Objective To suppress the expression of CCR5 and CXCR4,the co-receptors for human immunodeficiency virus type 1(HIV-1),and thus inhibit HIV-1 from entering cells.Methods DNA fragments encoding either CCR5 or CXCR4 were amplified from healthy human peripheral blood mononuclear cells(PBMCs)by reverse transcript polymerase chain reaction(RT-PCR)and sequencing was performed.Correct fragments were inserted into Shuttle plasmid inversely,which was recombined with backbone plasmid containing homologous adenoviral genome in E.coli BJ5183.The recombinant plasmids were transfected into 293 cells in which they were packaged and amplified.Recombinant adenoviruses containing antisense RNA of CCR5 or CXCR4 were obtained and identified by RT-PCR,and the titres of them were determined by cytopathic effect(CPE)method.The U937 and MT4 cells were infected by recombinant adenoviruses containing antisense RNA of CCR5(multiplicity of infection,MOI=100)and CXCR4(MOI=200),respectively.The expression of co-receptors on infected cell was measured by fluorescence activated cell sorter at 24,48,72 hours and 10 days after infection.In addition,the chemotactic activity and proliferation of infected cells were detected with Boyden chamber and 3H incorporation respectively.Results We constructed the recombinant plasmids and obtained the recombinant adenoviruses which contained antisense RNA of CCR5 or CXCR4 and were designated as pAd-antiR5 and pAd-antiX4 respectively.The titers of recombinant adenoviruses pAd-antiR5 and pAd-antiX4 were 5×10 11 PFU/ml and 7×10 10 PFU/ml,respectively.The expression rate of CCR5 on U937 cells decreased from 82.10%(blank control)to 1.12%(Ad-antiR5 infected),and that of CXCR4 on MT4 cells decreased from 42%(blank control)to 1.03%(Ad-antiX4 infected)24 hours later.The expression rates of CCR5 on Ad-antiR5 infected U937 cells were 1.02%,1.26%,1.23% at 48 hours,72 hours,and 10 days later,respectively.The expression rates of CXCR4 on Ad-antiX4 infected MT4 cells were 1.13%,1.17%,1.22% at 48 hours,72 hours,and 10 days later,respectively.Moreover,the recombinant adenovirus had no effects on chemotactic activity and proliferation of the cells.Conclusion The recombinant adenovirus containing antisense CCR5 or CXCR4 can remarkably decrease the expression of co-receptors for HIV-1 on U937 or MT4 cells without affecting their chemotactic activities and proliferative abilities.
To inhibit the expression of the target genes of peroxiredoxin (Prx) and thioredoxin reductase (TrxR) by RNA interference and evaluate its effect on the growth of Trichomonas vaginalis.Genomic DNA was extracted from cultured Trichomonas vaginalis with phenol-chloroform method and was transcribed to double stranded RNA (dsRNA). Short interference RNAs (siRNA, 21-23 bp) synthesized by digestion of dsRNA with RNase III and purified through filter cartridge, were transfected into the cells in three groups (A, B and C) to degrade the target genes of Prx, TrxR and Prx+TrxR through siPORT lipid, respectively, and the untransfected was selected as a control (group D). The levels of Prx and TrxR mRNA were determined 24 h and 48 h post-transfection by relative quantitative RT-PCR, and the growth of Trichomonas vaginalis was estimated under microscope 36 h post-transfection.Trichomonas vaginalis mRNA levels of Prx and TrxR decreased. Though the cell activity showed no significant difference (P > 0.05) in four groups as expected, a difference existed (P < 0.01) between the groups in the average of vells (7.2 x 10(7)/L, 14.2 x 10(7)/L, 3.8 x l0(7) L and 20.3 x 10(7)/L in groups A, B, C and D respectively).RNA interference inhibits the expression of the genes of Prx and TrxR and extended Trichomonas vaginalis cells cycle considerably, but showed no influence on the cell activity.
Objective To clone the coding sequence of Importin 8 (IPO8) cDNA from HeLa cells and construct a eukaryotic expression vector for its green fluorescent protein (GFP)-IPO8 fusion protein expression in HeLa cells.Methods The total RNA was extracted from HeLa cells and the full length cDNA of IPO8 was amplified using reverse transcription-polymerase chain reaction (RT-PCR),and cloned into green fluorescence protein vector phosphorylated enhanced green fluorescent protein (pEGFP)-C1 to construct recombinant expression plasmid pEGFP-IPO8.After recombinant plasmids were transfected into HeLa cells,the expression of IPO8 was observed by fluorescent microscope.Results DNA sequence analysis showed that the amplified rat IPO8 gene was in concordance with that published on Gene Bank.The results of enzyme digestion and sequence analysis demonstrated that the recombinant plasmid pEGFP-IPO8 was constructed successfully.Fluorescence microscopy revealed the fusion protein GFP-IPO8 was mainly located in the nucleus of HeLa cells.Conclusion The IPO8 gene was cloned successfully and the recombinant plasmid were constructed successfully.The fusion protein GFP-IPO8 was demonstrated to be mainly located in the nucleus of HeLa cells,which provides a basis for biological functional study of IPO8 gene.
Key words:
Importin 8 gene; Green fluorescent protein; Fusion protein; Eukaryotic vector
The penetration of distributed generation and electric vehicles requires advanced monitoring and control strategies to maintain the reliable operation of active distribution network (ADN). Phasor measurement unit (PMU), as an advanced measuring device, has been applied in the operation of transmission systems for decades. Recently, it is anticipated that PMUs can be adopted in the distribution network. In this paper, the optimal branch PMU (BPMU) placement is studied. First, an optimization model for the multi-stage BPMU placement is established considering the observability of ADN. Moreover, the weights of buses are designed to consider the influence of uncertain renewable energy generation and loads. Then, probabilistic load flow (PLF) is used to solve power flow with uncertainties, and weights of buses are obtained based on probability distributions of voltage magnitude. Finally, binary integer programming (BIP) is adopted to obtain the locations of BPMUs. The proposed method is tested on customized IEEE 33-bus and PG&E 69-bus distribution network, and the results are compared with those considering other methods.
Abstract Background: Transfer RNA-derived small RNAs (tsRNAs) are a recently discovered form of non-coding RNA capable of regulating myriad physiological processes. The role of tsRNAs in hMSC adipogenic differentiation, however, remains incompletely understood. Methods: In this study we conducted transcriptomic sequencing of hMSCs after inducing their adipogenic differentiation, and we were thereby able to identify tsRNA-16902 as a tsRNA that was potentially differentially regulated in this context by a series of molecular biology methods. Results: When we knocked down tsRNA-16902 expression, this impaired hMSC adipogenic differentiation and associated marker gene expression. Bioinformatics analyses further revealed tsRNA-16902 to target retinoic acid receptor γ (RARγ). Luciferase reporter assays also confirmed the ability of tsRNA-16902 to bind to the RARγ 3’-untranslated region. Consistent with this, RARγ overexpression led to impaired hMSC adipogenesis. Further analyses additionally revealed that Smad2/3 phosphorylation as increased in cells that either overexpressed RARγ or in which tsRNA-16902 had been knocked down. We also assessed the adipogenic differentiation of hMSCs in which tsRNA-16902 was knocked down and at the same time as Smad2/3 inhibitor was added to disrupt Smad2/3 phosphorylation. The adipogenic differentiation of hMSCs in which tsRNA-16902 was knocked down was further enhanced upon the addition of a Smad2/3 signaling inhibitor relative to tsRNA-16902 knockdown alone. Conclusions: Through a comprehensive profiling analysis of tsRNAs that were differentially expressed in the context of hMSC adipogenic differentiation, we were able to identify tsRNA-16902 as a previously uncharacterized regulator of adipogenesis. tsRNA-16902 is able to regulate hMSC adipogenic differentiation by targeting RARγ via the Smad2/3 signaling pathway. Together our results may thus highlight novel strategies of value for treating obesity.
Chemokine (C-C motif) receptor 5 (CCR5) is one of the major co-receptors for the macrophage (M)-tropic HIV-1. To prevent HIV-1 from entering into target cells, we inhibited CCR5 expression on target cell surface by recombinant adenovirus containing anti-sense CCR5 cDNA. A fragment of 653 bp cDNA located in the 5' region of CCR5 cDNA was reversely inserted into pAdTrack-CMV. Recombinant adenovirus containing antisense CCR5 cDNA (Ad-antiR5) was obtained by homologous recombination of resultant plasmid with the adenoviral backbone plasmid pAdEasy-2 in E. coli BJ5183 and then packed in AD-293 cells. Rate of positive CCR5 on U937 cell surface measured by flow cytometry was decreased from 89.53% to 1.88% after U937 cells infected with Ad-antiR5 for 24 hours, and this reduction lasted at least for 10 days. After challenged with HIV-1, the U937 cells infected with Ad-antiR5 produced much less p24 antigen in cultured medium than those infected with control recombinant adenovirus and the uninfected cells. The recombinant adenovirus had no effect on chemotactic activity and proliferation of the U937 cells. Therefore, the recombinant adenovirus containing anti-sense CCR5 cDNA can down-regulate CCR5 expression on U937 cells and protect the cells from HIV-1 infection without effects on their chemotaxis activity and proliferation function.
The ubiquitin-specific protease 22 (USP22) is an oncogene and its expression is upregulated in many types of cancer. In the nucleus, USP22 functions as one subunit of the SAGA to regulate gene transcription. However, the genome-wide USP22 binding sites and its direct target genes are yet clear. In this study, we characterized the potential genomic binding sites of UPS22 and GCN5 by ChIP-seq using specific antibodies in HeLa cells. There were 408 overlapping putative target genes bound by both USP22 and GCN5. Motif analysis showed that the sequences bound by USP22 and GCN5 shared two common motifs. Gene ontology (GO) and pathway analysis indicated that the genes targeted by USP22 and GCN5 were involved in different physiological processes and pathways. Further RNA-seq, GO and pathway analyses revealed that knockdown of UPS22 induced differential expression of many genes that participated in diverse physiological processes, such as metabolic process. Integration of ChIP-seq and RNA-seq data revealed that UPS22 bound to the promoters of 56 genes. These findings may provide new insights into the regulation of USP22 on gene expression during the development of cervical cancer.
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