The recombinaut porcine insulin precursor(PIP)produced by Pichia pastoris in shake-flask and 501.fermenter was investigated respectively.The results indicated that 60h induction time length and 2.0%～2.5% methanol addition every day was optimum in shake- flask.The process in 50L fermenter was consisted of batch,feed-batch and induction phases.The relationship between dry cell weight(y) and culture time (t) in growth phase(batch and feed-batch phase)could be described by model y=0.6525e~(0.1907t).Glycerol and ammonia were almost used for cell growth and maintain,and no by-product was observed in batch and fed-batch phase Only 80% ammonia and 70% methanol were used by cell in induction phase.By comparison the results of shake-flask and 50L fermenter,it was concluded that the limit- ing factor in the fermentation of shake-flask and 50L fermenter was dissolved oxygen(DO)and.carbon source,respectively.When scaling the result of shake-flask to 501.fermenter,the control strategy was adapted for 50L fermenter by increasing the feed rate of methanol and the maximum PIP concentration reached 1.72 g/L.

Immobilized penicillin acylase was used for bioconversion of penicillin G into 6-APA in aqueous two-phase systems consisted of a light-sensitive polymer PNBC and a pH-sensitive polymer PADB.Partition coefficients of 6-APA was found to be:about 5.78,in the presence of 1% NaCl.Enzyme kinetic showed that reaction reached equilibrium at 7h or so.The 6-APA mole yields were 85.3%(pH 7.8,and 20 ℃) and this value was about 20%higher than control in reaction of single aqueous phase buffer.Partition coefficient of penicillin G(Na) washardly changeable,while partition coefficient of product,6-APA and phenylacetate acid was significantly changeable.Reason is due to Donnan effect of phase systems andhydrophobicity of products.The change of partition coefficients of products also affects bioconversion yield of products.In the aqueous two-phase systems,substrate,penicillin G,products 6-APA and phenylacetate acid are biased in top phase,while immobilized penicillin acylase is completely partitioned in bottom.Substrate,penicillin G enters into bottom phase,and it is catalyzed into 6-APA and phenylacetate acid,then the products enter into top phase.Finally,inhibition of substrate and products is removed to result in improvement of products yield.Moreover,immobilized enzymehashigher efficiency than immobilized cells and occupy smaller volume.Comparing with free enzyme,immobilized enzymehashigher stability,longer use life,completely partitioned in bottom phase and recycle.Bioconversion in two-phase systems using immobilized penicillin acylase showed outstanding advantage.The light-sensitive copolymer forming aqueous two-phase systems could be recovered by laser radiation at 488 nm or filtrated 450 nm light,while pH-sensitive polymer PADB could be recovered by isoelectric point(pH 4.1).The recovery of the two copolymers was 95%~99%.

Optimization of the fermentation condition for human apolipoproteinA-I expression in recombinant Escherichia coli was investigated. The recombinant plasmid pBV220-ApoA-I was transformed respectively into different E.coli hosts such as JM109, BL21(DE3),DH5?, BMH7118,and TG1. The best host E.coli was DH5? in which the recombinant ApoA-I expression percentage was 21.2% corresponding to that in BL21(DE3) in flask shaker cultivation,while the ApoA-I expressed percentage in E.coli TG1 was 11%.Fed-batch cultivation was performed in FMG-5L fermentor,the optimum fermentation cultivation conditions were as following :optimum pH value was 7.0 in growth phase and 7.4 in the expression phase. The initial glucose concentration in batch phase was 3 g?L -1.The optimum C/N ratio was 2∶1.The recombinant ApoA-I reached about 40% of the total protein, and concentration of ApoA-I was 2.86 g?L -1.

The temperature effect on the recombinant protein production formation was investigated in present study. The culture temperature of growth phase is 30℃, and the culture temperature of induction phase was arranged according to three modes. Hign cell-density and high expression culture of E.coli to product recombinant human apolipoprotein A-I Milano by two temperature-shifted induction . Two temperature-shifted induction was carried out high density and high expression recombinant human ApoA-1 Milano. The recombinant protein ApoA-I Milano reached 4.8 g?L -1 with the final cell density of OD 600 150. And the two temperature-shifted induction avoided the acetic acid successfully to the influence of the high density and high expression. Two temperature-shifted induction was viable in high density culture and high expression of heterogenous protein in recombination E.coli.The sduty provides a basic work for production of recombinant ApoA-I Milano in scale.

On the base of element and metablism balancing, the mathematical model of the cultivation process with Streptomyces aureofaciens was developed, and the unknown parameters in the model were estimated with the method of nonlinear optimization. Firstly the energetic coefficient of CTC biosynthesis was gained, which was 1.8 - 2.8 mol-ATP x C-mol(-1). The macroscopic reaction rates were predicted in the process and compared with the experimental values. The results show that the model can preferably describe the relationships between several macroscopic reaction rates in the process and can supervise the optimization of CTC fermentation process theoretically.
Chlortetracycline ; metabolism ; Fermentation ; physiology ; Models, Theoretical ; Streptomyces aureofaciens ; growth & development ; metabolism

OBJECTIVETo investigate possible mechanism of angiogenesis in brain tissue of neonatal rat hypoxic-ischemic encephalopathy (HIE).

METHODSForty seven-day old neonatal rats were randomly assigned to hypoxic-ischemic (Model group) or sham treatment (Sham group), each group had 20 rats. Five rats from each group were sacrificed on days 1, 3, 7 and 14 after hypoxia-ischemia. Paraffin sections of the brain were stained with anti-endothelial cell, anti-proliferating cell nuclear antigen (PCNA) or anti-vascular endothelial growth factor (VEGF) by using single or double immunohistochemistry. The brain capillary density index (BCDI), brain proliferating capillary density index (BPCDI) and the expression of VEGF were analyzed under the microscope. The expression of VEGF and hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA in hypoxic-ischemic side of the brain was measured by RT-PCR.

RESULTSBCDI around infarct brain tissue in the model group began to rise on day 3 and remained higher than that of the sham group from day 3 to day 14 [day 3: (9.80 +/- 1.05)/HPF vs. (4.90 +/- 0.66)/HPF, P < 0.01;day 14: (13.29 +/- 3.90)/HPF vs. (6.08 +/- 1.50)/HPF, P < 0.01]. Occasional proliferating capillary was found in brain tissue of normal neonatal rats. The density of proliferating brain capillary on day 3 and day 7 of Model group [(0.54 +/- 0.15)/HPF vs. (0.90 +/- 0.25)/HPF] were significantly higher than those of Sham group [(0.12 +/- 0.05)/HPF vs. (0.13 +/- 0.07)/HPF, P < 0.01]. VEGF was mainly expressed in the cytoplasm of neurons, capillary endothelial cells and pial cells. Viable neurons and endothelial cells in the infarct areas also expressed VEGF. The expression of VEGF mRNA in hypoxic-ischemic brain tissue was significantly higher than that of normal control (P < 0.01) and temporally preceded angiogenesis. The expression of VEGF mRNA at 12 hours of HIE model was significantly higher than that of normal control (1.56 +/- 0.27 vs. 0.95 +/- 0.21, P < 0.05). It reached its peak on day 1 and day 3 (1.85 +/- 0.31 vs. 1.86 +/- 0.39), significantly higher than that of normal control (P < 0.01), and decreased by day 7 and day 14, without significant difference compared with normal control (P > 0.05). The expression of HIF-1alpha mRNA was also up-regulated after hypoxic-ischemic treatment. The expression of HIF-1alpha mRNA (1.07 +/- 0.21) was significantly higher than that of normal control (0.64 +/- 0.28, P = 0.048) at 3-hour of HIE model, reached its peak on day 1 (1.73 +/- 0.42, P < 0.01), remained at high expression level on day 3 (1.44 +/- 0.36, P < 0.05) and began to decline by day 7 and day 14 when it was not significantly different from normal control.

CONCLUSIONSAngiogenesis exists in the brain tissue of neonatal rat HIE model. Up-regulation of VEGF expression mediated by HIF-1 may play an important role in the process of angiogenesis.

Animals ; Animals, Newborn ; Brain ; blood supply ; Brain Diseases ; etiology ; genetics ; metabolism ; Disease Models, Animal ; Hypoxia-Inducible Factor 1, alpha Subunit ; Hypoxia-Ischemia, Brain ; complications ; Immunohistochemistry ; Neovascularization, Pathologic ; etiology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription Factors ; analysis ; genetics ; Vascular Endothelial Growth Factor A ; analysis ; genetics

Taking the typical metabolic control product-guanosine as an example, the method of metabolic flux shift investigation based on process multi-levels parameter correlation analysis was established. The metabolic pathway, multi-parameter correlation, accumulation of amino acid and organic acid during guanosine fermentation process were integratively analyzed. The metabolic flux shift from HMP to EMP was ascertained, which was assumed to be caused by the accumulation of ammonium ion. The subsequent optimization based on controlling flux distribution between EMP and HMP did improve the yield by 35% when the metabolic flux shift was prevented.
Amino Acids ; metabolism ; Ammonia ; metabolism ; Fermentation ; Guanosine ; metabolism

OBJECTIVETo evaluate the effects of antiviral agents on intrahepatic HBV covalently closed circular DNA (cccDNA) in HBeAg-positive chronic hepatitis B patients.

METHODSSeventy-one HBeAg positive chronic hepatitis B patients were enrolled in this study. Lamivudine was administered to 35 patients for 48 weeks, sequential therapy with lamivudine-IFN alpha-2b to 24 of the 71 patients for 48 weeks, and interferon alpha (IFN alpha-2b) was administered to 12 for 24 weeks. All subjects were followed-up for 24 weeks. Serum HBV DNA, intrahepatic HBV DNA and cccDNA were measured quantitatively by PCR. HBV genotypes were analyzed by PCR-RFLP.

RESULTSForty-eight weeks of sequential lamivudine-IFN alpha-therapy and lamivudine monotherapy and 24 weeks of IFN alpha monotherapy reduced the intrahepatic HBV DNA to (4.7+/-1.1) log10, (4.6+/-1.5) log10 and (5.6+/-1.5) log10, and cccDNA to (3.4+/-1.3) log10, (3.8+/-1.1) log10 and (5.0+/-1.5) log10, significantly lower than therapy (P < 0.05). Seventeen of the 71 patients developed HBeAg seroconversion, and the reduction of cccDNA in the HBeAg seroconverted patients was significantly more than that of the HBeAg positive patients (P < 0.05). After 24 weeks of antiviral therapy withdrawal, 18 patients achieved sustained virological response, and the baseline intrahepatic cccDNA in the patients with sustained virological response was significantly lower than that of patients with virological rebound (P < 0.05). The change in intrahepatic cccDNA correlated positively with the reduction in intrahepatic HBV DNA (P < 0.05). The cccDNA levels correlated with the serum HBeAg titers at the end of the treatment (P < 0.01). Of the total 71 cases, HBV genotype C accounted for 85.9% (n = 61), and genotype B for 14.1% (n = 10). There was no significant difference in the changes of intrahepatic HBV DNA and cccDNA levels between HBV genotypes C and B (P >0.05).

CONCLUSIONSBoth 48 weeks of sequential lamivudine-IFN alpha and lamivudine monotherapy strongly reduced intrahepatic HBV DNA and cccDNA more than 24 weeks of IFN alpha monotherapy. Low baseline intrahepatic cccDNA levels might predict a good long-term efficacy of antiviral treatment. The reduction of intrahepatic cccDNA correlated positively with the changes of intrahepatic HBV DNA, and intrahepatic cccDNA levels correlated with serum HBeAg titers. HBV genotypes had no obvious influence on intrahepatic HBV DNA load or cccDNA load.

Adult ; Antiviral Agents ; pharmacology ; therapeutic use ; DNA, Circular ; DNA, Viral ; Female ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; drug effects ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Interferon-alpha ; pharmacology ; therapeutic use ; Lamivudine ; pharmacology ; therapeutic use ; Male ; Middle Aged ; Recombinant Proteins ; Young Adult

Redox electrode was used to control redox potential at four different levels (-50 mV, -100 mV, -150 mV, - 230mV) for the study of ethanol fermentation. The result showed that there was notably influence on the yield of ethanol, the formation of glycerol, the secretion of organic acid, biomass and the death of cell by controlling redox potential at different levels. For example: the biomass of ORP at -50 mV was 1.26, 1.86, 2.59 times higher than ORP at -100 mV, -150 mV, -230 mV respectively, the final glycerol concentration was 1.2, 1.1, 1.7 times higher but final ethanol concentration was 0.87, 0.49, 0.51 times lower compared to the latest three ORP control level. And take biomass, ethanol yield, glycerol concentration, and unconsumed glucose into consider, we get the conclusion that it is very favorable for ethanol fermentation by control ORP at 150 mV. So it give us a apocalypse that we can use redox electrode to control the ethanol fermentation exactly on bioreactor scale.
Electrodes ; Ethanol ; metabolism ; Fermentation ; Industrial Microbiology ; methods ; Oxidation-Reduction ; Saccharomyces cerevisiae ; growth & development ; metabolism

Nanoporous ZnO was used as a carrier to prepare drug solid dispersion, the mechanism of which to improve the drug dissolution was also studied. Nanoporous ZnO, obtained through chemical deposition method, was used as a carrier to prepare indomethacin and cilostazol solid dispersions by melt-quenching method, separately. The results of scanning electron microscope, surface area analyzer, fourier transform infra-red spectroscopy, differential scanning calorimeter and X-ray diffraction showed that drugs were implanted into nanopores of ZnO by physical adsorption effect and highly dispersed into nanopores of ZnO in amorphous form, moreover, these nanopores strongly inhibited amorphous recrystallization in the condition of 45 degrees C and 75% RH. In addition, the results of the dissolution tested in vitro exhibited that the accumulated dissolutions of indomethacin and cilostazol solid dispersions achieved about 90% within 5 min and approximately 80% within 30 min. It was indicated in this study that the mechanism of drug dissolution improvement was associated with the effects of nanoporous ZnO carrier on increasing drug dispersion, controlling drug in nanopores as amorphous form and inhibiting amorphous recrystallization.
Anti-Inflammatory Agents, Non-Steroidal ; administration & dosage ; chemistry ; Calorimetry, Differential Scanning ; Drug Carriers ; Indomethacin ; administration & dosage ; chemistry ; Microscopy, Electron, Scanning ; Nanostructures ; Phosphodiesterase 3 Inhibitors ; administration & dosage ; chemistry ; Solubility ; Spectroscopy, Fourier Transform Infrared ; Tetrazoles ; administration & dosage ; chemistry ; X-Ray Diffraction ; Zinc Oxide ; chemistry