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The discovery of hydroxylases in the anticancer drug taxol biosynthesis pathway is a hotspot and difficulty in current research. In this study, a new hydroxylase gene TcCYP725A22 (GenBank accession number: MF448646.1) was used to construct a sub-cellular localization vector pCAMIBA1303-TcCYP725A22-EGFP to get the transient expression in onion epidermal cells. Laser confocal microscopy revealed that the protein encoded by this gene was localized in the cell membrane. Furthermore, the recombinant plant expression plasmid pBI121-TcCYP725A22 was constructed. After transient transformation to the Taxus chinensis mediated by Agrobacterium tumefaciens LBA4404, qRT-PCR and LC-MS were utilized to analyze the effects of TcCYP725A22 overexpression on the synthesis of taxol. The results showed that, in the TcCYP725A22 overexpressed cell line, expression levels of most defined hydroxylase genes for taxol biosynthesis were increased, and the yield of taxanes were also increased. It was concluded that the hydroxylase gene TcCYP725A22 is likely involved in the biosynthetic pathway of taxol.
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Biosynthetic Pathways , Mixed Function Oxygenases , Paclitaxel , Taxoids , TaxusABSTRACT
The NOD protein family include NOD1 and NOD2,both of which recognize intracellular bacterial peptidoglycan,induce inflammation and promote host defense.In this review article,we summarize the research progress on NOD family,including signaling and regulation.We will also discuss their cellular localization and the role in host defense.Understanding on their physiological function will help shed light on pathogenesis on related diseases.
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Objective To investigate expression of PP2CE in human hepatocellular carcinoma(HCC)tissues and the clinical pathological significance,and test its sub-cellular localization in liver cells and HCC cells.Methods A total of 50 samples of HCC tissues and their para-carcinoma tissues were collected in Tongji Hospital(Wuhan).PP2CE mRNA and protein levels were detected in HCC tissues and their para-carcinoma tissues by qRT-PCR and immunohistochemistry method.Immunofluorescent assay was performed to test the sub-cellular localization of PP2CE in the cells.Results PP2CE mRNA and protein expression levels were significantly decreased in HCC tissues as compared with those in para-carcinoma tissues(both P＜0.05).Immunohistochemistry analysis found that PP2CE expression was associated with tumor size,differentiated degree,portal vein invasion and clinical stage(both P＜0.05).Immunofluorescent assay suggested that PP2CE was mainly located in the cytoplasm and perinuclear area.Conclusion PP2CE is reduced in HCC and may be implicated in the development and progression of HCC.
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Objective The mutation of the ABCB6 gene is involved in a variety of diseases , including dyschromatosis univer-salis hereditaria (DUH).This study aimed to construct the expression vectors for the ABCB 6-DsRed fusion proteins, pDsRed-wt-AB-CB6 and pDsRed-L356P-ABCB6, detect its cellular localization in A375 cells, and thus facilitate future studies on the pathogenesis of ABCB6-related diseases . Methods The recombinant plasmids pDsRed-wt/L356 P-ABCB6 were constructed based on the previously constructed pIRES2-ZsGreen1-ABCB6 vector and then transfected into A 375 cells.At 48 hours after transfection , the expression of AB-CB6 was detected by Western blot and the cellular localization of ABCB 6 determined under the laser scanning confocal microscope . Results The expression vectors pDsRed-wt/L356P-ABCB6 were verified by colony PCR, enzyme digestion, and DNA sequencing. The expression of ABCB 6 was significantly increased in the A 375 cells after transfected with the recombinant plasmids .Confocal mi-croscopy showed the localization of both wild-type and mutant ABCB6 in the cytoplasm. Conclusion The expression vectors for wild-type and mutant ABCB6-DsRed fusion protein were successfully constructed and the localization of ABCB 6 in A375 cells was de-termined, which may serve as a basis for further studies of ABCB 6 and the pathogenesis of ABCB 6-related diseases .
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Objective To clarify the cellular localization of triptolide and to explore its in-cell action sites.Methods 4-(Bro-momethyl)-7-methoxycoumarin was employed to label triptolide,then labelled triptolide was incubated with human hepatoma carci-noma cells.Subsequently,incubated cells were subjected to stain with fluorescent dye DiI or PI,which were specific to cytoplasmic membrane system and nucleus,respectively.Results Compared with the non-triptolide control,coumarin labelled triptolide shown a light blue fluorescence under UV excitation;Co-localization with DiI showed that triptolide exist in cytoplasm and(or)on cell mem-brane;Co-localization with PI showed that triptolide located in cell nucleus.Moreover,microscopic observation indicated that the fluorescence intensity in nucleus was denser than that in cytoplasm.Conclusion The presnt study demonstrate that triptolide main-ly act in nucleus,followed by acting in cytoplasm and(or)on cell membrane.
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Objective To investigate the self-assembly and cellular localization patterns of fila-mentous temperature-sensitive protein Z (FtsZ) in Escherichia coli (E.coli) strains by using FtsZP74R, FtsZG77D and FtsZA81R mutants.Methods YFP or His labeled FtsZ proteins and the plasmids of FtsZ mu-tants were constructed by using molecular clone and site-directed mutagenesis methods.The targeted proteins were purified by affinity chromatography.FL37(△ftsZ-Cat) strains were constructed via linear DNA homol-ogous recombination.Living cell imaging was performed to observe the cellular localization patterns of FtsZ protein and its mutants in E.coli strains.The interactions between FtsZ-FtsZ/FtsZ mutants were examined by coi-mmunoprecipitation assay . The polymerization properties of FtsZ mutants were analyzed by light scattering.The activities of GTPase were monitored by using high performance liquid chromatography.Re-sults The P74, G77 and A81 amino acids were respectively replaced by different polar amino acids to change the amphipathicity of the helix within the domain of FtsZ ( 74-82 ) .The YFP-labeled FtsZP74R , FtsZG77D and FtsZA81R mutants failed to assemble into functional Z-ring structure in E.coli strains.The inter-actions between FtsZ protein and its mutants were weakened or completely disappeared.In addition, in vitro experiments showed that P74R, G77D and A81R mutations caused a decrease in the polymerization efficien-cy of FtsZ monomer.The activity of GTPase was significantly decreased in the FtsZA81R mutant. Conclusion The P74, G77 and A81 were critical amino acids in the function and assembly of FtsZ protein in E.coli strains.Moreover, A81 amino acid regulated the lateral interaction of FtsZ monomer and the activity of GTPase.
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Objective:To construct the recombinant eukaryotic expression vector pCDNA3.1-MT2A and to investigate the cellular localization of MT2A protein in 293T and SMCC7721cell lines.Methods: Gene synthesis method was used to synthetic gene MT2A,added a Kozak sequence and His tag sequence at the N-terminus,the amplified target gene was connected to the pcDNA3.1(+) vector which was double digested between the BamH Ⅰ and Not Ⅰ.After transformation to E.coli DH5α, the positive clones were picked for plasmid extraction then Electrophoretic and sequenced.The pCDNA3.1-MT2A plasmids which passed through electrophoretic and sequenced were transfected 293T and SMMC7721 cell lines by liposome method,and then observed their expression and localization in eukaryotic cells by laser confocal microscopy.Results: The recombinant plasmid pCDNA3.1-MT2A was confirmed by restriction analysis and DNA sequencing,the sequence of the target gene MT2A was entirely correct,eukaryotic expression vector was successfully constructed and cell lines which had transfected recombinants could see the expression of green fluorescent protein in the cytoplasm.Conclusion:Successfully constructed fusion gene of pCDNA3.1-MT2A and expressed in eukaryotic cells,we found that the MT2A was mainly localized in the cytoplasm of 293T and SMMC7721 cell lines.The findings can help us to lay the foundation for the functions of MT2A in hepatoma cells.
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Objective To study the intracellular localization and temporal expression of CPSIT_0271 in Chlamydia psittaci-infected cells; and to investigate the effects of recombinant GST-CPSIT_0271 protein on the expression of proinflammatory cytokines including IL-6, IL-1βand TNF-αby THP-1 cells.Methods The gene encoding CPSIT_0271 of Chlamydia psittaci was expressed as fusion protein ( GST-CPSIT_0271 ) in E.coli.The polyclonal antibody was prepared by immunizing BALB /c mice with the purified recombinant pro-tein.Antibody titer was determined by ELISA .Indirect immunofluorescence assay ( IFA) was performed to lo-cate the endogenous CPSIT_0271 protein in C.psittaci-infected cells .The expression characteristics of CPSIT_0271 protein were detected by Western blot in C.psittaci-infected HeLa cells at different time points .The levels of IL-6, IL-1βand TNF-αwere analyzed by ELISA after stimulating THP-1 cells with different concentrations of CPSIT_0271 protein.Results CPSIT_0271 protein was found to express in the chlamydia inclusion of C.psittaci-infected HeLa cells .The expression of CPSIT_0271 protein was detected firstly at 36 h and increased at 48 h after C.psittaci infection.The titer of anti-CPSIT_0271 specific antibody in GST-CPSIT_0271 immu-nized mice reached to 1 ∶16 000.GST-CPSIT_0271 protein increased the levels of IL-6, IL-1βand TNF-αin THP-1 cells in a dose-dependent manner in the range of 2 to 5 μg/ml.The levels of TNF-αand IL-1βreached their peaks at 24 h, and IL-6 level peaked at 48 h upon the stimulation by 5 μg/ml of GST-CPSIT_0271 pro-tein.Conclusion CPSIT_0271 expressed in inclusion bodies of Chlamydia psittaci in the infected cells , sug-gesting it might be a late expression gene .GST-CPSIT_0271 protein shows good immunogenicity and enhances the expressions of IL-6, IL-1βand TNF-αin THP-1 cells.
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Objective To construct mycobacterial membrane-anchored expression vector and to analyze expression level and sub-cellualr localization of exogenous target protein. Methods Based on the mycobacterial intracellular expression vector pMFA42 which contained a strong promoter of pfurAma mutant, the signal sequence of Mycobacterium tuberculosis(Mtb) 19×103 lipoprotein (19SS) was synthesized and was then cloned into the downstream of pfurAma mutant to generate the mycobacterial membrane-anchored expression vector pMFA42M. The coding gene of enhanced green fluorescent protein(EGFP) was amplified by PCR, and then sub-cloned into these two vectors described above to construct recombinant EGFP fused and membrane-anchored strains, respectively. The coding genes of Mtb immuno-dominant antigens Ag85A and its chimera Ag856A2 were then sub-cloned intothe membrane-anchored construct pMFA42MG to produce recombinant Mtb antigen EGFP fused-expression strains. After that, expression levels and sub-cellualr localization of exogenous target protein were further analyzed by Western blot and flow cytometry sorting(FCS), and the fluorescence intensities of recombinant EGFP- expressed strains were observed in vitro directly and after transfection of murine macrophage cell line RAW264.7. Results The novel mycobacterial membrane-anchored expression vector was constructed successfully by introduction of signal sequence of Mtb 19×103 lipoprotein. Using of EGFP as model antigen, exogenous target protein was demonstrated to be expressed with high level and could be anchored into cell membrane of recombinant mycobaterial strains. Conclusion A novel mycobacterial membrane-anchored expression vector was constructed successfully to research recombinant BCG and functions of mycobacterial membrane proteins, and the constructed EGFP-expressed recombinant strains could also be used to research cytophagy in cell model and mycobacterial colony and translocation in animal immunization as model indicator bacteria.
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Nuclear proteins often form punctiform structures, but the precise mechanism for this process is unknown. As a preliminary study, we investigated the aggregation of an HSV-1 immediate-early protein,infected-cell protein 22 (ICP22), in the nucleus by observing the localization of ICP22-EGFP fusion protein.Results showed that, in high-level expression conditions, ICP22-EGFP gradually concentrates in the nucleus,persists throughout the cell cycle without disaggregation even in the cell division phase, and is finally distributed to daughter cells. We subsequently constructed a mammalian cell expression system, which had tetracyclinedependent transcriptional regulators. Consequently, the location of ICP22-EGFP in the nucleus changed with distinct induction conditions. This suggests that the cellular location of ICP22 is also influenced by promoter regulation, in addition to its own structure. Our findings provide new clues for the investigation of transcriptional regulation of viral genes. In addition, the non-protease reporter system we constructed could be utilized to evaluate the role of internal ribosome entry sites (IRES) on transcriptional regulation.
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AIM: To identify the novel nuclear export signal by analyzing the DNA sequences and detecting the cell localization of different adenovirus ElA - associated protein BS69 isoforms. METHODS:BS69 DNA sequences in Emsebl database were blasted and the sequence of amino acids was aligned with the typical nuclear export signal. Different BS69 isoform fragments were cloned into pcDNA3.1 vector and transfected into Cos7 cells. The BS69 localization was observed by immunostaining and the function was verified by Western blotting. RESULTS: A novel nuclear export signal was found in BS69 isoform 2 but not in isoform 1.The isoform 2 was localized in cytoplasm and isoform 1 in nucleus, which was also consistent with the DNA sequence. The isoform 2 was involved in LMP1 - activated JNK phosphorylation whereas the isoform 1 was not. CONCLUSION: Different BS69 isoforms have different cellular localization. BS69 isoform 2 is localized in cytoplasm, interacting with Epstein - Barr virus latent membrane protein 1 and may be involved in nasopharyngeal carcinoma development.However, the isoform 1 is localized in nucleus and plays important roles in transcription.
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To examine the involvement of phospholipase D (PLD)isozymes in postnatal testis development, the expression ofPLD1 and PLD2 was examined in the mouse testis atpostnatal weeks 1, 2, 4, and 8 using Western blot analysisand immunohistochemistry. The expression of both PLD1and PLD2 increased gradually with development frompostnatal week 1 to 8. Immunohistochemically, PLDimmunoreactivity was detected in some germ cells in thetestis and interstitial Leydig cells at postnatal week 1.PLD was mainly detected in the spermatocytes andresidual bodies of spermatids in the testis after 8 weeksafter birth. The intense immunostaining of PLD in Leydigcells remained unchanged by postnatal week 8. Thesefindings suggest that PLD isozymes are involved in thespermatogenesis of the mouse testis.
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Animals , Female , Male , Mice , Blotting, Western , Immunohistochemistry , Isoenzymes , Mice, Inbred BALB C , Phospholipase D/biosynthesis , Spermatogenesis/physiology , Testis/enzymologyABSTRACT
Bimolecular fluorescence complementation (BiFC) assay is an innovative approach to investigate protein interactions, based on the reassembly of protein fragments of the fluorescent proteins which directly report interactions. The fluorescent proteins tolerate circular permutation and insertions of foreign proteins with maintenance of fluorescence. So when the proteins fused to the reporter fragments interact with each other, a direct readout of the association would be given from the facilitating reassembly of the active reporter protein. Moreover, with distinct spectra difference of the fluorescent protein family members, BiFC assay is expanded to multicolor BiFC assay which enables visualization of interactions between different proteins in the same cell and comparison of the efficiencies of complex formation with alternative interaction partners. From it first reported in Molecular Cell in 2002 till now, this approach has been used on the networks of protein interaction in mammal cells, plant cells or even E.coli, and researches on transcription factors, G protein ?? dimmers, protein ubiqutination and so on.
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APOBEC(apolipoprotein B mRNA-editing enzyme catalytic-polypeptide) family members were reported as innate immune molecules with anti-viral activity for many viruses, such as HIV and HBV.In order to understand the function of APOBEC, the APOBEC-3F and-3G were cloned, expressed, and the sub-cellular localization of them was detected.The genes of APBEC-3F and-3G were cloned from PHA-stimulated PMBC and expressed in the MDCK cell by transfection.The sub-cellular localization of APOBEC-3F and-3G were detected by immunofluorescence.APOBEC-3F and-3G were cloned by RT-PCR and confirmed by DNA sequencing.The immunofluorescence indicated APOBEC-3F and-3G were located in the cytosal.APOBEC-3F and-3G could inhibit HBV replication effectively in HepG2.2.15 cell.APOBEC-3F and-3G could not be trans-located into nuclear by nuclear location signal(NLS) or bi-NLS(B-NLS).These results will help the future research on the function of APOBEC.
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The α-crystallin-related stress protein HSP27, which promotes cellular resistance to different types of stress, is constitutively expressed during the growth of several primate tissue culture cells. Here, we report an analysis of the cellular localization of this protein in CV-1 monkey cells. Following cell lysis and fractionation in the absence of detergent about 2 5 % of the cellular content of HSP27 was recovered in the particulate fractions while the remaining of this protein was in the soluble cytoplasmic fraction. This association of HSP27 with particulate fractions was no more observed when cells were lysed in the presence of non-ionic detergent or when cells were pretreated with drugs, such as monensin and colcemid, that disrupt cytoskeletal architecture. Immunofluorescence analysis revealed that HSP27 is concentrated in a polarized perinuclear zone of CV-1 cells from where microtubules radiate. The particular locale of HSP27 was investigated in cells exposed to drugs or treatments, such as monensin, colcemid, cold stess and serum starvation, that disrupt the cellular architecture of microtubules. A correlation was observed between HSP27 cellular locale and microtubules integrity. Our results suggest a possible interaction of a fraction of HSP27 with cytoplasmic organelles or structures, different from the Golgi apparatus, whose distribution depends upon the organization of microtubules.