Nanog is a newly identified homeodomain gene that functions to sustain the pluripotency of embryonic stem cells.However,the molecular mechanism through which nanog regulates stem cell pluripotency remains unknown.Mous...Nanog is a newly identified homeodomain gene that functions to sustain the pluripotency of embryonic stem cells.However,the molecular mechanism through which nanog regulates stem cell pluripotency remains unknown.Mouse nanog encodes a polypeptide of 305 residues with a divergent homeodomain similar to those in the NK-2 family.The rest ofnanog contains no apparent homology to any known proteins characterized so far.It is hypothesized that nanog encodes a transcription factor that regulates stem cell pluripotency by switching on or off target genes.To test this hypothesis,we constructed fusion proteins between nanog and DNA binding domains of the yeast transcription factor Gal4 and tested the transactivation potentials of these constructs.Our data demonstrate that both regions N- and C- terminal to the homeodomain have transcription activities.Despite the fact that it contains no apparent transactivation motifs,the C-terminal domain is about 7 times as active as the N-terminal one.This unique arrangement of dual transactivators may confer nanog the flexibility and specificity to regulate downstream genes critical for both pluripotency and differentiation of stem cells.展开更多
Oral mucosal inflammatory responses to P. gingivalis and its key virulence factor, lipopolysaccharide (LPS), are characterized by a massive rise in proinflammatory cytokine production, up-regu- lation in mitogen-activ...Oral mucosal inflammatory responses to P. gingivalis and its key virulence factor, lipopolysaccharide (LPS), are characterized by a massive rise in proinflammatory cytokine production, up-regu- lation in mitogen-activated protein kinase (MAPK) cascade, and the induction in epidermal growth factor receptor (EGFR) activation. In this study, we report that stimulation of salivary gland acinar cells with P. gingivalis LPS leads to p38 MAPK-dependent release of soluble TGF-α ligand and the increase in EGFR phosphorylation. Further, we show that the LPS-induced TGF-α shedding and EGFR transactivation involve the activation of membrane-associated metalloprotease, TACE also known as ADAM17, through phosphorylation by p38 MAPK, and require Rac1 participation. Moreover, we demonstrate that blocking the Rac1 activation leads to the suppression in the membrane translocation of Rac1 as well as p38, thus indicating that the LPS-elicited p38 membrane recruitment for TACE phosphorylation requires colocalization with Rac1. Hence, our findings imply that Rac1 membrane translocation serves as an essential platform for the localization of p38 with TACE, TGF-α ectodomain shedding, and the EGFR activation.展开更多
Objective: To investigate the effect of Calpain inhibitor I on glucocorticoid receptor-dependent proteasomal degradation and its transcriptional activity. Methods: After Raw-264.7 cells were treated with Calpain inhib...Objective: To investigate the effect of Calpain inhibitor I on glucocorticoid receptor-dependent proteasomal degradation and its transcriptional activity. Methods: After Raw-264.7 cells were treated with Calpain inhibitor I, dexamethasone, or both for about 12 h, the change of glucocorticoid receptor was detected by western blot analysis. COS-7 cells were transfected with PRsh-GRα expression vector and glucocorticoid-responsive receptor pMAMneo-CAT, then the effect of Calpain inhibitor I on glucocorticoid receptor transcriptional activation ability was determined by CAT activity. Results: The glucocorticoid receptor levels decreased after RAW-264.7 cells were treated with dexamethasone for 12 hours, which effect can be inhibited by Calpain inhibitor I to some extent. CAT activity assay showed that Calpain inhibitor I enhance glucocorticoid receptor transcriptional activity. Conclusion: Calpain inhibitor I can inhibit the down-regulation of dexamethasone on glucocorticoid receptor, and enhances glucocorticoid receptor transactivation ability.展开更多
γ-amidobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and mediates fast synaptic inhibition through GABAA and GABAC
Hylocereus polyrhizus,also known as pitaya or dragon fruit,is a climbing cactus grown worldwide because of its excellent performance under drought stress and appealing red-purple fruits.In practice,accelerating flower...Hylocereus polyrhizus,also known as pitaya or dragon fruit,is a climbing cactus grown worldwide because of its excellent performance under drought stress and appealing red-purple fruits.In practice,accelerating flower formation and inducing more flowers usually result in higher yield.However,the genes for this purpose have not been well characterized in pitaya.Previously,FLOWERING BHLHs(FBHs)have been identified as positive regulators of flower formation.In the present work,a total of eight FBHs were identified in pitaya.This is a greater number than in beet and spinach,possibly because of the recent whole-genome duplication that occurred in the pitaya genome.The phylogenetic tree indicated that the FBHs could be divided into three groups.In TYPEⅡ,the genes of Caryophyllales encode atypical FBHs and are generated by dispersed duplication.The K_(a)/K_(s) ratios indicated that HpFBHs are under purifying selection.Promoter and expression analysis of HpFBHs revealed that they are spatiotemporally activated in flower-related tissues and responsive to multiple abiotic stresses.These results indicated that HpFBHs are involved in the flower formation of pitaya.Therefore,typical HpFBH1/3 from TYPEⅡI and an atypical HpFBH8 from TYPEⅡwere selected for functional verification.HpFBH3 was found to heterodimerize with HpFBH1 in the nucleus using subcellular localization,yeast two-hybrid and luciferase complementation assays.With bioinformatic analysis,all HpFBHs were predicted to transactivate downstream genes via binding to the E-boxes,which were frequently detected in the promoters of HpCOs,HpFTs and HpSOC1s.RNA-Seq datasets showed that these flowering accelerators were expressed in coordination with HpFBH3.Yeast one-hybrid and dual-luciferase reporter assays further verified that HpFBH3 transactivated HpCO7 by selectively binding to the E-boxes in the promoter.Moreover,ectopic overexpression of HpFBH3 accelerated flower formation in Arabidopsis.In summary,this study systematically characterized the typical HpFBHs,especially HpFBH3,as positive regulators of flower formation,which could be target genes for the genetic improvement of pitaya.展开更多
Hydrogen sulfide(H2S)is a signaling molecule that regulates plant hormone and stress responses.The phytohormone abscisic acid(ABA)plays an important role in plant adaptation to unfavorable environmental conditions and...Hydrogen sulfide(H2S)is a signaling molecule that regulates plant hormone and stress responses.The phytohormone abscisic acid(ABA)plays an important role in plant adaptation to unfavorable environmental conditions and induces the persulfidation of L-CYSTEINE DESULFHYDRASE1(DES1)and the production of H2S in guard cells.However,it remains largely unclear how H2S and protein persulfidation participate in the relay of ABA signals.In this study,we discovered that ABSCISIC ACID INSENSITIVE 4(ABI4)acts downstream of DES1 in the control of ABA responses in Arabidopsis.ABI4 undergoes persulfidation at Cys250 that is triggered in a time-dependent manner by ABA,and loss of DES1 function impairs this process.Cys250 and its persulfidation are essential for ABI4 function in the regulation of plant responses to ABA and the H2S donor NaHS during germination,seedling establishment,and stomatal closure,which are abolished in the ABI4Cys250Ala mutated variant.Introduction of the ABI4Cys250Ala variant into the abi4 des1 mutant did not rescue its hyposensitivity to ABA.Cys250 is critical for the binding of ABI4 to its cognate motif in the promoter of Mitogen-Activated Protein Kinase Kinase Kinase 18(MAPKKK18),which propagates the MAPK signaling cascade induced by ABA.Furthermore,the DES1-mediated persulfidation of ABI4 enhances the transactivation activity of ABI4 toward MAPKKK18,and ABI4 can bind the DES1 promoter,forming a regulatory loop.Taken together,these findings advance our understanding of a post-translational regulatory mechanism and suggest that ABI4 functions as an integrator of ABA and MAPK signals through a process in which DES1-produced H2S persulfidates ABI4 at Cys250.展开更多
Thymine DNA glycosylase CrDG）, an enzyme that initiates the repair of G/T and G/U mismatches, has been lately found crucial in em- bryonic development to maintain epigenetic stability and facilitate the active DNA de...Thymine DNA glycosylase CrDG）, an enzyme that initiates the repair of G/T and G/U mismatches, has been lately found crucial in em- bryonic development to maintain epigenetic stability and facilitate the active DNA demethylation. Here we report a novel role of TDG in Wnt signaling as a transcriptional coactivator of β-catenin/TCFs complex. Our data show that TDG binds to the transcriptional factor family LEF1/TCFs and potentiates β-catenin/TCFs transactivation, while TDG depletion suppresses Wnt3a-stimulated reporter activity or target gene transcription. Next, we show that CBP, a known coactivator, is also required for TDG function through forming a coopera- tive complex on target promoters. Moreover, there is an elevation of TDG levels in human colon cancer tissue, and knockdown of TDG inhibits proliferation of the colon cells. Overall, our results reveal that TDG, as a new coactivator, promotes β-catenin/TCFs transacti- vation and functionally cooperates with CBP in canonical Wnt signaUng.展开更多
Background:Our previous studies demonstrated that eyes absent homolog 4(EYA4),a member of the eye devel-opment-related EYA family in Drosophila,is frequently methylated and silenced in hepatocellular carcinoma(HCC)spe...Background:Our previous studies demonstrated that eyes absent homolog 4(EYA4),a member of the eye devel-opment-related EYA family in Drosophila,is frequently methylated and silenced in hepatocellular carcinoma(HCC)specimens and associated with shorter survival.The current work aimed to explore the mechanisms through which EYA4 functions as a tumor suppressor in HCC.Methods:Stable EYA4-expressing plasmid(pEYA4)transfectants of the human HCC cell lines Huh-7 and PLC/PRF/5(PLC)were established.Xenografts tumors were established via subcutaneous injection of the stable transfectants into BALB/c nude mice.Tissue samples were obtained from 75 pathologically diagnosed HCC patients.Quantitative real-time polymerase chain reaction,Western blotting and immunohistochemistry were performed to determine the expression of EYA4 in cell lines,xenografts and clinical specimens.The cell proliferation,colony formation,invasiveness and tumor formation of stable transfectants were studied.A gene expression microarray was utilized to screen genes regulated by EYA4 expression.The effect of EYA4 on nuclear factor-κB(NF-κB)/RAS-related protein 1(RAP1)signaling was demonstrated through the co-transfection of pEYA4 and Flag-tagged RAS-related protein 1A gene-expressing plasmid(Flag-RAP1A),functional studies,chromatin immunoprecipitation,immunofluorescence staining and cellular ubiquitination assay.Results:The restoration of EYA4 expression in HCC cell lines suppressed cell proliferation,inhibited clonogenic outgrowth,reduced cell invasion and restrained xenograft tumor growth,and Flag-RAP1A reversed the suppressive effects of pEYA4 in vitro.Activation of NF-κB with tumor necrosis factor-α(TNF-α)increased the binding of p65 to the RAP1A gene promoter and up-regulated RAP1 protein expression.The inhibition of NF-κB with BAY 11-7085 and p65 siRNA successfully blocked TNF-α-induced RAP1 up-regulation.EYA4 antagonized the TNF-α-induced phosphoryla-tion and ubiquitination of inhibitor of NF-κBα(IκBα)as well as the nuclear translocation and transactivation of p65,resulting in repressed NF-κB activity and RAP1 expression.Blocking the serine/threonine phosphatase activity of EYA4 with calyculin A notably abrogated its suppressive effect on NF-κB activity.In addition,EYA4 expression was inversely correlated with IκBα/RAP1 activity in clinical HCC specimens.Conclusion:Our findings provide a functional and mechanistic basis for identifying EYA4 as a bona fide tumor sup-pressor that disrupts aberrant activation of the NF-κB/RAP1 signaling pathway and thus orchestrates a physiological impediment to HCC growth and invasion.展开更多
Stimulation of G protein-coupled receptors(GPCRs) can lead to the transactivation of the epidermal growth factor receptors(EGFR). The cross-communication between the two signaling pathways regulates several important ...Stimulation of G protein-coupled receptors(GPCRs) can lead to the transactivation of the epidermal growth factor receptors(EGFR). The cross-communication between the two signaling pathways regulates several important physiological or pathological processes. However, the molecule mechanism underlying EGFR transactivation remains poorly understood. Here, we aim to study the GPCR-mediated EGFR transactivation process using the single-molecule fluorescence imaging and tracking approach.We found that although EGFR existed as monomers at the plasma membrane of resting cells, they became dimers and thus diffused slower following the activation of β2-adrenergic receptor(β2-AR) by isoproterenol(ISO). We further proved thatβ2-AR-mediated changes of EGFR in stoichiometry and dynamics were mediated by Src kinase. Thus, the observations obtained via the single-molecule imaging and tracking methods shed new insights into the molecular mechanism of EGFR transactivation at single molecule level.展开更多
AIM： To investigate the transactivating effect of pre-S2 protein of hepatitis B virus （HBV） and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridizat...AIM： To investigate the transactivating effect of pre-S2 protein of hepatitis B virus （HBV） and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization （SSH） technique, and to pave the way for elucidating the pathogenesis of HBV infection. METHODS： pcDNA3.1（-）-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1 （-）-pre-S21pSV-lacZ and empty pcDNA3.1（-）/pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase （β-gal）. SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1（-）-pre-S2 and pcDNA3.1（-） empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5α The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS： The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1（-）-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1 （-）-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid （P〈0.01）. The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positiveclones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein. CONCLUSION： The pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transcluction and cell apoptosis.This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.展开更多
AIM： To examine whether lysophosphatidic acid （LPA） induces phosphorylation of c-Met and epidermal growth factor receptor （EGFR）, both of which have been proposed as prognostic markers of colorectal cancer, and w...AIM： To examine whether lysophosphatidic acid （LPA） induces phosphorylation of c-Met and epidermal growth factor receptor （EGFR）, both of which have been proposed as prognostic markers of colorectal cancer, and whether LPA induces cyclooxygenase-2 （COX-2） expression in human colon cancer cells. METHODS： Using a human colon cancer cell line, LoVo cells, we performed immunoprecipitation analysis, followed by Western blot analysis. We also examined whether LPA induced COX-2 expression, by Western blot analysis. RESULTS： Immunoprecipitation analysis revealed that 10 μmol/L LPA induced tyrosine phosphorylation of c-Met and EGFR in LoVo cells within a few minutes. We found that c-Met tyrosine phosphorylation induced by LPA was not attenuated by pertussis toxin or a matrix metalloproteinase inhibitor, in marked contrast to the results for EGFR. In addition, 0.2-40 IJmol/L LPA induced COX-2 expression in a dose-dependent manner. CONCLUSION： Our results suggest that LPA acts upstream of various receptor tyrosine kinases （RTKs） and COX-2, and thus may act as a potent stimulator of colorectal cancer. 2005 The WJG Press and Elsevier Inc. All rights reserved.展开更多
Steroid hormone receptors （SHRs） act in cell type- and gene-specific manner through interactions with coregulatory proteins to regulate numerous physiological and pathological processes at the level of gene regulati...Steroid hormone receptors （SHRs） act in cell type- and gene-specific manner through interactions with coregulatory proteins to regulate numerous physiological and pathological processes at the level of gene regulation. Binding of steroid receptor modulator （SRM） ligand leads to allosteric changes in SHR to exert positive or negative effects on the expression of target genes. Due, in part, to the fact that current SRMs generally target ligand binding domain （LBD）/AF2 and neglect intrinsically disordered （ID） N-terminal domain （NTD）/AF1, clinically relevant SRMs lack selectivity and are also prone to the development of resistance over time. Therefore, to maximize the efficacy of SHR-based therapeutics, the possibility of developing unique modulators that act to control AF1 activity must be considered. Recent studies targeting androgen receptor＇s （AR＇s） ID AF1 domain for the castration-resistant prostate cancer has provided the possibility of therapeutically targeting ID NTD/AF1 surfaces by allosteric modulations to achieve desired effects. In this review article, we discuss how inter- and intra- molecular allosteric regulations controlled by AR＇s structural flexibility and dynamics particularly the ID NTD/AF1 is an emerging area of investigation, which could be exploited for drug development and therapeutic targeting of prostate cancer.展开更多
N-ethyl-N-nitrosourea （ENU） mutagenesis has led to the elucidation of several regulator genes for melanocyte and skin development. Here we characterized a mutant from ENU mutagenesis with similar phenotype as that o...N-ethyl-N-nitrosourea （ENU） mutagenesis has led to the elucidation of several regulator genes for melanocyte and skin development. Here we characterized a mutant from ENU mutagenesis with similar phenotype as that of Splotch mutant, including exencephaly, spina bifida and abnormal limbs in homozygotes as well as white belly spotting and occasionally loop-tail in heterozygotes. This novel mutant was named as SpxG. Through genome-wide linkage analysis in backcross progenies with microsatellite markers, the SpxG was confined to a region between DIMIT415 and DIMIT7 on chromosome 1, where notable Pax3 gene was located. Direct sequencing revealed that SpxG carried a nucleotide A894G missense transition in exon 6 of Pax3 gene that resulted in Asn to Asp substitution at amino acid 269 within the highly-conserved homeodomain （HD） DNA recognition module, which was the first point mutation found in this domain in mice. This N269D mutation impaired the transactivation capacity of Pax3 protein, but exerted no effect on Pax3 protein translation. The characterization of the new mutation expanded our understanding the transactivation and DNA-binding structure of Pax3 protein.展开更多
The multifunctional trans-activator Tat is an essential regulatory protein for HIV-1 replication and is characterized by high sequence diversity. Numerous experimental studies have examined Tat in HIV-1 subtype B, but...The multifunctional trans-activator Tat is an essential regulatory protein for HIV-1 replication and is characterized by high sequence diversity. Numerous experimental studies have examined Tat in HIV-1 subtype B, but research on subtype C Tat is lacking, despite the high prevalence of infections caused by subtype C worldwide. We hypothesized that amino acid differences contribute to functional differences among Tat proteins. In the present study, we found that subtype B NL4-3Tat and subtype C isolate HIV1084 i Tat exhibited differences in stability by overexpressing the fusion protein Tat-Flag. In addition, 1084 i Tat can activate LTR and NF-κB more efficiently than NL4-3 Tat. In analyses of the activities of the truncated forms of Tat, we found that the carboxylterminal region of Tat regulates its stability and transactivity. According to our results, we speculated that the differences in stability between B-Tat and C-Tat result in differences in transactivation ability.展开更多
Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors （TFs） has demonstrated that cell fates are not fixed and that cellular differentiation can be a t...Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors （TFs） has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to ＂read＂ genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivo counterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongeneticaUy by using drug.like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore; the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.展开更多
By using molccular doning technique,the E2 gene of human papillomavirus type 16 wasexpressed in E.coli.The 3.2 kb fragment containing the E2 gene was cut out from HPV16 genomeand blunted with nuelease S1.The plasmid p...By using molccular doning technique,the E2 gene of human papillomavirus type 16 wasexpressed in E.coli.The 3.2 kb fragment containing the E2 gene was cut out from HPV16 genomeand blunted with nuelease S1.The plasmid pBD2 DNA was linearized with Hind Ⅲ and bluntedwith nuclasc S1 too.Afler ligation,thc ligsted DNA was used to transform E.coli BMH 71-18.The positive colonies were screened by in situ hybridization technique,and proceedcd to DNA analy-sis and proton analysis.The purified expressed protein was used to run immunoclctrophoreesis withantiserum against pBD2.We concktudcd that thc expressed protcin was a fusion protein ofbeta-galae-sidasc-E2 protein.展开更多
BACKGROUND: Interferon-alpha (IFN-alpha) is an important cytokine with multiple functions, but the target genes transactivated by IFN-alpha remain largely unknown. A study of such genes will help to understand the mec...BACKGROUND: Interferon-alpha (IFN-alpha) is an important cytokine with multiple functions, but the target genes transactivated by IFN-alpha remain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-alpha. To isolate the gene transcripts specifically upregulated by IFN-alpha in HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis. METHODS: SSH was used to analyze the target genes transactivated by recombinant IFN-alpha protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-alpha (rIFN-alpha, 2000 IU/ml) for 16 hours as tester, and cells not treated with rIFN-alpha as driver. The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected, sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed significant homology to other known proteins. RESULTS: The subtractive cDNA library of genes upregulated by IFN-alpha was constructed successfully, rIFN-alpha upregulated the expression of the RAN binding protein 5 (RANBP5), NADH dehydrogenase, exosome component 3 (EXOSC3), zinc finger RNA binding protein, Dickkopf homolog 1 (DKK1) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). CONCLUSIONS: These results suggest that rIFN-alpha can upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-alpha.展开更多
A battery of tests was established to determine the oestrogenic, mutagenic and genotoxic potential of two categories of endocrine disrupting chemicals (EDCs), phthalates and alkylphenols. Diisononylphthalate (DINP), d...A battery of tests was established to determine the oestrogenic, mutagenic and genotoxic potential of two categories of endocrine disrupting chemicals (EDCs), phthalates and alkylphenols. Diisononylphthalate (DINP), diethylhexylphthalate (DEHP), dibutylphthalate (DBP), diisododecylphthalate (DIDP) and 4-nonylphenol (4-NP) were oestrogenic in the yeast estrogen screen (YES) assay and potently oestrogenic in the MVLN and E-SCREEN assays at environmentally relevant concentrations. DINP and 4-NP were mutagenic in the Ames assay and also induced significant levels of unscheduled DNA synthesis and DNA strand breakage. Significant induction in the percentage of cells containing micronuclei was observed after treatment with DINP, DEHP and 4-NP. In addition, sewage effluents from sewage treatment plants (STPs) in the Border, Midlands and Western (BMW) region of Ireland were significantly oestrogenic in the YES assay. Moreover, analysis of levels of phthalates and alkylphenol identified in Irish rivers receiving treated effluent showed potent oestrogenicity in the YES assay. The proliferative and genotoxic ability of the phthalates and alkylphenol, and the oestrogenicity of the treated effluents reported here, is significant as these EDCs and EDCs within the effluent may play a role in the etiology of human abnormalities.展开更多
AIM: To investigate the transactivating effect of complete S protein of hepatitis B virus (HBV) and to construct a subtractive cDNA library of genes transactivated by complete S protein of HBV by suppression subtracti...AIM: To investigate the transactivating effect of complete S protein of hepatitis B virus (HBV) and to construct a subtractive cDNA library of genes transactivated by complete S protein of HBV by suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivation activity, and to pave the way for elucidating the pathogenesis of hepatitis B virus infection. METHODS: pcDNA3.1(-)-complete S containing full-length HBV S gene was constructed by insertion of HBV complete S gene into BamH I/Kpn I sites. HepG2 cells were cotransfected with pcDNA3.1(-)-complete S and pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). Suppression subtractive hybridization and bioinformatics techniques were used. The mRNA of HepG2 cells transfected with pcDNA3.Incomplete S and pcDNA3.1(-) empty vector was isolated, and detected for the expression of complete S protein by reverse transcription polymerase chain reaction (RT-PCR) method, and cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptors 1 and 2, respectively. After tester cDNA had been hybridized with driver cDNA twice and underwent nested PCR twice, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out within E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR) amplification. RESULTS: The complete S mRNA could be detected by RT-PCR in HepG2 cells transfected with the pcDNA3.1(-)-complete S. The activity of β-gal in HepG2 cells transfected with the pcDNA3.1(-)-complete s was 6.9 times higher than that of control plasmid. The subtractive library of genes transactivated by HBV complete S protein was constructed successfully. The amplified library contains 86 positive clones. Colony PCR showed that 86 clones contained DNA inserts of 200-1 000 bp, respectively. Sequence analysis was performed in 35 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 33 coding sequences were obtained. These cDNA sequences might be target genes transactivated by complete S protein of HBV. Moreover, two unknown genes were discovered, full length coding sequences were obtained by bioinformatics techniques, one of them was named complete S transactivated protein 1 (CSTP1) and registered in GenBank (AY553877). CONCLUSION: The complete S gene of HBV has a transactivating effect on SV40 early promoter. A subtractive cDNA library of genes transactivated by HBV complete S protein using SSH technique has been constructed successfully. The obtained sequences may be target genes transactivated by HBV complete S protein among which some genes coding proteins are involved in cell cycle regulation, metabolism, immunity, signal transduction, cell apoptosis and formation mechanism of hepatic carcinoma.展开更多
AIM: To investigate the relationship between the polymorphism of class Ⅱ transactivator (CIITA) gene promoters and chronic hepatitis B (CHB). METHODS: Genomic DNA was prepared from peripheral blood leukocytes. Promot...AIM: To investigate the relationship between the polymorphism of class Ⅱ transactivator (CIITA) gene promoters and chronic hepatitis B (CHB). METHODS: Genomic DNA was prepared from peripheral blood leukocytes. Promoters Ⅰ,Ⅲ and Ⅳ of gene were analyzed respectively with polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) in 65 patients with CHB, 26 patients with acute hepatitis B (AHB) and 85 normal controls. RESULTS: No abnormal migration was found in PCR-SSCP analysis of the three promoters in the three groups. Also, no sequential difference was observed at the three promoters among the CHB patients, AHB patients and normal controls. CONCLUSION: No polymorphism in promoters I, III and IV of CIITA gene exists in CHB patients, ABH patients and normal controls, suggesting that the promoter of CIITA gene might be a conserved domain.展开更多
基金supported in part by the Tsinghua University BaiRen Scholar Program,NSFC 30270287the 973 Project--2001CB5101 from The Ministry of Science and Technology of China.
文摘Nanog is a newly identified homeodomain gene that functions to sustain the pluripotency of embryonic stem cells.However,the molecular mechanism through which nanog regulates stem cell pluripotency remains unknown.Mouse nanog encodes a polypeptide of 305 residues with a divergent homeodomain similar to those in the NK-2 family.The rest ofnanog contains no apparent homology to any known proteins characterized so far.It is hypothesized that nanog encodes a transcription factor that regulates stem cell pluripotency by switching on or off target genes.To test this hypothesis,we constructed fusion proteins between nanog and DNA binding domains of the yeast transcription factor Gal4 and tested the transactivation potentials of these constructs.Our data demonstrate that both regions N- and C- terminal to the homeodomain have transcription activities.Despite the fact that it contains no apparent transactivation motifs,the C-terminal domain is about 7 times as active as the N-terminal one.This unique arrangement of dual transactivators may confer nanog the flexibility and specificity to regulate downstream genes critical for both pluripotency and differentiation of stem cells.
文摘Oral mucosal inflammatory responses to P. gingivalis and its key virulence factor, lipopolysaccharide (LPS), are characterized by a massive rise in proinflammatory cytokine production, up-regu- lation in mitogen-activated protein kinase (MAPK) cascade, and the induction in epidermal growth factor receptor (EGFR) activation. In this study, we report that stimulation of salivary gland acinar cells with P. gingivalis LPS leads to p38 MAPK-dependent release of soluble TGF-α ligand and the increase in EGFR phosphorylation. Further, we show that the LPS-induced TGF-α shedding and EGFR transactivation involve the activation of membrane-associated metalloprotease, TACE also known as ADAM17, through phosphorylation by p38 MAPK, and require Rac1 participation. Moreover, we demonstrate that blocking the Rac1 activation leads to the suppression in the membrane translocation of Rac1 as well as p38, thus indicating that the LPS-elicited p38 membrane recruitment for TACE phosphorylation requires colocalization with Rac1. Hence, our findings imply that Rac1 membrane translocation serves as an essential platform for the localization of p38 with TACE, TGF-α ectodomain shedding, and the EGFR activation.
文摘Objective: To investigate the effect of Calpain inhibitor I on glucocorticoid receptor-dependent proteasomal degradation and its transcriptional activity. Methods: After Raw-264.7 cells were treated with Calpain inhibitor I, dexamethasone, or both for about 12 h, the change of glucocorticoid receptor was detected by western blot analysis. COS-7 cells were transfected with PRsh-GRα expression vector and glucocorticoid-responsive receptor pMAMneo-CAT, then the effect of Calpain inhibitor I on glucocorticoid receptor transcriptional activation ability was determined by CAT activity. Results: The glucocorticoid receptor levels decreased after RAW-264.7 cells were treated with dexamethasone for 12 hours, which effect can be inhibited by Calpain inhibitor I to some extent. CAT activity assay showed that Calpain inhibitor I enhance glucocorticoid receptor transcriptional activity. Conclusion: Calpain inhibitor I can inhibit the down-regulation of dexamethasone on glucocorticoid receptor, and enhances glucocorticoid receptor transactivation ability.
文摘γ-amidobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and mediates fast synaptic inhibition through GABAA and GABAC
基金supported by the National Natural Science Foundation of China(32160681 and 32060663)the National Guidance Foundation for Local Science and Technology Development of China(2023-009)+1 种基金the Guizhou Provincial Basic Research Program(Natural Science)(ZK[2022]YB132)the Foundation of Postgraduate of Guizhou Province,China(YJSKYJJ[2021]057)。
文摘Hylocereus polyrhizus,also known as pitaya or dragon fruit,is a climbing cactus grown worldwide because of its excellent performance under drought stress and appealing red-purple fruits.In practice,accelerating flower formation and inducing more flowers usually result in higher yield.However,the genes for this purpose have not been well characterized in pitaya.Previously,FLOWERING BHLHs(FBHs)have been identified as positive regulators of flower formation.In the present work,a total of eight FBHs were identified in pitaya.This is a greater number than in beet and spinach,possibly because of the recent whole-genome duplication that occurred in the pitaya genome.The phylogenetic tree indicated that the FBHs could be divided into three groups.In TYPEⅡ,the genes of Caryophyllales encode atypical FBHs and are generated by dispersed duplication.The K_(a)/K_(s) ratios indicated that HpFBHs are under purifying selection.Promoter and expression analysis of HpFBHs revealed that they are spatiotemporally activated in flower-related tissues and responsive to multiple abiotic stresses.These results indicated that HpFBHs are involved in the flower formation of pitaya.Therefore,typical HpFBH1/3 from TYPEⅡI and an atypical HpFBH8 from TYPEⅡwere selected for functional verification.HpFBH3 was found to heterodimerize with HpFBH1 in the nucleus using subcellular localization,yeast two-hybrid and luciferase complementation assays.With bioinformatic analysis,all HpFBHs were predicted to transactivate downstream genes via binding to the E-boxes,which were frequently detected in the promoters of HpCOs,HpFTs and HpSOC1s.RNA-Seq datasets showed that these flowering accelerators were expressed in coordination with HpFBH3.Yeast one-hybrid and dual-luciferase reporter assays further verified that HpFBH3 transactivated HpCO7 by selectively binding to the E-boxes in the promoter.Moreover,ectopic overexpression of HpFBH3 accelerated flower formation in Arabidopsis.In summary,this study systematically characterized the typical HpFBHs,especially HpFBH3,as positive regulators of flower formation,which could be target genes for the genetic improvement of pitaya.
基金supported by grants from the National Natural Science Foundation of China(31670255)the National Natural Science Foundation of China of Jiangsu Province(BK20200561,BK20200282,BK20161447)+3 种基金the National Science Fund for Outstanding Young Scholars(21922702)the China Postdoctoral Science Foundation(2019M661860)the Fundamental Research Funds for the Central Universities(KYZ201859)the European Regional Development Fund through the Agenda Estatal de Investigacion(grant no.PID2019-109785GB-IOO).
文摘Hydrogen sulfide(H2S)is a signaling molecule that regulates plant hormone and stress responses.The phytohormone abscisic acid(ABA)plays an important role in plant adaptation to unfavorable environmental conditions and induces the persulfidation of L-CYSTEINE DESULFHYDRASE1(DES1)and the production of H2S in guard cells.However,it remains largely unclear how H2S and protein persulfidation participate in the relay of ABA signals.In this study,we discovered that ABSCISIC ACID INSENSITIVE 4(ABI4)acts downstream of DES1 in the control of ABA responses in Arabidopsis.ABI4 undergoes persulfidation at Cys250 that is triggered in a time-dependent manner by ABA,and loss of DES1 function impairs this process.Cys250 and its persulfidation are essential for ABI4 function in the regulation of plant responses to ABA and the H2S donor NaHS during germination,seedling establishment,and stomatal closure,which are abolished in the ABI4Cys250Ala mutated variant.Introduction of the ABI4Cys250Ala variant into the abi4 des1 mutant did not rescue its hyposensitivity to ABA.Cys250 is critical for the binding of ABI4 to its cognate motif in the promoter of Mitogen-Activated Protein Kinase Kinase Kinase 18(MAPKKK18),which propagates the MAPK signaling cascade induced by ABA.Furthermore,the DES1-mediated persulfidation of ABI4 enhances the transactivation activity of ABI4 toward MAPKKK18,and ABI4 can bind the DES1 promoter,forming a regulatory loop.Taken together,these findings advance our understanding of a post-translational regulatory mechanism and suggest that ABI4 functions as an integrator of ABA and MAPK signals through a process in which DES1-produced H2S persulfidates ABI4 at Cys250.
文摘Thymine DNA glycosylase CrDG）, an enzyme that initiates the repair of G/T and G/U mismatches, has been lately found crucial in em- bryonic development to maintain epigenetic stability and facilitate the active DNA demethylation. Here we report a novel role of TDG in Wnt signaling as a transcriptional coactivator of β-catenin/TCFs complex. Our data show that TDG binds to the transcriptional factor family LEF1/TCFs and potentiates β-catenin/TCFs transactivation, while TDG depletion suppresses Wnt3a-stimulated reporter activity or target gene transcription. Next, we show that CBP, a known coactivator, is also required for TDG function through forming a coopera- tive complex on target promoters. Moreover, there is an elevation of TDG levels in human colon cancer tissue, and knockdown of TDG inhibits proliferation of the colon cells. Overall, our results reveal that TDG, as a new coactivator, promotes β-catenin/TCFs transacti- vation and functionally cooperates with CBP in canonical Wnt signaUng.
基金supported by National Natural Science Foundation of China(Grant Number 81472261)Natural Science Foundation of Guangdong Province(Grant Numbers 2014A030310033 and 2015A030313032)+1 种基金Science and Technology Planning Project of Guangdong Province(Grant Number 2013B021800122)Science and Technology Planning Projects of Guangzhou City(Grant Number 201604020044).
文摘Background:Our previous studies demonstrated that eyes absent homolog 4(EYA4),a member of the eye devel-opment-related EYA family in Drosophila,is frequently methylated and silenced in hepatocellular carcinoma(HCC)specimens and associated with shorter survival.The current work aimed to explore the mechanisms through which EYA4 functions as a tumor suppressor in HCC.Methods:Stable EYA4-expressing plasmid(pEYA4)transfectants of the human HCC cell lines Huh-7 and PLC/PRF/5(PLC)were established.Xenografts tumors were established via subcutaneous injection of the stable transfectants into BALB/c nude mice.Tissue samples were obtained from 75 pathologically diagnosed HCC patients.Quantitative real-time polymerase chain reaction,Western blotting and immunohistochemistry were performed to determine the expression of EYA4 in cell lines,xenografts and clinical specimens.The cell proliferation,colony formation,invasiveness and tumor formation of stable transfectants were studied.A gene expression microarray was utilized to screen genes regulated by EYA4 expression.The effect of EYA4 on nuclear factor-κB(NF-κB)/RAS-related protein 1(RAP1)signaling was demonstrated through the co-transfection of pEYA4 and Flag-tagged RAS-related protein 1A gene-expressing plasmid(Flag-RAP1A),functional studies,chromatin immunoprecipitation,immunofluorescence staining and cellular ubiquitination assay.Results:The restoration of EYA4 expression in HCC cell lines suppressed cell proliferation,inhibited clonogenic outgrowth,reduced cell invasion and restrained xenograft tumor growth,and Flag-RAP1A reversed the suppressive effects of pEYA4 in vitro.Activation of NF-κB with tumor necrosis factor-α(TNF-α)increased the binding of p65 to the RAP1A gene promoter and up-regulated RAP1 protein expression.The inhibition of NF-κB with BAY 11-7085 and p65 siRNA successfully blocked TNF-α-induced RAP1 up-regulation.EYA4 antagonized the TNF-α-induced phosphoryla-tion and ubiquitination of inhibitor of NF-κBα(IκBα)as well as the nuclear translocation and transactivation of p65,resulting in repressed NF-κB activity and RAP1 expression.Blocking the serine/threonine phosphatase activity of EYA4 with calyculin A notably abrogated its suppressive effect on NF-κB activity.In addition,EYA4 expression was inversely correlated with IκBα/RAP1 activity in clinical HCC specimens.Conclusion:Our findings provide a functional and mechanistic basis for identifying EYA4 as a bona fide tumor sup-pressor that disrupts aberrant activation of the NF-κB/RAP1 signaling pathway and thus orchestrates a physiological impediment to HCC growth and invasion.
基金supported by the National Basic Research Program of China (2013CB933701)the National Natural Science Foundation of China (81530009, 21127901, 91213305)Chinese Academy of Science
文摘Stimulation of G protein-coupled receptors(GPCRs) can lead to the transactivation of the epidermal growth factor receptors(EGFR). The cross-communication between the two signaling pathways regulates several important physiological or pathological processes. However, the molecule mechanism underlying EGFR transactivation remains poorly understood. Here, we aim to study the GPCR-mediated EGFR transactivation process using the single-molecule fluorescence imaging and tracking approach.We found that although EGFR existed as monomers at the plasma membrane of resting cells, they became dimers and thus diffused slower following the activation of β2-adrenergic receptor(β2-AR) by isoproterenol(ISO). We further proved thatβ2-AR-mediated changes of EGFR in stoichiometry and dynamics were mediated by Src kinase. Thus, the observations obtained via the single-molecule imaging and tracking methods shed new insights into the molecular mechanism of EGFR transactivation at single molecule level.
文摘AIM： To investigate the transactivating effect of pre-S2 protein of hepatitis B virus （HBV） and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization （SSH） technique, and to pave the way for elucidating the pathogenesis of HBV infection. METHODS： pcDNA3.1（-）-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1 （-）-pre-S21pSV-lacZ and empty pcDNA3.1（-）/pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase （β-gal）. SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1（-）-pre-S2 and pcDNA3.1（-） empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5α The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS： The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1（-）-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1 （-）-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid （P〈0.01）. The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positiveclones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein. CONCLUSION： The pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transcluction and cell apoptosis.This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.
基金Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a grant from the Ministry of Health, Labour and Welfare of Japan
文摘AIM： To examine whether lysophosphatidic acid （LPA） induces phosphorylation of c-Met and epidermal growth factor receptor （EGFR）, both of which have been proposed as prognostic markers of colorectal cancer, and whether LPA induces cyclooxygenase-2 （COX-2） expression in human colon cancer cells. METHODS： Using a human colon cancer cell line, LoVo cells, we performed immunoprecipitation analysis, followed by Western blot analysis. We also examined whether LPA induced COX-2 expression, by Western blot analysis. RESULTS： Immunoprecipitation analysis revealed that 10 μmol/L LPA induced tyrosine phosphorylation of c-Met and EGFR in LoVo cells within a few minutes. We found that c-Met tyrosine phosphorylation induced by LPA was not attenuated by pertussis toxin or a matrix metalloproteinase inhibitor, in marked contrast to the results for EGFR. In addition, 0.2-40 IJmol/L LPA induced COX-2 expression in a dose-dependent manner. CONCLUSION： Our results suggest that LPA acts upstream of various receptor tyrosine kinases （RTKs） and COX-2, and thus may act as a potent stimulator of colorectal cancer. 2005 The WJG Press and Elsevier Inc. All rights reserved.
文摘Steroid hormone receptors （SHRs） act in cell type- and gene-specific manner through interactions with coregulatory proteins to regulate numerous physiological and pathological processes at the level of gene regulation. Binding of steroid receptor modulator （SRM） ligand leads to allosteric changes in SHR to exert positive or negative effects on the expression of target genes. Due, in part, to the fact that current SRMs generally target ligand binding domain （LBD）/AF2 and neglect intrinsically disordered （ID） N-terminal domain （NTD）/AF1, clinically relevant SRMs lack selectivity and are also prone to the development of resistance over time. Therefore, to maximize the efficacy of SHR-based therapeutics, the possibility of developing unique modulators that act to control AF1 activity must be considered. Recent studies targeting androgen receptor＇s （AR＇s） ID AF1 domain for the castration-resistant prostate cancer has provided the possibility of therapeutically targeting ID NTD/AF1 surfaces by allosteric modulations to achieve desired effects. In this review article, we discuss how inter- and intra- molecular allosteric regulations controlled by AR＇s structural flexibility and dynamics particularly the ID NTD/AF1 is an emerging area of investigation, which could be exploited for drug development and therapeutic targeting of prostate cancer.
基金supported by the National Basic Research Program of China(No.2007CB947301)the National Natural Science Foundation of China(No.30800613)Pujiang Talent(No.08PJ1407200)
文摘N-ethyl-N-nitrosourea （ENU） mutagenesis has led to the elucidation of several regulator genes for melanocyte and skin development. Here we characterized a mutant from ENU mutagenesis with similar phenotype as that of Splotch mutant, including exencephaly, spina bifida and abnormal limbs in homozygotes as well as white belly spotting and occasionally loop-tail in heterozygotes. This novel mutant was named as SpxG. Through genome-wide linkage analysis in backcross progenies with microsatellite markers, the SpxG was confined to a region between DIMIT415 and DIMIT7 on chromosome 1, where notable Pax3 gene was located. Direct sequencing revealed that SpxG carried a nucleotide A894G missense transition in exon 6 of Pax3 gene that resulted in Asn to Asp substitution at amino acid 269 within the highly-conserved homeodomain （HD） DNA recognition module, which was the first point mutation found in this domain in mice. This N269D mutation impaired the transactivation capacity of Pax3 protein, but exerted no effect on Pax3 protein translation. The characterization of the new mutation expanded our understanding the transactivation and DNA-binding structure of Pax3 protein.
基金supported by grants from the National Natural Science Foundation of China (No.81571987 and 81371820)the Ph.D. Candidate Research Innovation Fund of Nankai University (2015) (No.68150003)
文摘The multifunctional trans-activator Tat is an essential regulatory protein for HIV-1 replication and is characterized by high sequence diversity. Numerous experimental studies have examined Tat in HIV-1 subtype B, but research on subtype C Tat is lacking, despite the high prevalence of infections caused by subtype C worldwide. We hypothesized that amino acid differences contribute to functional differences among Tat proteins. In the present study, we found that subtype B NL4-3Tat and subtype C isolate HIV1084 i Tat exhibited differences in stability by overexpressing the fusion protein Tat-Flag. In addition, 1084 i Tat can activate LTR and NF-κB more efficiently than NL4-3 Tat. In analyses of the activities of the truncated forms of Tat, we found that the carboxylterminal region of Tat regulates its stability and transactivity. According to our results, we speculated that the differences in stability between B-Tat and C-Tat result in differences in transactivation ability.
文摘Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors （TFs） has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to ＂read＂ genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivo counterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongeneticaUy by using drug.like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore; the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.
文摘By using molccular doning technique,the E2 gene of human papillomavirus type 16 wasexpressed in E.coli.The 3.2 kb fragment containing the E2 gene was cut out from HPV16 genomeand blunted with nuelease S1.The plasmid pBD2 DNA was linearized with Hind Ⅲ and bluntedwith nuclasc S1 too.Afler ligation,thc ligsted DNA was used to transform E.coli BMH 71-18.The positive colonies were screened by in situ hybridization technique,and proceedcd to DNA analy-sis and proton analysis.The purified expressed protein was used to run immunoclctrophoreesis withantiserum against pBD2.We concktudcd that thc expressed protcin was a fusion protein ofbeta-galae-sidasc-E2 protein.
文摘BACKGROUND: Interferon-alpha (IFN-alpha) is an important cytokine with multiple functions, but the target genes transactivated by IFN-alpha remain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-alpha. To isolate the gene transcripts specifically upregulated by IFN-alpha in HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis. METHODS: SSH was used to analyze the target genes transactivated by recombinant IFN-alpha protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-alpha (rIFN-alpha, 2000 IU/ml) for 16 hours as tester, and cells not treated with rIFN-alpha as driver. The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected, sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed significant homology to other known proteins. RESULTS: The subtractive cDNA library of genes upregulated by IFN-alpha was constructed successfully, rIFN-alpha upregulated the expression of the RAN binding protein 5 (RANBP5), NADH dehydrogenase, exosome component 3 (EXOSC3), zinc finger RNA binding protein, Dickkopf homolog 1 (DKK1) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). CONCLUSIONS: These results suggest that rIFN-alpha can upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-alpha.
文摘A battery of tests was established to determine the oestrogenic, mutagenic and genotoxic potential of two categories of endocrine disrupting chemicals (EDCs), phthalates and alkylphenols. Diisononylphthalate (DINP), diethylhexylphthalate (DEHP), dibutylphthalate (DBP), diisododecylphthalate (DIDP) and 4-nonylphenol (4-NP) were oestrogenic in the yeast estrogen screen (YES) assay and potently oestrogenic in the MVLN and E-SCREEN assays at environmentally relevant concentrations. DINP and 4-NP were mutagenic in the Ames assay and also induced significant levels of unscheduled DNA synthesis and DNA strand breakage. Significant induction in the percentage of cells containing micronuclei was observed after treatment with DINP, DEHP and 4-NP. In addition, sewage effluents from sewage treatment plants (STPs) in the Border, Midlands and Western (BMW) region of Ireland were significantly oestrogenic in the YES assay. Moreover, analysis of levels of phthalates and alkylphenol identified in Irish rivers receiving treated effluent showed potent oestrogenicity in the YES assay. The proliferative and genotoxic ability of the phthalates and alkylphenol, and the oestrogenicity of the treated effluents reported here, is significant as these EDCs and EDCs within the effluent may play a role in the etiology of human abnormalities.
基金Supported by the National Natural Science Foundation of China, No. C03011402, No. C30070690 the Science and Technique Foundation of PLA during the 9th Five-year Plan period, No. 98D063the Launching Foundation for Students Studying Abroad of PLA, No. 98H038the Youth Science and Technique Foundation of PLA during the 10th Five-year plan period, No. 01Q138the Science and Technique Foundation of PLA during the 10th Five-year Plan period, No. 01MB135
文摘AIM: To investigate the transactivating effect of complete S protein of hepatitis B virus (HBV) and to construct a subtractive cDNA library of genes transactivated by complete S protein of HBV by suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivation activity, and to pave the way for elucidating the pathogenesis of hepatitis B virus infection. METHODS: pcDNA3.1(-)-complete S containing full-length HBV S gene was constructed by insertion of HBV complete S gene into BamH I/Kpn I sites. HepG2 cells were cotransfected with pcDNA3.1(-)-complete S and pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). Suppression subtractive hybridization and bioinformatics techniques were used. The mRNA of HepG2 cells transfected with pcDNA3.Incomplete S and pcDNA3.1(-) empty vector was isolated, and detected for the expression of complete S protein by reverse transcription polymerase chain reaction (RT-PCR) method, and cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptors 1 and 2, respectively. After tester cDNA had been hybridized with driver cDNA twice and underwent nested PCR twice, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out within E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR) amplification. RESULTS: The complete S mRNA could be detected by RT-PCR in HepG2 cells transfected with the pcDNA3.1(-)-complete S. The activity of β-gal in HepG2 cells transfected with the pcDNA3.1(-)-complete s was 6.9 times higher than that of control plasmid. The subtractive library of genes transactivated by HBV complete S protein was constructed successfully. The amplified library contains 86 positive clones. Colony PCR showed that 86 clones contained DNA inserts of 200-1 000 bp, respectively. Sequence analysis was performed in 35 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 33 coding sequences were obtained. These cDNA sequences might be target genes transactivated by complete S protein of HBV. Moreover, two unknown genes were discovered, full length coding sequences were obtained by bioinformatics techniques, one of them was named complete S transactivated protein 1 (CSTP1) and registered in GenBank (AY553877). CONCLUSION: The complete S gene of HBV has a transactivating effect on SV40 early promoter. A subtractive cDNA library of genes transactivated by HBV complete S protein using SSH technique has been constructed successfully. The obtained sequences may be target genes transactivated by HBV complete S protein among which some genes coding proteins are involved in cell cycle regulation, metabolism, immunity, signal transduction, cell apoptosis and formation mechanism of hepatic carcinoma.
基金Supported by the National Natural Science Foundation of China,No. 30270597
文摘AIM: To investigate the relationship between the polymorphism of class Ⅱ transactivator (CIITA) gene promoters and chronic hepatitis B (CHB). METHODS: Genomic DNA was prepared from peripheral blood leukocytes. Promoters Ⅰ,Ⅲ and Ⅳ of gene were analyzed respectively with polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) in 65 patients with CHB, 26 patients with acute hepatitis B (AHB) and 85 normal controls. RESULTS: No abnormal migration was found in PCR-SSCP analysis of the three promoters in the three groups. Also, no sequential difference was observed at the three promoters among the CHB patients, AHB patients and normal controls. CONCLUSION: No polymorphism in promoters I, III and IV of CIITA gene exists in CHB patients, ABH patients and normal controls, suggesting that the promoter of CIITA gene might be a conserved domain.
