Background Primary open angle glaucoma(POAG) is a common type of glaucoma.It has been well known that a lot of factors are associated with the pathogenesis of POAG,but genetic factor plays a critical role.Optineurin (OPTN)gene is the second confirmed POAG-relevant gene,and screening its mutation in the population contribute to the deeply understanding of the pathogenesis of POAG.Objective The present study was to investigate the association between sequence variants of OPTN gene and POAG in Chinese patients.Methods DNA was isolated from peripheral blood of 100 POAG patients and 60 cataract individuals.The coding exons of OPTN gene were amplified by PCR.PCR products were then sequenced directly to assay the variants and contrasted to original sequence in GenBank.This study was approved by the Ethical Committee of Shenzhen Eye Hospital.All the subjects signed the written inform consent.Results A case-controlled study was designed.The mean intraocular pressure (IOP)of the POAG patients was (29.0±6.5)mmHg,and that of the cataract patients was (13.7 ±2.4)mmHg.Variant of synonymous coding T34T was found in 60 POAG patients.Genetic type frequencies of AA,GA and GG were 10％,50％ and 40％ in the POAG patients,and those of cataract patients were 0,25％ and 75％ respectively,showing significant difference between them (x2 =20.416,P =0.000).The allele frequencies of A and G were 35％ and 65％ in the POAG patients,and those of cataract patients were 12.5％ and 87.5％,with a statistically significant difference (x2 ＝ 19.464,P =0.000).The sequence changes of non-synonymous coding variants (M98K,691-692insA G,R545Q,H486R) were also found in both POAG and cataract patients,but no significant difference was seen in the genetype and allele frequencies between two groups (P＞0.05).Conclusions No obvious association of OPTN gene variant with POAG is verified.The variant of T34T maybe increase the risk of POAG.

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%.

OBJECTIVETo investigate whether resveratrol (RES) plays a protective role in hypothermic preserved isolated rat hearts and whether it is mediated by regulation of silent information regulator protein-1 (Sirt-1) expression.

METHODSThe Langendorff model of isolated rat heart was used. After stored in different Celsior solution at 4 degrees C for 9 h, SD rat hearts were randomly divided into 7 groups: blank control group;9 h group (soley hypothermic preservation for 9 h); RES group (3, 10, 30 micromol/L RES treatment plus hypothermic preservation for 9 h ), niacinamide (NAM) group (40 micromol/L NAM added in Celsior solution plus hypothermic preservation for 9 h), RES + NAM group (30 micromol/L RES and 40 micromol/L NAM were added in Celsior solution plus hypothermic preservation for 9 h). The morphological changes of cardiomyocytes were detected by the HE staining with the light microscope. The mRNA and protein expression levels of Sirt-1 were detected by Real-Time PCR and Western blot respectively.

RESULTS(1) Compared with the blank control group, myocardiocytes were injured remarkably in the 9 h group and the Sirt-1 mRNA and protein expression levels were decreased significantly (P < 0.01); (2) Compared with the 9 h group, rat myocardial injury was alleviated gradually in 3, 10, 30 micromol/L RES group and the Sirt-1 mRNA and protein expression levels were increased in a dose-dependent manner (P < 0.05); (3) The above protective effects of RES were attenuated by Sirt-1 inhibitor NAM.

CONCLUSIONRES can protect myocardiocytes from injury caused by long range hypothermic preservation and this protective effect maybe mediated by upregulation of Sirt-1 expression.

Animals ; Cryopreservation ; Heart ; drug effects ; Male ; Organ Preservation ; Rats ; Rats, Sprague-Dawley ; Sirtuin 1 ; metabolism ; Stilbenes ; pharmacology

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.

OBJECTIVETo understand the etiology of hand, foot and mouth disease (HFMD) in Guangzhou area in 2008.

METHODTotally 1023 clinical specimens were collected from pediatric patients suspected of HFMD in 2008. TaqMan real-time RT-PCR were used for detection of enterovirus 71 (EV71), Coxsackievirus A16 (CA16) and other enteroviruses. The specimens which were enterovirus positive by RT-PCR method with universal primer but EV71 and CA16 negative, were amplified and sequenced for 5'untranslated region.

RESULTEnterovirus was identified from 434 of 1023 samples and detection rate of enterovirus was 42.42%; of the 434 samples, 276 were positive for EV71 (63.6%), 126 for CA16 (29%), 4 samples for enterovirus 84, 3 for Echovirus 11, 2 for Echovirus 9, 3 for Coxsackievirus B3, 4 for Coxsackievirus A10, 3 for Coxsackievirus A6, 6 for Coxsackievirus A12 or A5, and for 7 samples typing was difficult.

CONCLUSIONThe major causative agents of HFMD in Guangzhou were EV71 and CA16 in 2008, and EV84, CA10, CA12, CA6, COSB3, ECHV11, ECHV9 were also the pathogens for smaller proportions of patients.

Child ; Child, Preschool ; China ; epidemiology ; Coxsackievirus Infections ; epidemiology ; DNA Primers ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Female ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans ; Infant ; Male ; RNA, Viral ; Reverse Transcriptase Polymerase Chain Reaction

Ex vivo expansion of hematopoietic progenitor cells (HPCs) is valuable for clinical application, however, traditional ex vivo culture negatively affects long-term hematopoietic reconstitution ability. In the hematopoietic system, the expression of Notch receptors and their ligands has been widely reported. Active Notch signal inhibits the differentiation of HSCs while promotes their expansion, suggesting that ex vivo expansion of hematopoietic progenitor cells could be enhanced by manipulating Notch signal pathways. In this article the Notch signal pathways, Notch signal and maintenance of hematopoietic progenitor cells, Notch signal and expansion of hematopoietic progenitor cells and molecular mechanism of Notch signal maintaining undifferentiation of hematopoietic progenitor cells were reviewed.
Animals ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Receptors, Notch ; metabolism ; Signal Transduction

To express the extracellular fragement of hepatoma associated antigen HAbl8G(HAb18GEF) in E. coli efficiently in a non-fusing way, the cDNA of HAb18GEF gene was inserted into prokaryotic expression vector pET21a + . The secondary structure and codon adaptation of translational initiation region (TIR, from-30 to + 39) in mRNA of recombinant vector HAb18GEF/ pET21a + was predicted simultaneously by computer-aided design. Stable Stem-Loop structures and many low-usage codons were detected in mRNA-TIR of non-optimized recombinant HAb18GEF/pET21a + vector. The stability of mRNA-TIR in recombinant HAb18GEF/pET21a + vector was reduced with following methods: (1) optimization of secondary structure (2) optimization of codon adaptation. These optimization were realized by non-continual site-directed mutagenesis without changing any amino acid sequence in TIR. After being checked through restriction endonuclease digestion and confirmed through nucleotide sequencing, the pre-optimized and post-optimized recombinant vectors were transformed into competent E. coli JM109-DE3. The resulted recombinant clones were selected randomly and induced by IPTG at 37 degrees C. The induced production of these recombinants was analyzed by SDS-PAGE, indirect ELISA, Western blot, and cell fractionation assay. The amount of HAb18GEF mRNA was also detected by RNA dot blot between pre-optimized recombinant and post-optimized recombinant. The results revealed that recombinant non-fused vectors HAb18GEF/pET21a + were successfully constructed and optimized in the secondary structure and codon adaptation of TIR respectively. The HAb18GEF was expressed efficiently in a non-fusing way in recombinant E. coli by secondary structure optimization or codon adaptation optimization. Whereas, no expression of HAb18GEF was detected in pre-optimized recombinants. The non-fused expression products-HAb18GEF, mainly as inclusion body in E. coli, yielded highly above 29.3%. A trait of expression HAb18GEF was also detected both in intermembrane space and in culture medium due to over-expression and cell leakage. Difference in non-fused expression level of HAb18GEF between secondary structure optimization and codon adaptation optimization was negligible. No difference in amount of transcribed mRNA of HAb18GEF between the pre-optimized and the post-optimized recombinants was detected. To sum up, it's feasible to express hepatoma associated antigen HAb18GEF in a non-fusing way by reducing the stability of TIR in mRNA.
Antigens, Neoplasm ; biosynthesis ; genetics ; Base Sequence ; Basigin ; biosynthesis ; genetics ; Carcinoma, Hepatocellular ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Extracellular Matrix Proteins ; metabolism ; Genetic Vectors ; genetics ; Humans ; Liver Neoplasms ; genetics ; immunology ; Molecular Sequence Data ; Nucleic Acid Conformation ; Protein Biosynthesis ; genetics ; RNA Stability ; RNA, Messenger ; biosynthesis ; genetics

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