Objective: To investigate the antitumor mechanism of EM-3, a ethanol extracts from Elephantopus mollis H.B.K, in nasopharyngeal carcinoma. Methods: The effects of EM-3 on the cell proliferative and migratory activity of CNE2, CNE2-S18 and L02 cells were detected by MTT assay, colony formation assay and cell scratch assay. Cell apoptosis was evaluated by Annexin V-FITC/PI double staining. Cell cycle was evaluated by PI single staining. The percentage of SP cells from CNE2-S18 cells was analyzed by fluorescence-activated cell sorting (FACS) assay. Expressions of apoptosis-relative proteins, cycle-relative, migration-relative proteins and cancer stem cells related proteins in CNE2 cells were measured by Western blotting. Result: The proliferation and migration of nasopharyngeal carcinoma was inhibited by EM-3 in a dose and time-dependent manner. Colony formation assays showed that EM-3 decreased colony formation compared with control. Flow cytometry analysis showed an increase of the percentage of apoptotic cells and G2/M phase in a dose-dependent manner treated with EM-3. FACS assays demonstrated that EM-3 decreased the percentage of CNE2-S18 stem-like SP cells. EM-3 downregulated the expression of xIAP, Bcl-2, Cyclin D1, MMP2(high drug concentration), MMP9, p-Met, Oct4(high drug concentration) and Sox2 with different concentrations. In addition, active bands of Caspase-9, Caspase-3 and PARP could be detected using Western blotting. But the expression of Cyclin B1 and Bax was upregulated. Conclusions: EM-3 induces the apoptosis and G2/M cycle arrest by down-regulation of Stat3 signaling pathway. In addition, EM-3 inhibits cell migration by MMPS pathway and can effectively reduce the maintenance of CNE2-S18 stem-like SP cells.
Peroxiredoxin6 (Prdx6) is a bifunctional protein that includes glutathione peroxidase and calcium-independent phospholipase A2. A SSH cDNA library of buffy coat from Ovis aries was constructed through infecting with virulent and avirulent strains of Brucella, and a differentially expressed Prdx6 was cloned.It indicated that Prdx6 might take part in the infection of brucella. In order to study the Prdx6 protein function, here total RNAs from the mice macrophage cell line Raw 264.7 were extracted, cDNAs were reverse transcribed, and the open read frame (ORF) of mouse Prdx6 gene was cloned using the specific primes by means of RT-PCR. Then, the prokaryotic expression vector of Prdx6 was constructed, and the recombinant Prdx6 protein with a His tag was expressed by induction of IPTG, and purified by affinity chromatography of Ni-NTA. A specific polyclonal antibody against mouse Prdx6 was obtained by immunizing a rabbit with the purified Prdx6, which would benefit the further study on the functions of Prdx6 protein in the future.
Objective:To investigate the effect on lipolysis of 3T3-L1 adipocytes by down-regulating Perilipin 1 gene expression. Methods: Three shRNA recombinant vectors targeting mouse PLIN1 gene and one negative control were designed, synthesized and detected by bacterium liquid PCR reaction and DNA sequencing. Western blot analysis was performed to verify the transfection effects.Bodipy 493/503 staining was used to observe the lipid droplets size, and the content of cellular triglyceride and glycerol were detected after transfection of adipocytes. The adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and phosphorylase HSL (p-HSL) were detected by Western blot, and the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) were measured by enzyme linked immunosorbent assay (ELISA). Results: Every recombinant vector was constructed successfully, and the three positive vectors could significantly inhibit the protein expression of PLIN1A (P<0.05). Knockdown of PLIN1 gene expression decreased lipiddroplets size and cellular triglyceride level and increased glycerol level. In addition, the level of ATGL and HSL was increased significantly (P<0.05), and the level of cellular cAMP and PKA had no obvious change (P>0.05). Conclusion: Down-regulating PLIN1 gene could promote lipolysis in 3T3-L1 adipocytes by up-regulating the level of ATGL and HSL, and cAMP-PKA signaling pathway had no significant effect on it.
Objective: Providing a theoretical basis for the further study on the mechanism of miR-335 in cancer, bioinformatics was utilized to investigate miR-335 expression in tumor tissues, and the functions of miR-335 predicted target genes were analyzed as well. Methods: Sequence alignment of miR-335 in multiple species was used to analyze its conservation. The miRNA microarray data of tumor tissues were applied for detecting miR-335 expression level between the tumor and normol tissues. Predicting miR-335 target genes by several on-line tools. DAVID was used to analyze the function and signal pathway of miR-335 target genes. Results: The sequence of miR-335 was highly conserved among different species. Compared with the normal tissues, miR-335 was low-expression in the tissues of liver cancer, lung cancer, breast cancer, intrahepatic cholangiocarcinoma and liposarcoma(P<0.05). 34 target genes were predicted, and the function of these target genes mainly enriched in cell division, cell migration, regulation of apoptosis, protein binding, regulate transcription factor activity and protein amino acid phosphorylation(P<0.05). In KEGG pathway, the predicted target genes set was involving in axon guidance signal, melanoma diseases pathway, focal adhesion and TGF-beta signaling pathway(P<0.05). Conclusion: Abnormal gene expression of miR - 335 was discovered in variety of tumor tissues. The target genes of miR-335 closely related to multiple biological processes and signal transduction pathways in cancer.
In order to study the control characteristic of carbon metabolism repressor CRE to the cellulases produced by Rhizopus stolonifer TP-02, a full-length gene cre was extracted from the genomic DNA. An antibiotic resistance gene hygB was inserted into the cre by overlapping PCR, which not only broken the normal transcription of cre but also provided a selective maker for the work follow-up. The pUCm-T-cre-hygB was transformed into the germinal spores of R.stolonifer TP-02 by electroporation, and then a mutant △CRE was screened by the hygromycin resistance. RT-qPCR was utilized to analyze the transcription features of cellulases secreted by the mutant. It was found that the transcription of eg, bg, cbh1 and cbh2 are improved than the wild type, the values are 48.75%, 26%, 5.6% and 38.6%, respectively. Meanwhile, the mutant was preceded in the expression level cellulases, in which the activity of endoglucanases was improved 58.62%. CRE has a specificity in the control of cellulases genes, expecially to the endoglucanase gene eg.Destruction of CRE could weaken the effect of carbon repression. In addition, the mutant △CRE could maintain a high yields of cellulases within the 3% sugar concentration, which may lay a foundation for the subsequent enzyme system optimization and industrial production.
To generate genetic selective marker for constructing super-producing lycopene strains, chemical mutagenesis was applied in mutagenizing Mucor circinelloides MU616. Five uracil auxotroph strains of Mucor circcinelloides were isolated with nitrosoguanidine and selected by resistance to 5-fluoroorotic acid, and these mutants were unable to grow in the minimal medium even after 5 days' culture. Plasmid pEPM1 which harbors the pyrG gene encoding orotidine-5'-monophosphate decarboxylase in Mucor circinelloides were used to transformed the representative uracil auxotroph strains, the result demonstrated that mutants Mt1, Mt4 and Mt5 were deficient in pyrG gene. However, the lycopene content analysis shown that only Mt4 maintained the capacity of accumulating lycopene[(1 648±185)μg/g and (3 234±281)μg/g under dark and light conditions respectively], in addition, the biomass[(9.0±0.6)g/L] and growth properties of Mt4 is also similar to the original strain MU616. It suggested that Mt4 could be used as the receipt strain for the subsequent molecular manipulation. The acquisition of the pyrG mutant strain is very important for construction of the super-producing lycopene strains by molecular genetic manipulation.
A gene lipaseB5 encoding a lipase, was cloned from the genome of Pseudonocardia antitumoralis SCSIO 01299 isolated from the deep sea of the South China Sea. LipaseB5 contains 972bp and encodes a putative lipase of 323 amino acids. LipaseB5 exhibits 98% similarity with a putative lipase from Pseudonocardia sp. HH130629-09. LipaseB5 was cloned into expression vector pET28a(+), expressed in E.coli BL21(DE3) and further purified using Ni-NTA affinity chromatography. The optimal substrate tested of lipaseB5 was p-NPD, the optimal working temperature was 30℃, the optimal working pH was 7.5. Vmax and Km were 109.8μmol/min and 0.976mmol/L, respectively. The hydrolysis activity of lipaseB5 toward olive oil was 32.019 7U/mg. LipaseB5 could remain high activities under pH 7.5~8.5 and 10~20℃. LipaseB5 was thermal-stable at 10~40℃. Multiple metal ions could not affect the activities of LipaseB5 and low-concentration Li+ and Mg2+ could stimulate the activity of LipaseB5. LipaseB5 could stand high concentration of NaCl and could still remain 103% of its original activity in the presence of 100mmol/L NaCl. LipaseB5 could also stand many short-chain alcohols and surfactants. TritonX-100, Tween-80 and Tween-20 could even stimulate the activities of LipaseB5.
The nucleosomes constitute the basic structural unit of eukaryotic chromatin. The variant H2A.Z and H3.3, both of which are highly conserved evolutionarily, have been proposed to play crucial and specific roles in the regulation of chromatin dynamics and transcription. In vivo eukaryotic nucleosome formation will be subject to interference from a variety of factors, and in vitro assembling nucleosome structure by DNA and histone variants H2A.Z and H3.3 is one of basic method to study the nucleosome positioning and chromatin structure. The H2A, H2B, H3, H4, H2A.Z and H3.3 histones have been expressed and assembled to form octamer containing histone variants. Based on 10 base periodicity and sequence motif on nucleosome DNA, CS1, CS2 and CS3, which could have high affinity to histones, was designed to assembly the nucleosome structure. Then the DNA sequences labeling Cy3 are obtained through using PCR and gel extraction method. After a salt dilution method employing to assembly nucleosome, reconstituted nucleosome was analyzed by Cy3 labeling, EB staining and Coomassie blue staining. The results show that the nucleosome structure can be effectively reconstituted in vitro and the formation efficiency of nucleosome was positively related to the ratio of octamer to DNA. The quantitative result by Cy3 labeling can be used to compute the Gibbs free energy of the reconstituted assay. The work laid the foundation for further study of epigenetic and structural biology associated with histone variant H2A.Z and H3.3.
Objective: To investigate cold-adapatation mechanisms of outer membrane protein folding of psychrotrophic Gram-negative bacterium by constructing the in vitro refolding system. Methods: The outer membrane protein Omp74 from a psychrotrophic bacterial Shewanella sp. was overexpressed as inclusion body in E. coli, purified, dissolved in surfactant or high concentrations of urea, and refolded with non-ionic surfactant. OmpA from E. coli was used as a control from mesotrophic bacteria. Results: Being different from OmpA, the refolding of Omp74 was not sensitive to temperature, and low concentration of sarkosyl facilitated the folding of wild type Omp74 but showed no effect on OmpA folding; C-terminal periplasmic domain inhibited the folding; complete refolding this recombinant OMP in vitro in 1%DDM mixed with 0.4% sarkosyl.
A novel method for direct determining the broth methanol concentration online from the fermentation exhausted gas with electronic nose sensor, established an auto-feedback model for methanol concentration control during glucoamylase fermentation by genetic Pichia pastoris were investigated, the model could be used for precise control in the fermentation process. Methanol concentration controlling strategies through electronic nose showed that when the broth methanol concentration in stability controlled at (890±35)ppm, the highest glucoamylase activity reached 8 153U/ml at 128h of fermentation, which was 48.8% higher than that with the methanol concentration controlled at (350±26)ppm. Online methanol detection and feedback control models needn't pre-treatment and contact with the broth, has the rapid and accuracy advantages. It will be approved as an effective and important role on the online methanol concentration analysis and feedback control on industrial enzyme or aim protein fermentation with genetic Pichia pastoris.
Recombinant antibodies have good prospects in anti-tumor, anti-autoimmune disease and other fields, the market demand is increasing year by year. However, the study of recombinant antibody are facing with many problems, such as the low yields and poor stability. The recombinant antibody was often expressed in CHO cell as a secret protein, for which the signal peptide is very important. The signal peptide is an amino acid sequence that attach to the N-terminus of immature protein, it can affect the expression of protein by controlling the secretory pathway. The structure, mechanism of the signal peptide andits functions on the recombinant protein secretion pathway were briefly outlined. In addition, the optimizing strategy of signal peptide was summarized based on a variety of researches.
Fibroblast growth factor-17 (FGF17) is one of the fibroblast growth factor (FGF) family members and it makes up FGF8 subfamily along with FGF8 and FGF18. It is an important role in embryo and acts predominantly on multiple tissues and organs. Current evidence suggests that FGF17 is not only involved in the development of brain and neurogenesis, but also participate in many other biological processes, including developmental skeleton and arteries and cancer. The following review summarizes the current knowledge on FGF17 with special emphasis on its characteristics and functions on development of embryo, nervous system, cancer and so on.
TCR genes adoptive therapy is a clinical promise approach for the treatment of cancer. Using ex vivo gene transfer, T cells isolated from patients can be genetically engineered to express a novel TCR, and then the engineered T cells re-infuse back into the patient to specifically recognize a tumor-associated antigen and thereby selectively kill tumor cells. One of the current challenges of TCR gene therapy is the optimization of TCR α and β transgene pairing to enhance the functional avidity of therapeutic T cells and avoid the off-target toxicity. Recently, various genetically modified TCRs have been developed that enhance TCR pairing and minimize mispairing. The strategies of optimization the transgenic TCR pairing and the clinical trials with TCR gene therapy were summarized.
The tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family was originally identified as signaling adaptors that directly bind to the cytoplasmic regions of receptors of the TNF-R superfamily. TRAFs have also been identified to function in signaling for TLRs, NLRs, RLRs, etc. TRAF7, the most recently identified member, acts as an E3 ubiquitin ligase with its conserved RING finger domain, essential for signal transduction pathways mediated by TNFR and TLR2. In addition, TRAF7 also regulates the activation of cellularstress pathways, as well as unconventional ubiquitination events, the differentiation of muscle tissue and tumorigenesis. The most recent advances in the understanding of TRAF7 function and the biological processes this protein is involved in.
Hemicellulose is an abundant and renewable plant biomass yet untapped utilization. The bioconversion of hemicellulose to sugars is essential in the production of xylitol and other chemicals. Acetyl xylan esterase is the key enzyme in hydrolyzing hemicellulose, which can deacetylate on the O-2 and O-3 positions of xylopyranosyl residue. It has broad application prospect in agriculture and industry. The recent research progresses on catalysis mechanism, structure, characterization, gene cloning and synergetic hydrolysis of acetyl xylan esterase were addressed.
Industrial fungi are economically important microorganisms used for enzyme production. Strain improvements towards these organisms have great application value. The application of the fungi for enzyme industry, the development of mutagenesis breeding technologies as well as the mechanisms for high-efficiency enzyme production, mostly derived from the genome-level analysis including genomics, transcriptomics and proteomics are introduced. The recent application of mutagenesis mechanisms for strain improvement also be summarized.
