Up-regulation of ErbB3-binding protein 1 inhibits the growth of esophageal carcinoma cells / 生理学报

The objective of this study was to investigate the role of ErbB3-binding protein 1 (Ebp1) in the growth of esophageal squamous cell carcinoma (ESCC) cells and the underlying mechanism. Eca109 and KYSE150 cells were transfected with lentiviral vector carrying Ebp1 gene. The mRNA levels of Ebp1 in esophageal cancer tissues and paired adjacent normal tissues were examined by real-time PCR. The growth and viability of esophageal carcinoma cells were assessed using MTT and crystal violet assays, respectively. Clone-forming abilities of Eca109 and KYSE150 cells were analyzed by soft agar assay. Apoptotic rates of esophageal carcinoma cells were detected by flow cytometry, and expression levels of the proteins involved in apoptosis were assessed by Western blot. Tumorigenicity of Eca109 cells were detected by nude mouse transplantation tumor experiment. The results indicated that the mRNA levels of Ebp1 in esophageal cancer tissues was down-regulated compared with paired adjacent normal tissues. The growth and viability of Eca109 and KYSE150 cells were all suppressed by Ebp1 overexpression. Besides, Ebp1 overexpression induced apoptosis, increased Rb and P53 expressions, and decreased CyclinD1 expression in Eca109 and KYSE150 cells. In addition, Ebp1 overexpression inhibited the tumorigenesis of Eca109 cells in vivo. These results suggest that Ebp1 may suppress the growth of esophageal carcinoma cells in vitro and in vivo by inducing apoptosis.

Bioinformatic identification and analysis of Schistosoma japonicum adenine phosphoribosyltransferase / 南方医科大学学报

<p><b>OBJECTIVE</b>To identify adenine phosphoribosyltransferases in Schistosoma japonicum and analyze their structural features.</p><p><b>METHODS</b>Based on the accessible transcriptome and proteomic data, the S. japonicum adenine phosphoribosyl transferases were identified using bioinformatics approaches including bi-directional homology comparison, domain search and phylogenetic analysis. Homology modeling was also performed to describe the structural features of the proteins.</p><p><b>RESULTS AND CONCLUSION</b>Two homologue sequences of adenine phosphoribosyltransferase were obtained from S. japonicum, and the EST abundance, physico-chemical properties and three-dimensional structures of them were also acquired.</p>

Synthesis of methotrexate-poly (ethylene glycol) conjugate and their anti-tumor activity in vitro / 药学学报

To improve the physical property and bioactivity of methotrexate, this paper investigated the new formation of conjugate methotrexate-poly (ethylene glycol) and in vitro anti-tumor activity of the synthesized conjugate. The conjugate of methotrexate-poly (ethylene glycol), which was verified by the spectroscopy analysis of UV, IR and 13C NMR, was synthesized by chemical catalysis and micro-wave irritation. The determination for the conjugate of solubility in water and distribution coefficient in octanol-water system of the conjugate was done to examine its deliquescence property. The solubility in water and the distribution coefficient of the conjugate was greatly improved, which was increased by 128 folds and 5 folds, respectively. The in vitro anti-tumor activity of the conjugate was tested by mouse L(1210) leukaemia cells, and the synthesized conjugate showed the same anti-tumor activity as the original methotrexate. Compared to the reported literature, the modification of methotrexate by poly (ethylene glycol) is more rapid and convenient.

Preparation of recombinant HIV-TATm-survivin (T34A) protein and its pro-apoptosis activity to cancer cells in vitro / 生物工程学报

As a novel member of the IAP (Inhibitor of apoptosis protein) family, survivin was observed to be expressed in most human cancerous cells. Fusion protein TATm-survivin (T34A) has drawn considerable attention because it is a potential anti-tumor protein that can be transduced into cancer cell with the help of HIV-TAT domain. In this study, the cDNA encoding survivin was cloned by RT-PCR from human breast cancer cell lines B-Cap-37. An expression vector of pRSET-B-HIV-tatm-survivin (T34A) was constructed by PCR after survivin (T34A) was mutated by site-directed mutagenesis. Subsequently, the resultant plasmid was transformed into E. coli BL21 (DE3). Recombinant HIV-TATm-Survivin (T34A) protein was expressed efficiently with 0.5mM IPTG as inducer, reaching a yield of 650mg/liter (as inclusion body) in fermentation culture. The inclusion bodies were solubilized, refolded and purified to a purity of 96% by ion exchange chromatograghy and size-exclusion chromatography. Remarkable effects of the purified recombinant HIV-TATm-Survivin (T34A) on the morphology of cell line SW1990 and B-Cap-37 were observed after being administrated for 4h. MTT assay showed recombinant HIV-TATm-survivin (T34A) protein could inhibit significantly cell proliferation of SW1990 and B-Cap-37 and SSMC-7721 in vitro. Apoptosis rate and cell circle of SW1990 and B-Cap-37 that had been treated with target protein (final concentration 30 microg/mL) were detected with flow cytometry. Results revealed that more than 65% cancer cells were arrested at G1 phase. The study suggested that TATm-survivin (T34A) protein was a hopeful protein drug in the treatment of cancers by facilitating apoptosis of cancer cells. Key words recombinant HIV-TATm-Survivin (T34A), expression and purification, pro-apoptosis bioactivity, SW1990 and B-Cap-37 cancer cell lines

Optimization of cultural condition of genetic engineering strain for antibiotic peptide adenoregulin and research on its fed-batch cultivation / 生物工程学报

33 amino acid antibiotic peptide adenoregulin (ADR), which were firstly isolated from the skin of South America arboreal frog Phyllomedusa bicolor, forms alpha-helix amphipathic structure in apolar medium and has a wide spectrum of antimicrobial activity and high potency of lytic ability. Adr gene was cloned in pET32a and transformed into Escherichia coli BL21(DE3) . The cultural and inductive conditions of E. coli BL21(DE3)/pET32a-adr have been optimized. The effect of three factors which were time point of induction, concentration of IPTG in the culture and time of induction on the expression level of Trx-ADR was investigated. The results indicated that the expression level was affected by the time point of induction most predominantly. 9 veriaties of media in which BL21 (DE3)/pET32a-adr was cultured and induced were tested to achieve high expression level of target protein. It was found that glucose in the medium played an important role in keeping stable and high expression level of Trx-ADR. The optimal inductive condition is as follows: the culture medium is 2 x YT + 0.5% glucose, the time point of induction is OD600 = 0.9, the final concentration of IPTG in the culture is 0.1 mmol/L and the induction time is 4 h. BL21 (DE3)/pET32a-adr was cultivated according to the strategy of constant pH at early stage and exponential feeding at later stage to obtain high cell density. During the entire fed-batch phase, by controlling the feeding of glucose, the specific growth rate of the culture was controlled at about 0.15 h(-1), the accumulation of acetic acid was controlled at low level (<2 g/L), but the plasmid stability could not be maintained well. At the end of the cultivation, 40% of the bacteria in the culture lost their plasmids. As a result, the expression level of the target protein declined dramatically, but 90% of Trx-ADR was in soluble form. The expressed fusion protein showed no antibacterial activity, while the native form of ADR lysed from Trx-ADR showed distinct antibacterial activity.

Study on HPLC fingerprint and quality control of Propolis / 中国中药杂志

<p><b>OBJECTIVE</b>To study on HPLC fingerprint of Propolis and to control it's quality.</p><p><b>METHOD</b>The chromatographic fingerprints of seven samples from different producing areas were determined by RP-HPLC.</p><p><b>RESULT</b>The chromatograms of Propolis from different producing areas were very similar.</p><p><b>CONCLUSION</b>The quality of Propolis can be controlled by determination the HPLC fingerprint.</p>

High-cell density cultivation of recombinant Escherichia coli for production of TRAIL by using a 2-stage feeding strategy / 生物工程学报

Escherichia coli was genetically engineered to produce recombinant tumor necrosis factor-related apoptosis inducing ligand (Apo2L/TRAIL) using a temperature-inducible expression system. To create a fed-batch culture condition that allows efficient production of TRAIL, different feeding strategy including discontinuous, DO-stat and pH-stat feeding strategies were compared. Then, a special 2-stage feeding strategy was developed. High concentration of biomass (300g wet cell weight per liter of culture broth) and active soluble TRAIL protein (1.1g/L) was obtained by applying a high-cell-density cultivation procedure with the 2-stage feeding strategy. Cultivation of recombinant E. coli was started as a batch process at 30 degrees C and then followed by fed-batch culture when the dissolved oxygen concentration presented a steep increase resulted from the exhaustion of glucose in the medium. At the first phase of fermentation (batch phase), agitation rate was enhanced to control dissolved oxygen at 30 percent. When glucose in the medium was used up, indicated by a sudden rise in pH value and dissolved oxygen, the second phase (fed-batch phase) was started with glucose and nitrogen resource being supplied automatically. At the beginning of fed-batch operation, stirrer rate was cascaded with dissolved oxygen signals to keep it at 20 percent (DO-stat). During the fed-batch phase, glucose was limited to control the specific growth rate under the critical value microcrit, to avoid acetic acid excretion. When the stirrer speed arrived at its up-limit, the flow rate of feed was kept constant. In the inducing phase(42 degrees C for 4h) glucose was fed as a pH regulating agent (pH-stat) and the specific growth rate and dissolved oxygen decreased sharply. Aqueous ammonia was used for maintaining pH value at 7.0 throughout the first two phases. In the whole fermentation, acetic acid concentration didn't exceed 2.9 g/L. At the end of the high-cell-density cultivation process, no acetic acid could be detected in the medium. These results indicated that our fed-batch strategy was able to prevent acetate accumulation significantly. Although high cell density has been achieved, the induction process was not optimized satisfactorily and much work should be done further. Furthermore, since no special ways, like pure oxygen, pressure, has been used in our experiments, this efficient approaches would be useful not only in a pilot scale but also in an industry scale. Finally, simple purification procedure based on immobilized metal affinity column (IMAC) and CM-Sepharose column was implemented to isolate the TRAIL. Yields of more than 800mg TRAIL per liter of culture broth were obtained, the final purity reaching more than 95%. The purified TRAIL showed strong cytotoxity activity against human pancreatic 1990 tumor cells, with ED50 about 1.6 microg/mL.

Protective effects of Cleistocalyx operculatus on lipid peroxidation and trauma of neuronal cells / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the protective effects of Cleistocalyx operculatus on lipid peroxidation in rat liver microsomes and on the trauma of PC12 cells induced by H2O2.</p><p><b>METHOD</b>The mouse liver homogenate lipid peroxidation assay and PC12 Cell culture and Cell viability (MTT assay) were applied.</p><p><b>RESULT</b>Cleistocalyx operculatus showed strong protective effects on lipid peroxidation in rat liver microsomes in a dose-dependent manner and exhibited potent protective effects on the trauma of PC12 cells induced by H2O2 (200 micromol x L(-1)) when the concentration reached 1.00 g x L(-1).</p><p><b>CONCLUSION</b>Cleistocalyx operculatus may be used as antioxidant to prevent or delay the pathogenesis of neural cell diseases.</p>

Studies on lipase-catalyzed synthesis of ethylglucoside lactate in non-aqueous phase / 生物工程学报

Ethylglucoside lactate, a novel Alpha-Hydroxy Acids Derivative, was synthesized by transesterification in non-aqueous phase using immobilized lipase as biocatalyst. Based on the studies of the factors effecting initial rate and conversion under atmospheric pressure (solvent, acyl donor, different immobilized lipase, substrate concentration, enzyme concentration and temperature), the results show that solvent-free medium using butyllactate as acyl donor is suitable to the ester synthesis. The reaction conditions have been optimized as the following: the amount of enzyme = 75 g/L, the ethylglucoside concentration = 0.4 mol/L, 70 degrees C, 200 r/min, 50 h, which the conversion was 71%. A 90% conversion and a 60.7 mmol.L-1.h-1 initial rate can be obtained under reduced pressure, which the conditions are enzyme 75 g/L, ethylglucoside 0.35 mol/L, 65 degrees C, 200 r/min and 40 h. The product purified by extraction and SIO2 chromatography was identified by infrared spectroscopy and 1H NMR.