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

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.

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.

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.

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.

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.

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

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.

Exploring the developmental potential of adult mammalian somatic cells

In the traditional views on developmental biology, the process of a mammal from a zygote to. an adult individual follows continuous changes of space and time environments and is the result of different expressions of target genes. It has long been known that this process is irreversible and the terminal differentiated adult cells, such as cardiac myocytes and neurons, will not divide and differentiate. But recent reports on the two hottest fields - cloning medicine and stem cell biology doubted these concepts. This may lead to a further understanding of the potentiality of mammal development and may provide great chances for commercial and clinical practice.

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.

TRANSCRIPTION ACTIVATION BY HBV PRE SI PROTEIN

L One or more hepatitis B virus(HBV)gene products may contribute to hepatocarcinogenesis.Whether Pre S1 protein of HBV involves this process is not known.Hybrid proteins that contain the DNA binding domain of yeast GAL4 and full-length or portions of pre S1 sequence have been used to show that the pre S1 protein contains a transcription-activating sequence that functions in yeast. The domain of pre S1 protein responsible for transcription activation is located between residues 12 and 71.Experiments on transcription activation by pre S1 protein in mammalian cells are in progress.The preliminary results indicate that transcription activation by pre S1 protein is a possible mechanism for hepatocarcinogenesis.

Temperature effect on CRISPR-Cas9 mediated genome editing

Zinc-finger nuclease（ZFN）, transcription activator-like effector nuclease（TALEN）, and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9（CRISPR-Cas9） are the most commonly used genome editing tools. Previous studies demonstrated that hypothermia treatment increased the mutation rates induced by ZFNs and TALENs in mammalian cells. Here, we characterize the effect of different culture temperatures on CRISPR-Cas9 mediated genome editing and find that the genome editing efficiency of CRISPR-Cas9 is significantly hampered by hypothermia treatment, unlike ZFN and TALEN. In addition, hyperthermia culture condition enhances genome editing by CRISPR-Cas9 in some cell lines, due to the higher enzyme activity and sg RNA expression level at higher temperature. Our study has implications on CRISPR-Cas9 applications in a broad spectrum of species, many of which do not live at 37 C.

Interrogating genome function using CRISPR tools: a narrative review

Genome editing serves as a powerful approach to interrogate the functions of both coding and noncoding sequences.In particular,clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR associated protein 9(Cas9)system-based editing tools have revolutionized the way we study genome function in mammalian cells,and are being widely used for interrogating critical genes and DNA elements essential for many biological processes.Here,we review CRISPR/Cas9-based genetic tools with an emphasis on CRISPR-mediated high throughput genetic screens in the mammalian genome.

CHO cell cytotoxicity and genotoxicity analyses of disinfection by-products： An updated review

The disinfection of drinking water is an important public health service that generates high quality, safe and palatable tap water. The disinfection of drinking water to reduce waterborne disease was an outstanding public health achievement of the 20 th century.An unintended consequence is the reaction of disinfectants with natural organic matter,anthropogenic contaminants and bromide/iodide to form disinfection by-products（DBPs）.A large number of DBPs are cytotoxic, neurotoxic, mutagenic, genotoxic, carcinogenic and teratogenic. Epidemiological studies demonstrated low but significant associations between disinfected drinking water and adverse health effects. The distribution of DBPs in disinfected waters has been well defined by advances in high precision analytical chemistry. Progress in the analytical biology and toxicology of DBPs has been forthcoming.The objective of this review was to provide a detailed presentation of the methodology for the quantitative, comparative analyses on the induction of cytotoxicity and genotoxicity of103 DBPs using an identical analytical biological platform and endpoints. A single Chinese hamster ovary cell line was employed in the assays. The data presented are derived from papers published in the literature as well as additional new data and represent the largest direct quantitative comparison on the toxic potency of both regulated and emerging DBPs.These data may form the foundation of novel research to define the major forcing agents of DBP-mediated toxicity in disinfected water and may play an important role in achieving the goal of making safe drinking water better.

Investigation of nuclear enzyme topoisomerase as a putative molecular target of monohaloacetonitrile disinfection by-products

Disinfection by-products occur widely as the unintended effect of water disinfection and are associated with toxicity and adverse human health effects. Yet the molecular mechanisms of their toxicity are not well understood. To investigate the molecular basis of hyperploidy induction by monohaloacetonitriles, the interaction of monohaloacetonitriles with topoisomerase Ⅱ in Chinese hamster ovary cells was examined. We showed a concentration-dependent inhibition of DNA decatenation activity of topoisomerase under acellular conditions while in vitro monohaloacetonitrile treatment expressed mixed results. The working hypothesis, that topoisomerase Ⅱ is a molecular target of monohaloacetonitriles, was only partially supported.Nevertheless, this research serves as a starting point toward molecular mechanisms of toxic action of monohaloacetonitriles.

Engineering synthetic optogenetic networks for biomedical applications

Background： Recently, optogenetics based on genetically encoded photosensitive proteins has emerged as an innovative technology platform to revolutionize manipulation of cellular behavior through fight stimulation. It has enabled user defined control of various cellular behaviors with spatiotemporal precision and minimal invasiveness, creating unprecedented opportunities for biomedical applications. Results： This article reviews current advances in optogenetic networks designed for the treatment of human diseases. We highlight the advantages of these optogenetic networks, as well as emerging questions and future perspectives. Conclusions： Various optogenetic systems have been engineered to control biological processes at all levels using light and applied for numerous diseases, such as metabolic disorders, cancer, and immune diseases. Continued development of optogenetic modules will be necessary to precisely control of gene expression magnitude towards clinical medical practice in the context of real-world problems.