This study was based on live yeast cell derivative (LYCD), which was produced by live yeast cell stressed with high temperature and H 2O 2. The results showed that pretreating of low dose(37℃and 0.2mmol/L H 2O 2) could increase the content of GSH and the activity of SOD and CAT. These pretreatment could induce the resistance to lethal concentration of H 2O 2. LYCD was produced by yeast treated with 37℃ and 0.2mmol/L H 2O 2. And it was found that the survival of yeast treated with lethal concentration of H 2O 2 obviously increased, while LYCD was added in yeast culture. It indicated that LYCD could have resistance to oxidative condition.

To obtain a number of penicillinases and degrade penicillin in milk by using the penicillinases,the gene encoding penicillinase was amplified by PCR from Bacillus cereus ATCC10987,cloned into pET28a(+) ,transformed into E. coli BL21;analysis of SDS-PAGE and penicillinase activity of the recombinant protein were done under induction of IPTG and the result showed that the maximum penicillinase activity reached 480 U/mL;the purity of penicillinase purified by Ni2+ Purification System was more than 90%;the immobilized penicillinases were obtained by sodium periodate method and the residual quantity of penicillin in milk(containing 0.5 U penicillin G/mL) was less than 4 ppb after degraded by the immobilized penicillinase.

To improve 3-ketosteroid-△1-dehydrogenase(KSDH) activity and the transformation level for androst-4-ene-3,17-dione, 3-ketosteroid-△1-dehydrogenase gene(ksdD) from Arthrobacter simplex was cloned into plasmid pWB980 and expressed in B. subtilis WB600 under the control of promoter P43. The molecular weight of expressed enzyme was about 55kDa by SDS-PAGE analysis. The activitities assayed by spectrophotometrical method of intracellular and extracellular soluble enzyme were 110?0.5mU and 15?0.6mU per milligram of protein respectively. The transformation rate of androst-4-ene-3,17-dione by the B. subtilis recombinant cells was 45.3%. Compared with Arthrobacter simplex, the enzyme activity of KSDH expressed in B. subtilis was improved about 30 fold, and the transformation level of androst-4-ene-3,17-dione by the B. subtilis recombinant cells was improved about 10 fold. The recombinant B. subtilis cells used in biotransformation of steroids provided a new way for steroid medicines production.

To obtain high-yield avilamycin-producing strains,low energy N~+ ion implantation technology and screening of streptomycin-re- sistant mutants are used in the study on breeding mutation.The results show that,“saddle”region,which range is from 3?10~(15) to 5?10~(15) ions/cm~2,has got better induced mutation action.It also means that the strain's resistant mutation and yield mutation closely correlate to each other,and the method of streptomycin resistant screening is feasible.We have isolated a high-yield strain SVT-45 which the productivi- ty is 195% higher than the original strain's in the rotation-flask experiments.These results showed that the ion implantation was an effective method for microbe mutagensis.

The promoter and signal peptide sequence of sacB gene (sacR gene) has been amplified by PCR.An inducible expression and secretion vector pHP13SN has been constructed with this amplified sequence,which was ligated with the pro-peptide and mature peptide of neutral protease gene on the vector pHP13.Transforming Bacillus subtilis DB104 with the vector pHP13SN, and the recombinant strain DB104(pHP13SN) can be got.The neutral protease gene has been expressed by the inducement of sucrose and the regulation of sacR,and the production has been secreted with bioactivity.

Objective To explore the expressions of Survivin and VEGF and relationship between them in hepatocellular carcinoma(HCC).Methods The expressions of Survivin protein and VEGF protein in 50 HCC.30 cirrhosis and 10 normal tissues were assessed by immunohistochemical method.The expressions of Survivin mRNA and VEGF mRNA in 50 HCC,30 cirrhosis and 10 normal tissues were assessed by in situ hybridization.Results The expressions of Survivin and VEGF in cancer tissues,cirrhosis tissues,normal tissues weresignificantly different. The expression of Survivin in HCC tissues was stronger than that in cirrhosis,but the expreesion of VEGF in cirrho- sis was stronger than that in HCC tissues.Conclusion The expression of survivin.is closely associated with the ex- pression of VEGF in HCC and they take positive correlation.The abnormal expressions of Survivin and VEGF are closely associated with the development of HCC.They may play important roles in the development of HCC.

The effects of different physical and chemical factors on hairy root growth and flavonoids production were studied in suspension culture of Saussurea medusa hairy root in 1/2 MS medium. The results showed that the following culture conditions, nitrogen concentratiaon (involved NH4+ and NO3-), 30 mmol/L; the ratio of ammonium to nitrate, 5:25; the combination of 2% sucrose and 3% glucose; 0.5 mg/L GA3; 0.5 mg/L IBA; initial pH 5.8; light cycle, 18 h/d (3500lx); temperature, 24 degrees C; shaker revolutions per minute, 100 r/min, were favourable to hairy root growth and flavonoids production. Under the above culture conditions, up to 12.8 g/L (DW) of hairy root and 1922 mg/L of flavonoids were obtained after 21 days of culture. The content of total flavonoids in hairy root was 15%, which was about 25 times as that in the wild plantlet.
Culture Media ; Flavonoids ; biosynthesis ; Plant Roots ; growth & development ; metabolism ; Saussurea ; growth & development ; metabolism ; Temperature ; Tissue Culture Techniques

OBJECTIVETo establish osteoblast apoptosis model induced by gingipains, and to examine the expression of pro-apoptotic protein Bcl-2 interacting mediator (Bim), Bcl-2 associated X protein (Bax) and Bcl-2 antagonist/killer (Bak).

METHODSGingipain and gingipain acticity were extracted and measured. Mouse osteoblast cell line MC3T3-E1 cells were cultured in the presence of 0.453, 0.906, 1.812 U/L gingipains for 0, 16, 24 and 48 h. Apoptosis was examined by 4',6-diamidino-2-phenylindole (DAPI) staining or annexin V/propidine iodide (PI) staining.Protein expression of Bim, Bax and Bak was determined by Western blotting after osteoblasts were cultured with 1.812 U/L gingipain for 0, 4, 8, 16, 24 and 48 h. Osteoblasts were cultured with 1.812 U/L gingipain which had been inhibited with N-alpha-tosyl L-lysyl-chlorom ethylketone (TLCK). Western blotting was used to detect Bim expression and DAPI staining to measure apoptosis.

RESULTSArginine-specific proteinases (Rgp) activity was (18.11 ± 2.11) U/L and specific proteinases (Kgp) was (1.02 ± 0.25) U/L. Percentage of osteoblast apoptosis induced by 1.812 U/L gingipain rose to (6.31 ± 0.37)% after 16 h, and reached (11.20 ± 0.35)% at 24 h and (10.80 ± 0.46)% after 48 h with DAPI staining. Annexin V/PI staining supported the result from DAPI staining.Bim protein level increased during osteoblast apoptosis, the relative fold rose to (0.31 ± 0.03) after 4 h (about 2 fold compared to control), peaking at 24 h (0.57 ± 0.05, 3-4 fold compared to control). Proteinase inhibitor TLCK effectively blocked the activity of gingipain and inhibited up-regulation of Bim induced by gingipains from (0.58 ± 0.04) to (0.14 ± 0.03). The percentage of osteoblast apoptosis decreased from (11.20 ± 0.35)% to (4.31 ± 0.38)% in the presence of TLCK. Expression of Bax remained unchanged when cells were cultured with or without gingipains. Bak was under the detectable level in MC3T3-E1.

CONCLUSIONS1.812 U/L gingipains induced osteoblast apoptosis. Protein expression of Bim was up-regulated during cell apoptosis and was down-regulated when gingipain inhibited with TLCK, suggesting that Bim was involved in osteoblast apoptosis induced by gingipain. Inhibition of Bim protein expression protected osteoblast from apoptosis.

Adhesins, Bacterial ; pharmacology ; Animals ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Bcl-2-Like Protein 11 ; Cell Line ; Cysteine Endopeptidases ; pharmacology ; Humans ; MCF-7 Cells ; Membrane Proteins ; metabolism ; Mice ; Osteoblasts ; cytology ; metabolism ; Proto-Oncogene Proteins ; metabolism ; Tosyllysine Chloromethyl Ketone ; pharmacology ; bcl-2 Homologous Antagonist-Killer Protein ; metabolism ; bcl-2-Associated X Protein ; metabolism

AIMTo study the effects of Cu2+ on growth hormone (GH) secretion of pig pituitary cells in vivo.

METHODSGland pituitary cells of 32-35 days old pigs were incubated for 48 h in presence of 10% fetal calf serum in DMEM. Cells were treated for 36 h with various concentrations of Cu2+ (0 mg/L, 0.025 mg/L, 0.1 mg/L, 0.4 mg/L, 1.6 mg/L) in serum-free DMEM. Culture medium was collected in 12 h, 24 h, 36 h thereafter for GH measurement and the radioimmunoassay kits were provided by Linco.

RESULTSThe GH concentration was higher in treatments of 0.025 mg/L, 0.1 mg/L, 0.4 mg/L than that of 0 mg/L when the cells were treated with Cu2+ for 24 h, and it was significantly different (P < 0.05) between treatment 0.1 mg/L and treatment 0 mg/L.

CONCLUSIONIt was concluded that the presence of Cu2+ stimulated pig pituitary cells to secret GH in vivo.

Animals ; Cells, Cultured ; Copper ; pharmacology ; Culture Media ; Growth Hormone ; secretion ; Pituitary Gland ; cytology ; drug effects ; metabolism ; Swine

Objective To discuss and analyze the diagnosis,management and surgical techniques for cranioplasty of skull vault defects and skull base reconstruction in order to raise the therapeutic effect. Methods The clinical data of 169 cases of skull vault and skull base defects treated with cranioplasty of skull vault defect and skull base reconstruction were retrospectively analyzed.Results Overlay tech- nique or inlay technique with imported titanium alloy mesh was used in 160 cases,and homologous bone was used in 9 cases.The surgical time ranged from 3 months to 8 years after injury.Eight cases presented with complications such as hematoma,subcutaneous effusion,infection and epilepsy postoperatively,but no operative death occurred.Conclusion For patients with skull vault defect with the diameter≥3 cm the best operative time is 3 months after injury,and for patients with intracranial and extracranial communica- ting tumors,skull base reconstruction can be performed when tumors are removed.Much attention should be paid to perioperative management and surgical skills.