Isolation, purification and antitumor activity of Bacillomycin D from Bacillus amyloliquefaciems Q-426 / 生物工程学报

Cyclic lipopeptide has extensive application prospect in the field of medicine due to its unique chemical structure and biological activity. This study aims to obtain high purity of cyclic lipopeptide monomer from Bacillus amyloliquefaciems strain Q-426, and illuminate preliminary antitumor mechanism of C-15 Bacillomycin D and C-16 Bacillomycin D. Firstly, crude cyclic lipopeptide solution was prepared by two-steps purification of acid precipitation and double-resins chromatography. In order to obtain purer product preparative HPLC was utilized to separate and purify cyclic lipopeptide. Component 1 and component 2 were detected as C-15 Bacillomycin D and C-16 Bacillomycin D by HPLC-MS and ESI-MS/MS. Secondly, the effect of C-15 Bacillomycin D, C-16 Bacillomycin D and their mixture (1:1, mol:mol) on cell proliferation was measured using human cancer cells (Hela, MG, Hep-G2 and HT-29). The cyclic peptide showed a dose dependent manner on the cell proliferation inhibition of Hela and MG cells. Finally, the results of the scratch wound healing assay and FACS analysis revealed that C-16 Bacillomycin D can effectively influence the cells migration and the cells treated with C-16 Bacillomycin D showed typical apoptotic morphology with the increase of drug concentration in the early apoptosis, late apoptosis percentage increased, and G₀G₁ arrest was induced significantly.

Simplification and optimization of the preparation of Escherichia coli extract for cell-free protein expression / 生物工程学报

Cell-free protein expression system is a new method to express target protein in vitro and has been widely applied to the study of protein structure, protein function and other related fields. Preparation of cell extract is one of the key factors that affect the efficiency of the cell-free system. To improve the efficiency and economical feasibility of cell-free protein synthesis, we discussed the parameters during the preparation of the cell extract. These parameters include centrifugation speed, pre-incubation, and dialysis. We used the green fluorescent protein as the reporter protein, and obtained a simple procedure for the preparation of Escherichia coli cell extract. A simple centrifugation step (12 000 x g, 10 min) followed by a brief incubation was sufficient for the preparation of an active cell extract to support protein expression with higher productivity (209 microg/mL). Compared to the traditional E. coli S30 procedure, the processing time was reduced by 62%, and the productivity was increased by 2.6 times. The new procedure will make the advantage of cell-free technology more obvious, and promote its wider application.

Expression profiling of genes involved in Taxuyunnanine C biosynthesis in cell suspension cultures of Taxus chinensis by repeated elicitation with a newly synthesized jasmonate and sucrose feeding / 生物工程学报

Taxus suspension cell culture has the potential to provide a sustainable source of anticancer drug paclitaxel (Taxol) and other taxoids. In the cell culture of Taxus chinensis, Taxuyunnanine C (Tc) is the primary taxoid. To design a rational strategy for redirecting the precursor fluxes from other taxoids into paclitaxel production, we employed Real-time Quantitative PCR (RQ-PCR) to understand the dynamic profiling of key biosynthetic pathway genes of palcitaxel and taxoids during the culture process. Six genes (TASY, TDAT, T5alphaH, TalphaH, T10betaH and T14betaH) were quantified under the process condition of double elicitation by 2,3-dihydroxylpropanyl jasmonate (DHPJA) (100 micromol/L on day 7 and day 12), and sucrose feeding (20 g/L) on day 7. This process treatment led to a high accumulation of Tc at (554.46 +/- 21.28) mg/L 8 days after the first elicitation. Then 9 days after the second elicitation, Tc production was as high as (997.72 +/- 1.51) mg/L. The early pathway genes TASY and TDAT were significantly up-regulated by 182-fold and 98-fold, respectively for the first DHPJA elicitation and by 208-fold and 131-fold, respectively for the second elicitation. The induction occurred after each elicitation lasted for about 24 h before their abundances decreased. Things are somewhat different in the case of the other four genes T5alphaH, TalphaH, T10betaH and T14betaH. For gene TalphaH, it was highly up-regulated by 3061-fold for the first DHPJA elicitation and by 1016-fold for the second elicitation. For the other three genes T5alphaH, T10betaH, T14betaH, they were up-regulated by 13-fold, 38-fold and 20-fold, respectively for the first DHPJA elicitation and by 7-fold, 16-fold and 6-fold, respectively for the second elicitation. The RQ-PCR results showed that there is tight correlation between gene expression and Tc accumulation. Gene expression was in accordance with Tc yield. Elicitation could improve expression of six genes. While along with culture course, high expression of the genes weakened. Elicitation for the second time would promote high expression of the genes again.

Factors influencing the in vitro culture of osteoblasts / 中国组织工程研究

OBJECTIVE: Osteoblasts are essential for osteogenesis and bone metabolism, the in vitro culture of osteoblasts is the foundation for studies on bone metabolism and osteogenetic mechanism. Therefore, it is of great significance to study the related factors affecting it.DATA SOURCES: Related literature about the influencing factors of in vitro culture of osteoblasts were searched for in Medline from January 1980 to December 2004 with retrieval words of "osteoblasts, culture in vitro, in fluencing factors", with the language limited to English. Meanwhile, it was also searched in the CBM between January 1995 and December 2004 with the retrieval words of "osteoblasts, in vitro culture, influencing factors",with the language of the articles limited to Chinese.STUDY SELECTION: After preliminary examination, literature that met the need of this study was searched for the full text. Inclusion criteria: Factors influencing the in vitro culture of osteoblasts included ① physical factors; ② microelements; ③ growth factors; and ④ hormones.Reviews were removed from this study because of summary or repetitive research.DATA EXTRACTION: A total of 105 articles related to the influencing factors of the in vitrocul ture of osteoblasts were obtained, articles of repetitive and similar researchwere removed; thereby 17 articles were included in the study.DATA SYNTHESIS: ① Physical factors: Ionizing radiation, microgravity,external force, and oxygen pressure. ② Microelements: microelement deficiency would hinder the skeletal growth, or even lead to malformation. Osteoblastic proliferation is closely related to some microelements, mainly including zinc, aluminum, fluorine, copper, manganese, calcium, and magnesium. ③ Growth factors closely related to osteogenesis mainly consist of bone morphogenetic protein, platelet-derived growth factor, fibroblast growth factor, transforming growth factor-beta, insulin like growth factor,and osteogenic growth peptide (OGP). ④ Hormones capable of promoting the proliferation and differentiation are growth hormone, estrogen, thyroxine, parathyroxine, and glucocorticosteroid.CONCLUSION: Multiple factors are involved in the in vitro culture of osteoblasts. It is helpful to understand these influencing factors to seek an effective way for the in vitro culture ofosteoblasts that is applied in tissue engineering.