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.

Objective To study the risk factors and prognosis of post-transplant diabetes mellitus (PTDM) in recipients with steatotic donor livers.Method The clinical data of 182 patients who underwent liver transplantation from donors with liver steatosis in Tianjin First Central Hospital from June 2002 to December 2010 were retrospectively analyzed.There were sixteen patients with PTDM and one hundred sixtysix without PTDM.The clinical data of these patients were compared and the risk factors were evaluated by COX regression analysis.The 1-,3-,5-year cumulative survival rates were analyzed after liver transplantation.Result The variables which included sex,pretransplant serum creatinine level,model for end-stage liver disease (MELD) score,intraoperative red blood cell transfusion,and post-transplant biliary complications were significantly different between the two groups.Multivariate Cox regression analysis showed that living-donor,pretransplant fasting blood glucose and post-transplant biliary complications could affect the survival time of patients in PTDM group.The 1-,3-and 5-year cumulative survival rates in the PTDM group were 81.3％,68.8％ and 62.5％,which were significantly lower than those in the non-PTDM group (95.2％,86.1％ and 80.7％ respectively,P ＜ 0.05).Conclusions Living-donation,pretransplant fasting blood glucose and post-transplant biliary complications had a worse prognosis in the PTDM group.A comparatively better long-term survival after liver transplantation can be achieved by reducing the risk factors and the occurrence of PTDM.

Objective To evaluate the effect of berberine on fatty liver ischemia-repeffusion (I/R) injury and the relationship with endoplasmic reticulum stress in liver tissues.Methods Thirty-two male Wistar rats,aged 4 weeks,weighing 100-150 g,were divided into 4 groups (n=8 each) using a random number table method:control group (group C),fatty liver group (group FL),I/R group and berberine group (group BBR).Rats were fed a normal fat diet for 12 weeks,normal saline 3.5 ml was given intragastrically for 4 weeks starting from 9th week,and rats only underwent simple laparotomy in group C.Rats were fed a high-fat diet (45％ energy originating from fat) for 12 weeks,and the other treatments were similar to those previously described in group C.Rats were fed a high-fat diet (45％ energy originating from fat) for 12 weeks,normal saline 3.5 ml was given intragastrically for 4 weeks starting from 9th week,and then the model of liver I/R injury was established in group I/R.Rats were fed a high-fat diet (45％ energy originating from fat) for 12 weeks,berberine solution (300 mg/kg) 3.5 ml was given intragastrically for 4 weeks starting from 9th week,and then the model of liver I/R injury was established in group BBR.Hepatic ischemia was induced by clamping the portal vein,hepatic artery,right gastric vein,and supra-and inferior-hepatic vena cava to perform cold perfusion with 4 ℃ lactated Ringer's solution lasting for 30 min,followed by reperfusion.The serum triglyceride (TG) concentrations were determined after 4,8 and 12 weeks of diet.Blood samples were collected at 6 h of reperfusion for measurement of serum aspartate transminase (AST) and alanine transaminase (ALT) concentrations.Livers were removed after blood sampling at 6 h of reperfusion and liver tissues were obtained and stained with oil red O and haematoxylin and eosin for examination of pathological changes and for determination of the expression of Bip,CCAAT/enhancer-binding protein homologous protein (CHOP),protein kinase RNA-like endoplasmic reticulum kinase (PERK) and phosphorylated PERK (p-PERK) (by Western blot).p-PERK/PERK ratio was calculated.Results Compared with group C,the serum TG,ALT and AST concentrations were significantly increased,the expression of Bip and CHOP was up-regulated,p-PERK/PERK ratio was increased (P＜0.05),lipid deposition was increased,and liver steatosis was found in group FL.Compared with group FL,the serum AST and ALT concentrations were significantly increased,the expression of Bip and CHOP was up-regulated,pPERK/PERK ratio was increased (P＜0.05),and the pathological changes of liver tissues were accentuated in group I/R.Compared with group I/R,the serum TG,ALT and AST concentrations were significantly decreased,the expression of Bip and CHOP was down-regulated,p-PERK/PERK ratio was decreased (P＜ 0.05),lipid deposition was reduced,and the pathological changes of liver tissues were significantly attenuated in group BBR.Conclusion Berberine can ameliorate fatty liver I/R injury,and the mechanism may be related to inhibiting endoplasmic reticulum stress in liver tissues of rats.

Objective To explore the effect of berberine (BBR) on steatotic liver ischemia reperfusion injury and analyze the role of endoplasmic reticulum stress and autophagy .Methods Thirty-four Wistar rats were fed with a high-fat diet for 12 weeks and 2 rats were randomly selected after 8 weeks to observe pathological changes and confirm the model of steatotic liver successfully .Then before opening and closing abdominal cavity ,32 rats were divided into I/R group (normal saline was intragastrically 4 weeks before performing cold I/R treatment) ,BBR group (normal saline was replaced by BBR ,BBR was intragastrically at a dose of 300 mg·kg-1 ·d-1 weeks and others were the same as I/R group) and TG group (TG was intraperitoneally at a dose of 0 .2 mg·kg-124h pre-operation and others were the same as BBR group ) .Then the rats were sacrificed at 6h post-reperfusion .Blood samples were collected from inferior vena cava and hepatic tissues harvested .The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected ,histopathologic changes observed by Hematoxylin & Eosin (HE) staining ,oxidative stress and inflammation determined by ELISA kit and the expressions of p-PERK ,CHOP ,Bip ,LC3 ,Beclin-1 and p62 detected by Western blot .Results As compared with Sham group ,the serum levels of ALT and AST were significantly higher in I/R ,BBR and TG groups (P < 0 .05) .And hepatic histological changes were severe and oxidative stress increased in parallel with the enhancement of pro-inflammation (P< 0 .05) .In BBR group ,the level of hepatic enzymes declined ,liver injury was milder ,oxidative stress decreased and pro-inflammation was lesser compared with I/R and TG groups (P< 0 .05) .Additionally ,as compared with sham group ,the expressions of p-PERK ,CHOP ,Bip ,LC3 ,Beclin-1 and p62 were up-regulated in I/R and BBR groups (P < 0 .05) .TG group increased the levels of LC3 ,Beclin-1 and p62 (P< 0 .05) .Interestingly ,compared with I/R group ,BBR pretreatment down-regulated the expressions of p-PERK ,CHOP ,Bip ,LC3 ,Beclin-1 and p62 (P< 0 .05) .TG group had the higher expressions of LC3 ,Beclin-1 and p62 than those of BBR group (P< 0 .05) .Conclusions BBR pretreatment can protect steatotic liver ischemia reperfusion injury .And the mechanisms may be attributed to the inhibitions of endoplasmic reticulum stress and autophagy .

OBJECTIVETo assess the therapeutic effects of low tension, anti-reflux Roux-y sigmoid neobladder.

METHODSA total of 21 patients (7 male and 14 female) were included, aged 43-87 years. All cases received radical cystectomy and low tension Roux-y sigmoid neobladder procedure for invasive bladder cancer were included in this study. The period of follow-up was from 8 to 79 months (the average was 36 months). Evaluations included urinary flow rate, post voiding residual and filling cystometry.

RESULTSThe mean maximum urinary flow rate, the voiding time and the post voiding residual were 28.1 ml/s (21.4-38.4 ml/s), 17 s(9-28 s) and 0 ml respectively. The cystometric capacity was 480 m1 (350-560 ml). The volume of desire to void was 330 ml (120-410 ml). The bladder pressure was from 14.2 to 18.6 cm H2O (the average bladder pressure was 16.4 cm H2O) at high filling volumes. The maximum voiding pressure was 45.0 cm H2O (23.6-63.4 cm H2O).

CONCLUSIONSThe Roux-y sigmoid neobladder has an adequate capacity at low pressure with a satisfactory continence, and it is an effective method for continent urinary diversion.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Transitional Cell ; surgery ; Colon, Sigmoid ; surgery ; Cystectomy ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Treatment Outcome ; Urinary Bladder Neoplasms ; surgery ; Urinary Diversion ; methods ; Urodynamics

Adolescent ; Body Height ; Chromosomes, Human, X ; Female ; Growth Hormone ; deficiency ; Humans ; Karyotyping ; Monosomy ; Mosaicism ; Turner Syndrome ; genetics ; metabolism ; pathology

Apolipoprotein AI (apo AI), the major protein component of human high-density lipoprotein (HDL), is a single-chain polypeptide of 243 amino acids. Several epidemiological studies have shown that the plasma concentrations of HDL has the role of reverse cholesterol transport (RCT) and inversely correlated with the incidence of coronary artery disease. Because apo AI lacks post-translational modifications, it is convenient to express human apo AI in Escherichia coli expression system. However, there is a poor stability of the mRNA and the apo AI protein in E. coli, it is difficult to express mature apo AI in recombinant bacteria, moreover, even as a fusion protein, apo AI is still sensitive to degradation and can not be cleaved efficiently from the fusion tags. In contrast, proapolipoprotein AI (proapo AI, having an additional polypeptide containing the amino acids Arg-His-Phe-Trp-Gln-Gln at the amino-teminal of the mature protein) proved stable and undegraded in Escherichia coli, and therefore, in this research, an expression system of E. coli including a plasmid of P(R)P(L) tandem promoter was adapted to produce proapo AI. Furthermore, site-directed mutagenesis of the proapo AI cDNA was performed to generate a Clu8Asp mutation in the amino-terminal sequence of proapo AI which created an acid labile Asp-Pro peptide bond between amino acid 8 and 9, and permitted specific chemical cleavage to remove pro-peptide. After inducing with a shift of temperature, yields of recombinant proapo AI achieved about 40% of total cell protein and the recombinant proapo AI expressed proved as a form of inclusion body in cells, so protein need to renature. First of all, the protein was dissolved in buffer with denaturant, and renaturation was carried out on a hydrophobic interaction column (Phenyl Sepharose), ion-exchange chromatography and gel-filtration chromatography were then used to further purify the protein. The purified recombinant apo AI was detected by a set of tests including Western-blotting, Circular dichroism spectra and lipid-binding test, the results shown that recombinant apo AI has similar structural and lipid-binding properties identical to those of native plasma apo AI, which facilitates further research and application.
Apolipoprotein A-I ; biosynthesis ; genetics ; Chromatography, Ion Exchange ; methods ; Escherichia coli ; genetics ; metabolism ; Humans ; Mutagenesis, Site-Directed ; Mutation ; Protein Precursors ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo identify gene mutations of a pedigree with inherited factor V (FV) deficiency.

METHODSThe activated partial thromboplastin time (APTT), prothrombin time (PT), FV activity (FV:C) and FV antigen (FV:Ag) tests were performed for phenotypic diagnosis. The genomic DNA was extracted from the peripheral blood of the proband and all the 25 exons and their flanks of FV gene were amplified by polymerase chain reaction (PCR). The PCR products were screened by direct sequencing and the mutations were further confirmed by restriction enzyme digestion.

RESULTSAPTT, PT, TT, FV:C, FV:Ag of the proband were 249.2 s, 46.6 s, 17.9 s, 0.1% and 1.5%, respectively. FII, FVII, FVIII, FIX, FX activities, vWF and Fg were within normal ranges. Taking the GenBank Z99572 sequence as the reference, four mutations were identified in FV gene of the proband. They were a heterozygous two bases deletion in exon 13 (2238 approximately 2239delAG) introducing a frameshift and a premature stop at codon 689, and a heterozygous missense mutation in exon 23 (G6410T) resulting in the substitution of Gly for Val at codon 2079, respectively. The proband's father and mother were heterozygous for G6410T and for 2238 approximately 2239delAG, respectively.

CONCLUSIONThe severe FV deficiency of the proband is caused by a frameshift mutation of 2238 approximately 2239delAG and a missense mutation of G6410T, which haven't been identified before.

Adult ; Base Sequence ; DNA Mutational Analysis ; Exons ; genetics ; Factor V ; genetics ; metabolism ; Factor V Deficiency ; genetics ; Female ; Frameshift Mutation ; Heterozygote ; Humans ; Infant ; Male ; Mutation, Missense ; Partial Thromboplastin Time ; Pedigree ; Phenotype ; Prothrombin Time ; Thrombin Time

OBJECTIVESTo identify the FXI gene mutations in two Chinese pedigrees of congenital factor XI deficiency.

METHODSThe peripheral blood samples were collected from the probands and their family members and the plasma FXI:C and FXI:Ag were determined. All the exons and exon-intron boundries of FXI gene were amplified with PCR and sequenced thereafter.

RESULTSA nonsense mutation Trp228stop and two missense mutations Glu323Lys and Leu172Pro were disclosed in the two pedigrees. All mutations existed in a heterozygous state.

CONCLUSIONThe FXI gene mutations Trp228stop, Glu323Lys and Leu172Pro attribute to the pathogenesis of the congenital factor XI deficiency in Chinese. The Leu172Pro is identified for the first time.

Adult ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child ; Factor XI ; genetics ; Factor XI Deficiency ; genetics ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation ; Pedigree