Relationship between polymorphism of class Ⅱ transactivator gene promoters and chronic hepatitis B

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.

Lnc-RP5 Regulates the mi R-129-5p/Notch1/PFV Internal Promoter Axis to Promote the Expression of the Prototype Foamy Virus Transactivator Tas

Prototype foamy virus(PFV)is a unique retrovirus that infects animals and humans and does not cause clinical symptoms.Long noncoding RNAs(lncRNAs)are believed to exert multiple regulatory functions during viral infections.Previously,we utilized RNA sequencing(RNA-seq)to characterize and identify the lncRNA lnc-RP5-1086 D14.3.1-1:1(lnc-RP5),which is markedly decreased in PFV-infected cells.However,little is known about the function of lnc-RP5 during PFV infection.In this study,we identified lnc-RP5 as a regulator of the PFV transcriptional transactivator(Tas).Lnc-RP5 enhanced the activity of the PFV internal promoter(IP).The expression of PFV Tas was found to be promoted by lnc-RP5.Moreover,mi R-129-5 p was found to be involved in the lnc-RP5-mediated promotion of PFV IP activity,while the Notch1 protein suppressed the activity of PFV IP and the expression of Tas.Our results demonstrate that lnc-RP5 promotes the expression of PFV Tas through the miR-129-5 p/Notch1/PFV IP axis.This work provides evidence that host lnc RNAs can manipulate PFV replication by employing mi RNAs and proteins during an early viral infection.

Generation and Characterization of a Transgenic Zebrafish Expressing the Reverse Tetracycline Transactivator

Conditional expression of a target gene during zebrafish development is a powerful approach to elucidate gene functions. The tetracycline-controlled systems have been successfully used in the modulation of gene expression in mammalian cells, but few lines of zebrafish carrying these systems are currently available. In this study, we had generated a stable transgenic zebrafish line that ubiquitously expressed the second-generation of reverse Tet transactivator （rtTA-M2）. Southern blotting analysis and high-throughput genome sequencing verifed that a single copy of rtTA-M2 gene had stably integrated into the zebrafish genome. After induction with doxycycline （Dox）, a strong green fluorescent protein （GFP） was seen in rtTA-transgenic eggs injected with pTRE--EGFP plasmids. The fluorescent signal gradually decreased after the withdrawal of Dox and disappeared. However, leaky expression of GFP was undetectable before Dox- induction. Additionally, transgenic embryos expressing rtTA-M2 exhibited no obvious defects in morphological phenotypes, hatching behavior and expression patterns of developmental marker genes, suggesting that rtTA-M2 had little effect on the development of transgenic zebrafish. Moreover, expressed Dickkopf-1 （DKK1） in pTRE-DKKl-injected embryos led to alterations in the expression of marker genes associated with Wnt signaling. Thus, this rtTA-transgenic zebrafish can be utilized to dissect functions of genes in a temporal manner.

In Vitro Biological Activity of Anti-C Ⅱ TA Hammerhead Ribozyme——A Novel Approach for Autoimmune Diseases

This study investigated the feasibility of using an hammerhead ribozyme against C Ⅱ TA, a major regulator of MHC Ⅱ antigens, to repress the expression of MHC Ⅱ molecules on Hela cells. A hammerhead ribozyme (Rz464) specific to 463-465 GUC triplet of C Ⅱ TA and its target gene were transcribed, then mixed up and incubated in vitro . The cleavage products were analyzed by PAGE and silver staining. Rz464 was then inserted into the pIRES2 EGFP vector (pRz464). Stable transfectants of Hela with pRz464 were tested for class Ⅱ MHC induction by recombinant human interferon gamma (IFN γ). mRNA of C Ⅱ TA was measured by RT PCR. Our results showed that Rz464 could exclusively cleave C Ⅱ TA RNA. When induced with IFN γ, the expression of HLA DR, DP, DQ on pRz464 + Hela was induced, and the mRNA content of C Ⅱ TA decreased too. It is concluded that Rz464 could inhibit C Ⅱ TA and thus the family of genes was regulated by C Ⅱ TA:MHC Ⅱ molecules. These results provided insight into the future application of Rz464 as a new nucleic acid drug against auto immune diseases.

Establishment of a tetracycline-off and heat shock-on gene expression system in tobacco

The tetracycline （Tet）-off gene expression regulation system based on the TetR-VP16/Top10 construct has not been widely utilized in plants, for its highly expressed TetR-VP16 activator is toxic to some plants and repeatedly replenishing tetracycline to turn off the constitutively active system is a tedious process. To solve these problems, a Tet-off and heat shock （HS）-on gene expression regulation system was constructed in this study. This system is composed of a chimeric transactivator gene TetR-HSF that is derived from a Tet repressor （TetR） and a HS transcription factor （HSF） controlled by a HS promoter HSP70m, and a Tet operator containing hybrid promoter, Om35S, that drives expression of the β-glucuronidase （GUS） gene. The resultant system yields a GUS expression pattern similar to that of the HSP70m promoter under inducing temperatures and at 35 and 40℃ drives GUS expression to a similar level as the Cauliflower mosaic virus （CaMV） 35S promoter. Further examination revealed that the TetR-HSF and GUS genes were induced by HS, reaching peak expression after 1 and 6 h treatment, respectively, and the HS induction of the expression system could be inhibited by Tet. This system will provide a useful tool for transgenic studies of plants in the laboratory and in the field, including transgene function analysis, agronomic trait improvement, biopharmaceutical protein production and others.

The enhancement of astrocytic-derived monocyte chemoattractant protein-1 induced by the interaction of opiate and HIV tat in HIV-associated dementia

HIV-associated dementia(HAD)is a public health problem and is particularly prevalent in drug abusers.The neuropathogenesis of human immunodeficiency virus(HIV)infection involves a complex cascade of inflammatory events,including monocyte/macrophage infiltration in the brain,glial immune activation and release of neurotoxic substances.In these events,astrocytic-derived monocyte chemoattractant protein-1(MCP-1)plays an important role,whose release is elevated by HIV transactivator of transcription(HIV tat)and could be further elevated by opiates.This review will also consider some critical factors and events in MCP-1 enhancement induced by the interactions of opiate and HIV tat,including the mediating role of mu opioid receptor(MOR)and CCR2 as well as the possible signal transduction pathways within the cells.Finally,it will make some future perspectives on the exact pathways,new receptors and target cells,and the vulnerability to neurodegeneration with HIV and opiates.

Functional study of p38 mitogen-activated protein kinase based on cell-penetrating peptide delivery system

Objective p38 Mitogen-activated protein kinase （MAPK） is a crossing center of various pathways. In this study, protein transduction system based on human immunodeficiency virus （HIV）-1 transactivator of transcription （TAT）, which is an efficient delivery peptide of the foreign proteins into cells, was employed to study p38 MAPK functions in eukaryotic cells. Methods p38 And its dominant negative form, p38AF, were constructed into pET-His-TAT vector correctly to verify that the recombinant plasmids were well-founded through restriction enzyme digestion and DNA sequencing. The two proteins, His-TAT-p38 and His-TAT-p38AF, were expressed and purified in Escherichia coli by SDS-PAGE. Then they were incubated with ECV304 cells respectively and readily transduced into cells in a time-dependent and dose-dependent manner. The cells were stimulated by sorbitol. Activating transcription factor （ATF） 2 phosphorylation level was checked using Western blot to assess the activity of endogenous p38. Results Compared with controls, it was found that His-TAT-p38 increased the level ofATF2 phosphorylation in sorbitol-stimulated ECV304 cells, while His-TAT-p38AF inhibited it, indicating p38 MAPK protein delivery system based on TAT was constructed successfully. TAT-p38 and its dominant negative form possessed high biological activity after transduction into ECV304 cells by TAT protein delivery system. The results showed that p38AF fused with TAT could inhibit the transduction of endogenous p38 signal pathway in part, and other pathway might regulate p38 phosphorylation. Conclusions Our study provides a novel pathway to inhibit p38 signal pathway and establish a new method to study p38 function.

Expression of CⅡTA Gene in Five Human Malignant Hematological Cell Lines and Its Significance

The role of MHC class Ⅱ transactivator (CⅡTA) in constitutive or IFN-γ inducible expression of HLA molecules in human malignant hematological cell lines was investigated. The expression of HLA molecules and CⅡTA protein was detected by Western blot, immunohistochemistry and flow cytometry. The expression of CⅡTA gene was determined by RT-PCR. The capability of peripheral blood T cell reaction stimulated by tumor cells was monitored by mixed lymphocyte reaction. It was found that the HLAⅡ-positive tumor cells expressed the CⅡ TA quite well, and the expression of HLAⅠ+Ⅱ was increased in the tumor cells with constitutive or inducible expression of CⅡ TA after induced by IFN-γ. The tumor cells which did not express CⅡ TA after induced by IFN- γ were not response to the expression of HLAⅡ promoted by IFN- γ. It suggests a correlation between the inability of some malignant hematological cell lines in response to IFN-γ for HLA expression and the deficiency in the inducible expression of CⅡTA, indicating CⅡ TA might take part in the regulation of HLA Ⅰ+Ⅱ expression in the tumor cells, which might play an important role in tumor immunologic escape.

Identification of two distinct transactivation domains in the pluripotency sustaining factor nanog

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.

Study of transactivating effect of pre-S2 protein of hepatitis B virus and cloning of genes transactivated by pre-S2 protein with suppression subtractive hybridization

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.

Lysophosphatidic acid transactivates both c-Met and epidermal growth factor receptor, and induces cyclooxygenase-2 expression in human colon cancer LoVo cells

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.

Transactivating effect of complete S protein of hepatitis B virus and cloning of genes transactivated by complete S protein using suppression subtractive hybridization technique

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.

<i>Porphyromonas gingivalis</i>-Stimulated TACE Activation for TGF-<i>α</i>Ectodomain Shedding and EGFR Transactivation in Salivary Gland Cells Requires Rac1-Dependent p38 MAPK Membrane Localization

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.

Effect of Calpain inhibitor I on glucocorticoid receptor-dependent degradation and its transactivation ability

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.

Subcloning and Expression of Human Papillomavirus Type 16 E2 Gene in E.Coli

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.

Identification of genes upregulated by recombinant interferon-alpha in HepG2 cells by suppressive subtractive hybridization analysis

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.

A Toxicological Assessment of Endocrine Disrupting Chemicals Found in the BMW (Border, Midland and Western) Region of Ireland

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.

Fusion of a rice endogenous N-methylpurine DNA glycosylase to a plant adenine base transition editor ABE8e enables A-toK base editing in rice plants

Engineering of a new type of plant base editor for simultaneous adenine transition and transversion within the editing window will greatly expand the scope and potential of base editing in directed evolution and crop improvement.Here,we isolated a rice endogenous hypoxanthine excision protein,N-methylpurine DNA glycosylase(OsMPG),and engineered two plant A-to-K(K=G or T)base editors,rAKBE01 and rAKBE02,for simultaneous adenine transition and transversion base editing in rice by fusing OsMPG or its mutant mOsMPG to a plant adenine transition base editor,ABE8e.We further coupled either OsMPG or mOsMPG with a transactivation factor VP64 to generate rAKBE03 and rAKBE04,respectively.Testing these four rAKBEs,at five endogenous loci in rice protoplasts,indicated that rAKBE03 and rAKBE04 enabled higher levels of A-to-G base transitions when compared to ABE8e and ABE8e-VP64.Furthermore,whereas rAKBE01 only enabled A-to-C/T editing at one endogenous locus,in comparison with rAKBE02 and rAKBE03,rAKBE04 could significantly improve the A-to-C/T base transversion efficiencies by up to 6.57-and 1.75-fold in the rice protoplasts,respectively.Moreover,although no stable lines with A-to-C transversion were induced by rAKBE01 and rAKBE04,rAKBE04 could enable simultaneous A-to-G and A-to-T transition and transversion base editing,at all the five target loci,with the efficiencies of A-to-G transition and A-to-T transversion editing ranging from 70.97 to 92.31%and 1.67 to 4.84%in rice stable lines,respectively.Together,these rAKBEs enable different portfolios of editing products and,thus,now expands the potential of base editing in diverse application scenario for crop improvement.

TNF-α Induces Transient Resistance to Fas-Induced Apoptosis in Eosinophilic Acute Myeloid Leukemia Cells

Tumor necrosis factor α （TNF-α） has been recognized as an activator of nuclear factor kB （NF-kB）, a factor implicated in the protection of many cell types from apoptosis. We and others have presented evidence to suggest that Fas-induced apoptosis may be an important aspect of the resolution of inflammation, and that delayed resolution of inflammation may be directly associated with NF-kB-dependent resistance to Fas. Because TNF-α activates NF-kB in many cell types including inflammatory cells such as eosinophils, we examined effects of TNF-α signaling on the Fas-mediated killing of an eosinophilic cell line AML14. While agonist anti-Fas （CHII） treatment induced apoptosis in AML14 cells, no significant cell death occurred in response to TNF-α alone. Electrophoretic mobility shift assay （EMSA） revealed that TNF-α induced NF-kB transactivation in AMLI4 cells in a time- and dose-dependent fashion, and subsequent supershift assays indicated that the translocated NF-kB was the heterodimer p65 （RelA）/p50. Pre-treatment of cells with TNF-α dramatically decreased the CHll-induced cell death in a transient fashion, accompanied by suppression of activation of caspase-8 and caspase-3 activation. Inhibition of NF-kB transactivation by inhibitors, BAY 11-7085 and parthenolide, reversed the suppression of Fas-mediated apoptosis by TNF-α. Furthermore, TNF-α up-regulated X-linked inhibitor of apoptosis protein （XIAP） transiently and XIAP levels were correlated with the temporal pattern of TNF-α protection against Fas-mediated apoptosis. This finding suggested that TNF-α may contribute to the prolonged survival of inflammatory cells by suppression of Fas-mediated apoptosis, the process involved with NF-kB transactivation, anti-apoptotic XIAP up-regulation and caspase suppression. Cellular ＆ Molecular Immunology. 2007;4（1）：43-52.

Role of vitamin D receptor in the regulation of CYP3A gene expression

Vitamin D3(VD3)is a multifunctional nutrient which can be either synthesized or absorbed from the diet.It plays a pivotal role in systemic calcium and phosphate homeostasis,as well as in various physiological and pathological processes.VD3 is converted to the active form,1α,25-dihydroxy vitamin D3(1,25-D3),by cytochrome P4502R1(CYP2R1)/CYP27A1 and CYP27B1 sequentially,and deactivated by multiple enzymes including CYP3A4.On the other hand,1,25-D3 is capable of activating the transcription of CYP3A genes in humans,mice and rats.The vitamin D receptor(VDR)-mediated transactivation of human CYP3A4 and CYP3A5 resembles that known for pregnane X receptor(PXR).Activated VDR forms a heterodimer with retinoid X receptorα(RXRα),recruits co-activators,translocates to the cell nucleus,binds to the specific vitamin D responsive elements(VDRE),and activates the gene transcription.In mice,intestinal Cyp3a11 mRNA levels,but not those of hepatic CYP3As,were induced by in vivo administration of VDR and PXR agonists.In rats,intestinal Cyp3a1 and Cyp3a2mRNAs were induced by 1,25-D3 or lithocholic acid(LCA),whereas hepatic Cyp3a2,but not Cyp3a1and Cyp3a9,was modulated to 1,25-D3 treatment.In general,the VDR-mediated regulation of CYP3A presents species and organ specificity.