Cytotoxicity of Captafol in Mammalian Cells

The cytotoxicity of captafol, a phthalimide-derived fungicide, was evaluated in IB-RS-2 cells. Captafol at 0. 12-1 .0μg/ml blocks the cell multiplication. This effect is concentrationdependent, only partially rcversible and the degree of inhibition increases with time. The synthesis of DNA and RNA is inhibited in parallel by increasing concentrations of the chemical

Expression of Recombinant Baculovirus Carrying Schistosoma japonicum 26 ku GST in Mammalian Cells

In order to construct recombinant baculovirus carrying Schistosoma japonicum 26 ku glutathione S-transferase gene （Sj26）, and observe the expression of Sj26 in mammalian cells, the Sj26 gene was amplified with plasmid pGEX-3X as template by PCR, and then recombined into T vector for sequencing. Sj26 gene was inserted into the downstream of CMV promoter of donor plasmid pFBDGC, and the recombinant donor plasmid pFBDGC-Sj26 transformed into DH10Bac, then the recombinant bacmid AcCMVSj26 was isolated and transfected into Sf9 cells, The recombinant baculovirus was harvested and final titer of vAcCMVSj26 was measured. BHK cells were transducted with recombinant baculovirus in vitro, By using Western blot, the expression of 26 ku glutathione S-transferase （GST） was detected. The results showed that after enzyme digestion and sequencing, the donor plasmid was successfully constructed. PCR confirmed that pFBDGC-Sj26 and Bacmid homologous recombination occurred in E. coli. After transfection of Sf9 cells with recombinant Bacmid, recombinant baculovirus was replicated in Sf9 cells and expressed green fluorescent protein. PCR further revealed recombinant baculovirus contained Sj26. The titer of the harvested baculovirus was 1.24×10^8. Western blot demonstrated that recombinant baculovirus could express 26 ku GST in BHK cells, It was concluded that Sj26 recombinant baculovirus was successfully constructed, and the 26 ku GST was expressed in mammalian cells.

Research Progress on the Large-scale Culture Technology of Mammalian Cells

The culture of mammalian cells is closely related to the development of biotechnology, which has been used extensively in the research and application fields of biology and medical science. In this article, various factors affecting cell cultivation and the application of microcarrier and bioreactor on large-scale culture of mammalian ceils were reviewed.

Localization and Functional Analysis of SeMNPV IE1 in Mammalian Cells

In this paper, the function of the iel gene from baculovirus Spodoptera exigua multiple nucleopolyhedrovirus （SeMNPV）, belonging to group II nucleopolyhedrovirus, was studied in mammalian cells We amplified the SeMNPV iel gene and expressed it by fusing to the C terminal of enhanced GFP protein in HEK 293 cells. Confocal microscopy revealed that the IE1-GFP fusion protein was localized in the nucleus of the mammalian cells. The promoter sequences of AcMNPV gp64, SeMNPV F protein and Drosophila hsp70 were also analyzed, to further study the function of SeMNPV IE1. The results showed that, in the absence of the hr sequence, IE1 improved the expression of the F promoter but didn＇t influence the gp64 promoter significantly, but IE1 moderately stimulated the hsp70 promoter.

Construction and identification of a vector expressing TRAM siRNA in mammalian cells

Objective:To construct and identify a vector expressing TRAM siRNA in mammalian cells.Methods:It was constructed that the vector named R-pSUPER-EGFP used to transcribe functional TRAM siRNA. Two of pair 64 nt TRAM gene specific target sequences were inserted into the downstream of the H1 promoter. The recombinants were transformed into E. coli JM109, and finally their veracity was confirmed by double cutting with the enzymes and sequencing. R-pSUPER-EGFP was transfected into RAW264.7 cell by using Lipofectamine TM2000, and the expression of TRAM was detected by Western blotting. Results:Two different recombinant plasmids containing corresponding TRAM gene specific target sequences were constructed, transfected into RAW264.7 cell line successively, which can specifically restrain expression of TRAM protein. Conclusion:The optimizing method in constructing the recombinant vector serves other plasmid-based RNA interference research. Therefore, the recombinant vectors establish the basis for research on the function of TRAM gene.

Lactate Dehydrogenase Isoenzymes in Mammalian Cells Exposed to Isophenphos

The cytotoalcity of isophenphos, an organophosphorus insecticide that has the potential to cause delayed polyneuropathy, was evaluated in GBK and V79 ce1ls. A 72 h time course following isophenphos exposure indicated a dose-dependent growth inhibition as dctermined by cell counts. The administration of isophenphos (20g/ml) to GBK cells cultured at high densities indicated a decrease in the activities of LDH isocnzymes. Analysis of V79 cells revealed a decrease of LDH3, the only LDH isocnzyme detected in these cells.

Comparison of transient and stable expression of foot-and-mouth disease virus capsid proteins in mammalian cells

Foot-and-mouth disease is a highly contagious disease that produces severe economic losses in the livestock industry. This disease is being controlled by the use of an inactivated vaccine. However, the use of recombinant empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production. A plasmid containing the capsid precursor P12A and protease 3C sequences of foot-and-mouth disease virus (FMDV) was constructed and used to compare transient and stable expression in mammalian cells. When BHK-21 cells were transfected with the recombinant vector, protease 3C cleaved the capsid precursor P12A into the structural proteins VP0, VP1 and VP3. A sucrose gradient demonstrated that the structural proteins assembled into different subviral particles. Attempts to generate a stable cell line only allowed isolating low-level-expressing clones, probably due to the effect of protease 3C on the cells. Moreover, the recombinant protein yield achieved in transient expression assays was much higher than the one achieved in stable expression assays. Results indicate that mammalian cells are a good strategy to produce recombinant FMDV subviral particles. However, the alternative approach of transient gene expression in scalable systems should be used instead of the standard method that involves the generation of a stable cell line.

Relationship between cellular radio-sensitivity and naked DNA damage in mammalian cells exposed to heavy ions

The relationship between deoxyribonucleic acid (DNA) damage and the cell death induced by 12C ions irradiation was examined in four kinds of cells, Melanoma B16, cervical squamous carcinoma HeLa, Chinese hamster V79 and hepatoma SMMC-7721. Cell survival was determined by a colonogenic assay, and the sensitivity was described by D50 (the dose of radiation necessary to reduce the survival to 50%). For all cell lines, D50 ranged from 0.74 Gy to 3.85 Gy, among them B16 was the most radiosensitive to 12C ions, and V79 and HeLa cells had almost the same radio-sensitivity, SMMC-7721 was the last. The induction of deproteinized DNA double-strand breaks induced by 12C ions were measured by pulsed-field gel electrophoresis (PFGE), and the initial yield of the deproteinized DNA dsbs per unit dose was expressed as the DNA double break level (L). A linear dose-response curve was seen for initial DNA dsbs for all cell lines (slopes range from 0.40-0.98 (DSBs/100Mbp/Gy)). V79 was the steepest, B16 was the last. There was an inverse relationship between the initial DNA dsb and D50 if the B16 cell line was not considered, but there was no relativity even excludes the B16 cell line. The present results indicate that there is no relationship between cellular sensitivity and initial DNA dsb, even exclude the effects of chromatin structure.

Low dose effects on cultured mammalian cells...

The low dose effects induced by carbon ions on Chinese hamster V79 cells and murine melanoma B16 cells were investigated in this paper. Both cell lines were divided into four groups for irradiation: (1) control, (2) 0.02 Gy or 0.05 Gy(D1), (3) 1 Gy(D2), (4) D1+D2. The survivors and micronuclei were studied as biological endpoints. The results of group (1) and group (2) showed that there were no obvious differences on micronucleus frequency but there were significant increases when irradiation dose was 0.02Gy on colony formation efficiency. Although low dose ion irradiation could not contribute to DNA damages, it could enhance the colony formation efficiency. In the study of group (3) and (4), when the ion dose was 0.02 Gy, there were evident increases on surviving fraction and decreases on micronucleus frequency, but there were no statistical changes on these endpoints when the ion dose was 0.05Gy. This meant that high LET radiation could induce the adaptive response of cultured cells, furthermore, in the range of inducing ion dose , low dose irradiation was more profitable than high dose one.

High-level expression, purification and characterization of codon-optimized recombinant hemagglutinin 5 proteins in mammalian cells

Background Numerous Asian cases of avian influenza virus infection, especially the highly pathogenic strain H5N1, in humans have raised the concern that another influenza pandemic is close. However, there are no effective therapeutic drugs or preventative vaccines available. Hemagglutinin is the membrane glycoprotein of avian influenza virus responsible for receptor binding to human cells and the main immunogenic protein that elicits a strong immune response. Although this protein is of great importance to the study of pathogenesis and vaccine development, its expression and purification are difficult due to high levels of glycosylation. Methods In this study, we expressed codon-optimized, full-length hemagglutinin 5 （H5） protein fused with a human IgG Fc tag （H5-Fc） in HEK293 cells. To enhance secretion of this protein, we also deleted the transmembrane domain and the intracellular domain of the H5 protein （H5ATM-Fc）. Purified proteins were obtained using a protein A column. Results ELISA revealed that the yield of soluble H5ATM-Fc protein in the supernatant was about 20 mg/L. Western blotting and fluorescence activated cell sorter （FACS） indicated that the purified H5 protein was correctly folded and biologically active. Conclusion Purification of H5 proteins from mammalian cells could be used for large-scale production of recombinant H5 protein for basic scientific research or the development of vaccines.

Improving Baculovirus Transduction of Mammalian Cells by Incorporation of Thogotovirus Glycoproteins

Baculovirus can transduce a wide range of mammalian cells and is considered a promising gene therapy vector. However,the low transduction efficiency of baculovirus into many mammalian cells limits its practical application. Co-expressing heterologous viral glycoproteins(GPs), such as vesicular stomatitis virus G protein(VSV G), with baculovirus native envelope protein GP64 is one of the feasible strategies for improving virus transduction. Tick-borne thogotoviruses infect mammals and their GPs share sequence/structure homology and common evolutionary origins with baculovirus GP64.Herein, we tested whether thogotovirus GPs could facilitate the entry of the prototype baculovirus Autographa californica multiple multiple nucleopolyhedrovirus(AcMNPV) into mammalian cells. The gp genes of two thogotoviruses, Thogoto virus and Dhori virus, were inserted into the AcMNPV genome. Both GPs were properly expressed and incorporated into the envelope of the recombinant AcMNPVs. The transduction rates of recombinant AcMNPVs expressing the two thogotovirus GPs increased for approximately 4–12 fold compared to the wild type AcMNPV in six of the 12 tested mammalian cell lines. It seemed that thogotovirus GPs provide the recombinant AcMNPVs with different cell tropisms and showed better performance in several mammalian cells compared to VSV G incorporated AcMNPV. Further studies showed that the improved transduction was a result of augmented virus-endosome fusion and endosome escaping, rather than increased cell binding or internalization. We found the AcMNPV envelope protein GP64-mediated fusion was enhanced by the thogotovirus GPs at relatively higher p H conditions. Therefore, the thogotovirus GPs represent novel candidates to improve baculovirus-based gene delivery vectors.

GroupⅠbut not GroupⅡNPV induces antiviral effects in mammalian cells

Nucleopolyhedrovirus(NPV) is divided into Group I and Group II based on the phy-logenetic analysis.It has been reported that Group I NPVs such as Autographa californica multiple NPV(AcMNPV) can transduce mammalian cells,while Group II NPVs such as Helicoverpa armigera single NPV(HaSNPV) cannot.Here we report that AcMNPV was capable of stimulating antiviral ac-tivity in human hepatoma cells(SMMC-7721) manifested by inhibition of Vesicular Stomatitis virus(VSV) replication.In contrast,the HaSNPV and the Spodoptera exigua multiple NPV(SeMNPV) of group II had no inhibitory effect on VSV.Recombinant AcMNPV was shown to induce interferons al-pha/beta even in the absence of transgene expression in human SMMC-7721 cells,while it mediated transgene expression in BHK and L929 mammalian cells without an ensuing antiviral activity.

Metallothionein Genes in the Nematode Caenorhabditis elegans and Metal Inducibility in Mammalian Culture Cells

Genomic DNAs of metallothionein Ⅰ and Ⅱ in Caenorhabditis elegans (CeMT-Ⅰand CeMT-Ⅱ) were isolated by YAC library/polytene filter hybridization followed by subcloning of corresponding cosmid clones. Both genes are mapped at chromosome V. Although the similarities of 5'-flanking regions and coding regions have shown only 55-58%, the introns are split at the same position in both genes, indicating that these two genes are originally from the same gene. While several metal responsive elements are conserved among eukaryotes, only one metal responsive element was found in the promoter region in CeMT-Ⅱ and not in CeMT-Ⅰ. Indced, neither of 5'-flanking regions of CeMT-Ⅰ nor CeMT-Ⅱ connected to chloramphenicol acetyltransferase reporter gene is responsive to heavy metals in mammalian culture cells by transient transfection analysis. These results would suggest that the metal regulatory factors in C.elegans might be different from those conserved in invertebrates and vertebrates, although the MTs in C elegans revealed the similarities to mammalian MTs in several points

Baculovirus-mediated gene transfer into mammalian cells

A kind of cytotoxinic gene barnase was cloned downstream of immediate early promoter from cytomegalovirus (CMV). Recombinant virus vAcCMVBar was constructed using Bac\|to\|Bac system by insertion of the expression cassette into the polyhedrin gene locus of Autographa californica nuclear polyhedrosis virus (AcNPV). Cytotoxinic effect caused by expression of barnase was observed in four mammalian cells after vAcCMVBar infection. Dose\|dependent analysis revealed that one of the cells HICAM was the most sensitive to the virus infection. It was shown that AcNPV could be used as gene transfer vector in mammalian cells and that barnase is the suitable report gene which could be detected more easily than that commonly used.

Co-genotoxic Effects of Diesel Particulate Extract and Ultraviolet A Radiation in Mammalian Cells

Diesel exhaust (Diesel exhaust particles, DEPs, and their extracts, DPE) and ultraviolet A are two ubiquitous environmental factors that have been identified as essential risk factors

A Genetically Encoded Toolkit for Probing the Post Translational Modifications of Lysine in Mammalian Cells

Post translational modifications (PTMs) of lysine play a crucial role on modulating the activity and stability of essential proteins in eukaryotic cells including the tumor

Secretory expression of the fusion protein composed of human IL-2 and PreS antigen of hepatitis B virus in mammalian cells

In order to make entire HBV preSAg secrete from mammalian cells, we constructed an eukaryotic expression vector by using leader sequence of human interleukin-2 (IL-2) as secretory signal peptide, and using high hydrophilic amino acids as the linker between IL-2 C end and preSAg N end. As a result, the IL-2preS fusing protein could be secreted from mammalian cells transfected with the reconstructed vector and the expression efficiency was identical to that of natural IL-2. It was considered that the retentive effect of preS1Ag could be successfully bypassed. The results not only laid a theoretical and practical foundation for constructing specific gene vaccine against HBV persistent infection, but also supplied experimental evidence for studying modulation of protein secretory expression.

Efficient expression of histidine-tagged large hepatitis delta antigen in baculovirus-transduced baby hamster kidney cells

AIM： To study the baculovirus/mammalian cell system for efficient expression of functional large hepatitis delta antigen （L-HDAg）. METHODS： A recombinant baculovirus expressing histidine-tagged L-HDAg （L-HDAgH） was constructed to transduce baby hamster kidney （BHK） cells by a simplified transduction protocol. RESULTS： The recombinant baculovirus transduced BHK cells with efficiencies higher than 90% as determined by flow cytometry. The expression level was significantly higher than that obtained by plasmid transfection and was further enhanced 3-fold to around 19 pg/cell by the addition of 10 mmol/L sodium butyrate. Importantly, the expressed L-HDAgH was localized to the cell nucleus and correctly isoprenylated as determined by immunofluorescence labeling and confocal microscopy. Moreover, L-HDAgH interacted with hepatitis B surface antigen to form virus-like particles. CONCLUSION： The fusion with histidine tags as well as overexpression of L-HDAgH in the baculovirus-transduced BHK cells does not impair the biological functions. Taken together, the baculovirus/mammalian cell system offers an attractive alternative for high level expression of L-HDAgH or other proteins that require extensive posttranslational modifications.

Evaluation of diffusion in gel entrapment cell culture within hollow fibers

AIM: To investigate diffusion in mammalian cell culture by gel entrapment within hollow fibers. METHODS: Freshly isolated rat hepatocytes or human oral epidermoid carcinoma (KB) cells were entrapped in type I collagen solutions and statically cultured inside microporous and ultrafiltration hollow fibers. During the culture time collagen gel contraction, cell viability and specific function were assessed. Effective diffusion coefficients of glucose in cell-matrix gels were determined by lag time analysis in a diffusion cell. RESULTS: Significant gel contractions occurred in the collagen gels by entrapment of either viable hepatocytes or KB cells. And the gel contraction caused a significant reduction on effective diffusion coefficient of glucose. The cell viability assay of both hepatocytes and KB cells statically cultured in hollow fibers by collagen entrapment further confirmed the existence of the inhibited mass transfer by diffusion. Urea was secreted about 50% more by hepatocytes entrapped in hollow fibers with pore size of 0.1 μm than that in hollow fibers with MWCO of 100 ku. CONCLUSION: Cell-matrix gel and membrane pore size are the two factors relevant to the limited mass transfer by diffusion in such gel entrapment of mammalian cell culture.

High-Affinity Decoy PD-1 Mutant Screened from an Epitope-Specific Cell Library

Immunotherapy with anti-programmed cell death protein-1(PD-1)/programmed cell death ligand-1(PDL1)monoclonal antibodies has become routine in the treatment of many kinds of human cancers,such as lung cancer,intestinal cancer,and melanoma.The PD-1/PD-L1 pathway inhibits T cell activation in the micro-environment,making it an attractive target against cancer.Wild-type(WT)PD-1 ectodomain has been shown to have difficulty blocking PD-1/PD-L1 mixture formation due to its low affinity.The present work uses three-dimensional(3D)crystal complex structures to analyze the interaction by which PD-1 binds to PD-L1 or PD-L2.It also reports on a theoretical study of the binding mode between PD-1 and its clinical antibody Opdivo.Based on the theoretical binding analysis of PD-1 and its ligands(i.e.,PD-L1 and PD-L2)or antibody(Opdivo),a small-content,epitope-oriented mammalian cell library was established for PD-1.After three rounds of cell sorting,the decoy PD-1 mutant 463,which presented a higher affinity than WT PD-1 to the PD-L1(the affinity has increased by almost three orders of magnitude)was screened out.It exhibited an inhibitory effect against PD-1 to prevent it from forming mixtures with PD-L1,which was similar to the effect of the commercial anti-PD-L1 antibody atezolizumab(ATE).The median effective concentration(EC50)value of the decoy mutant was 0.031 μg·mL^(-1) in comparison with 0.063 μg·mL^(-1) for ATE;both values were much lower than that of WT PD-1,at 2.571 μg·mL^(-1).The 463 decoy mutant reversed the inhibitory function of PD-1 in T cell activation;furthermore,10 mg·kg^(-1) of 463 inhibited about 75%of tumor growth in vivo in a MC38 transgenic xenograft mice model,which was similar to the activity of ATE.More interestingly,an even lower dose of 463(2 mg·kg^(-1))showed a better effect than 10 mg·kg^(-1) of WT PD-1.This work offers the decoy 463 with an improved curative effect,which holds potential to become a good option against PD-1/PD-L1-related cancers.