat3-GFP respectively between the C-terminus of Stat3 and the N-terminus of GFP to create Stat3-NLS-GFP and Dstat3-NLS-GFP. With NLS-GFP as the positive control, Expression of the Stat3-NLS-GFP without IL-6 stimulation and Stat3-GFP with IL-6 stimulation resulted in a predominantly nuclear localization in 293T cell. Expression of Stat3-GFP and Dstat3-NLS-GFP without IL-6 stimulation resulted in predominantly cytoplasm localization in 293T cell. The results suggest that latent Stat3 is not anchored in the cytoplasm, and that nuclear localization in response to IL-6 is facilitated by gain of an NLS function.
Active Transport, Cell Nucleus
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Cell Nucleus
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metabolism
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Nuclear Localization Signals
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Plasmids
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metabolism
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STAT3 Transcription Factor
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metabolism
2.Serine residues at position 63-84 are important for CITED1 nuclear translocation and osteoblast differentiation.
Zhen LIN ; Liang YUAN ; Yue MENG ; Ruiqiang FENG ; Zhaozong FU ; Dehong YANG
Journal of Southern Medical University 2013;33(3):326-331
OBJECTIVETo determine the role of serine residues at position 63-84 of CITED1 in the nuclear translocation of CITED1 and osteoblast differentiation.
METHODSWe engineered all the 9 phosphorylated serine residues of CITED1 with a serine-to-alanine mutation at position 63-84. MC3T3E1 cells transfected with pCDNA3-CFP-CITED1 63-84 (9S>A), pCDNA3-CFP-CITED1, and vehicle plasmid were examined with confocal laser scanning microscopy before and after treatment with 100 nmol/L parathyroid hormone [PTH(1-34)] to observe the changes in the intracellular localization of CITED1. The transfected cells were induced for osteoblastic differentiation with mineralized solution in the absence or presence of 10 nmol/L PTH(1-34), and the changes in ALP activity and Ca(2+) concentration were measured; RT-PCR was used to detect the changes in ALP2, RUNX2, and OC gene expressions after the treatments.
RESULTSs PTH(1-34) promoted the nuclear translocation of CITED1 in MC3T3-E1 cells. The (63-84) 9S>A mutation of CITED1 obviously suppressed its translocation and increased ALP activity and Ca(2+) levels in the cells, which led to enhanced mineralization in the cells with also increased expressions of ALP2, RUNX2, and OC.
CONCLUSIONThe serine residues at position 63-84 of CITED1 play a vital role in the nuclear translocation of CITED1 and osteoblast differentiation.
Active Transport, Cell Nucleus ; Animals ; Cell Differentiation ; Cell Line ; Cell Nucleus ; Mice ; Mice, Inbred C57BL ; Mutation ; Nuclear Proteins ; metabolism ; Osteoblasts ; cytology ; Plasmids ; Serine ; metabolism ; Trans-Activators ; metabolism
3.Studies on dihydrotestosterone-binding capacity in prostate tissue.
Hong-Tao JIANG ; Zhao-Dian CHEN
National Journal of Andrology 2003;9(8):589-593
OBJECTIVETo explore the effect of the binding ability of the dihydrotestosterone(DHT) in prostate.
METHODSTwenty-two normal prostate tissues taken from accident-death corpses without serious diseases, and cytosolic and nuclear fractions were prepared with all the endogenous hormone removed from the cytosolic and nuclear fractions by ether stripping. The content of the bound 3H-DHT was assayed by adding 3H-DHT.
RESULTSThe average DHT-binding capacity of the DHT-binding protein in prostate was (0.0263 +/- 0.0047) nmol/g wet tissue. The DHT-binding capacities of cytosolic and nuclear fractions were (0.0103 +/- 0.0015) nmol/g wet tissue and (0.0155 +/- 0.0035) nmol/g wet tissue respectively, and the difference between them was very significant(P < 0.01).
CONCLUSIONSThe DHT-binding capacity of the DHT-binding protein in prostate is high and maintaining the high DHT level facilitates the effect of DHT.
Adult ; Cell Nucleus ; metabolism ; Cytoplasm ; metabolism ; Dihydrotestosterone ; metabolism ; Humans ; Male ; Prostate ; metabolism ; Protein Binding
4.A novel method for studying nuclear localization signal-mediated nuclear translocation.
Peng DENG ; Xiaowei GONG ; Yong JIANG
Journal of Southern Medical University 2012;32(8):1148-1150
OBJECTIVETo establish a new method for studying the mechanism of nuclear localization signal (NLS)-mediated nuclear translocation in living cells.
METHODSThe cells were treated with 67 mg/L 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), followed by incubation with 1 g/L wheat germ agglutinin (WGA), and their effects on interferon- γ (IFN-γ)-induced nuclear translocation of signal transducer and activator of transcription 1 (STAT1) were observed.
RESULTSTreatment with CHAPS alone had no effect on IFN-γ-induced nuclear translocation of STAT1, while this process was blocked by further WGA incubation.
CONCLUSIONWe established a new, simple but effective method for studying the mechanism of NLS-mediated nuclear translocation in living cells by perforating the cell membrane with CHAPS treatment.
Active Transport, Cell Nucleus ; Cell Nucleus ; metabolism ; Cholic Acids ; Cytological Techniques ; HeLa Cells ; Humans ; Interferon-gamma ; metabolism ; Nuclear Localization Signals ; metabolism ; STAT1 Transcription Factor ; metabolism ; Signal Transduction
5.Quantitative Analysis of Immuno-fluorescence of Nuclear Factor-κB Activation.
Min XIU ; Feng HE ; Yuanlei LOU ; Lu XU ; Xiong JIEQI ; Ping WANG ; Sisun LIU ; Fei GUO
Journal of Biomedical Engineering 2015;32(3):669-674
Immuno-fluorescence technique can qualitatively determine certain nuclear translocation, of which NF-κB/ p65 implicates the activation of NF-κB signal pathways. Immuno-fluorescence analysis software with independent property rights is able to quantitatively analyze dynamic location of NF-κB/p65 by computing relative fluorescence units in nuclei and cytoplasm. We verified the quantitative analysis by Western Blot. When we applied the software to analysis of nuclear translocation in lipopolysaccharide (LPS) induced (0. 5 h, 1 h, 2 h, 4 h) primary human umbilical vein endothelial cells (HUVECs) , we found that nuclear translocation peak showed up at 2h as with calculated Western blot verification results, indicating that the inventive immuno-fluorescence analysis software can be applied to the quantitative analysis of immuno-fluorescence.
Active Transport, Cell Nucleus
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Cell Nucleus
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metabolism
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Cytoplasm
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metabolism
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Fluorescent Antibody Technique
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Human Umbilical Vein Endothelial Cells
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Humans
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NF-kappa B p50 Subunit
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metabolism
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Software
6.Research progress of nanosecond pulsed electric field applied to intracellular electromanipulation.
Chenguo YAO ; Dengbin MO ; Caixin SUN ; Xin CHEN ; Zheng'ai XIONG
Journal of Biomedical Engineering 2008;25(5):1206-1209
In recent years, many experts have done some researches on experiment and mechanism of intracellular electromanipulation (IEM) under nanosecond pulsed electric field (nsPEF). The experiment results have shown that nsPEF could not induce electroporation of cell membrane, but could induce intracellular effects such as apoptosis, calcium release, enhancement of gene expression, and fragmentation of DNA and chromosome. In order to account for the phenomenon, researchers believe that when the pulse width of the pulsed electric field is larger than the charging time of plasma membrane, the pulsed electric field mainly targets on the outer membrane of cell; and that the effect of the pulsed electric field on nucleus and nuclear membrane increases with the decrease of the pulse width. It is also believed that the effect of electroporation changes from the outer membrane to intracellular electromanipulation when the pulse width decreases to a value being smaller than the charging time of plasma membrane.
Apoptosis
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Calcium
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metabolism
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Cell Membrane
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metabolism
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Cell Nucleus
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metabolism
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Cell Physiological Phenomena
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Electromagnetic Fields
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Electroporation
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Gene Expression
7.Characterization of nucleohistone and nucleoprotamine components in the mature human sperm nucleus.
Yan LI ; Claudia LALANCETTE ; David MILLER ; Stephen A KRAWETZ
Asian Journal of Andrology 2008;10(4):535-541
AIMTo simultaneously determine the localization of histones and protamines within human sperm nuclei.
METHODSImmunofluorescence of the core histones and protamines and fluorescence in situ hybridization of the telomere region of chromosome 16 was assessed in decondensed human sperm nuclei.
RESULTSImmunofluorescent localization of histones, protamine 1 (PRM1) and protamine 2 (PRM2) along with fluorescence in situ hybridization localization of chromosome 16 telomeric sequences revealed a discrete distribution in sperm nuclei. Histones localized to the posterior ring region (i.e. the sperm nuclear annulus), whereas PRM1 and PRM2 appeared to be dispersed throughout the entire nucleus.
CONCLUSIONThe co-localization of the human core sperm histones with the telomeric regions of chromosome 16 is consistent with the reorganization of specific non-protamine regions into a less compacted state.
Cell Nucleus ; metabolism ; Chromosomes, Human, Pair 16 ; metabolism ; Histones ; metabolism ; Humans ; Male ; Protamines ; metabolism ; Spermatozoa ; metabolism ; Telomere ; metabolism
8.Subcellular localization of human endothelial-overexpressed lipopolysaccharide-associated factor 1 protein.
Min LUO ; Zi-Wen LIANG ; Zong-Cheng YANG ; Xiang-Dong LUO
Chinese Journal of Burns 2010;26(6):444-447
OBJECTIVETo study the subcellular localization of human endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1) protein in endothelial cells.
METHODSHuman umbilical vein endothelial cell strain ECV304 were cultured in vitro. The fusion protein of enhanced green fluorescent protein (EGFP)-EOLA1 expressing plasmid was constructed. Empty plasmid with EGFP at N side (pEGFP-N2) and fusion protein expressing plasmid EGFP-EOLA1 was respectively transfected into ECV304 cells with liposome. After being cultured for 48 hours, the expression levels of EGFP and fusion protein EGFP-EOLA1 in cells were detected with Western blot. The subcellular localization of EOLA1 protein was detected by laser scanning confocal microscope and immunoelectron microscopy.
RESULTSThe EGFP-EOLA1 coexpression plasmid was verified to be successfully constructed by enzyme cutting and gene sequencing. The fusion protein of EGFP-EOLA1 was observed to express in transfected cells through Western blot. Green fluorescence scattered all over the ECV304 cells transfected with empty plasmid and cells transfected with fusion protein expressing plasmid, and it gathered obviously in the nuclei in the latter cells. Immune deposits were observed in the matrix of cells transfected with fusion protein expressing plasmid but not in the cells transfected with empty plasmid.
CONCLUSIONSEOLA1 protein is localized in the nucleus and the matrix of ECV304 cell, and it plays its role as a signal transduction factor.
Cell Line ; Cell Nucleus ; metabolism ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lipopolysaccharides ; metabolism ; Membrane Proteins ; metabolism ; Signal Transduction
9.Complex structures and diverse functions of nuclear bodies: a review.
Xinyuan LIU ; Lian-Feng LI ; Hua-Ji QIU
Chinese Journal of Biotechnology 2021;37(7):2223-2231
Nuclear bodies are membrane-free nuclear substructures that are localized in the mammalian nuclear matrix region. They are multiprotein complexes that recruit other proteins to participate in various cellular activities, such as transcription, RNA splicing, epigenetic regulation, tumorigenesis and antiviral defense. It is of great significance to clarify the functions and regulatory mechanisms of nuclear bodies to probe related diseases and virus-host interactions. This review takes several nuclear bodies associated proteins as examples, summarizes the formation process, structure and functions of nuclear bodies, and focuses on their important roles in antiviral infection. It is expected to provide new insight into host antiviral mechanisms.
Animals
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Cell Nucleus
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Epigenesis, Genetic
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Intranuclear Inclusion Bodies/metabolism*
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Nuclear Proteins/metabolism*
10.A molecular brake that modulates spliceosome pausing at detained introns contributes to neurodegeneration.
Dawei MENG ; Qian ZHENG ; Xue ZHANG ; Xuejiao PIAO ; Li LUO ; Yichang JIA
Protein & Cell 2023;14(5):318-336
Emerging evidence suggests that intron-detaining transcripts (IDTs) are a nucleus-detained and polyadenylated mRNA pool for cell to quickly and effectively respond to environmental stimuli and stress. However, the underlying mechanisms of detained intron (DI) splicing are still largely unknown. Here, we suggest that post-transcriptional DI splicing is paused at the Bact state, an active spliceosome but not catalytically primed, which depends on Smad Nuclear Interacting Protein 1 (SNIP1) and RNPS1 (a serine-rich RNA binding protein) interaction. RNPS1 and Bact components preferentially dock at DIs and the RNPS1 docking is sufficient to trigger spliceosome pausing. Haploinsufficiency of Snip1 attenuates neurodegeneration and globally rescues IDT accumulation caused by a previously reported mutant U2 snRNA, a basal spliceosomal component. Snip1 conditional knockout in the cerebellum decreases DI splicing efficiency and causes neurodegeneration. Therefore, we suggest that SNIP1 and RNPS1 form a molecular brake to promote spliceosome pausing, and that its misregulation contributes to neurodegeneration.
Spliceosomes/metabolism*
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Introns/genetics*
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RNA Splicing
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RNA, Messenger/genetics*
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Cell Nucleus/metabolism*