The plasma levels of TNF alpha was determined dynamically using TNF ELISA; and TNF mRNA in spleen cells from varied groups was determined using Northern Blot. The results showed that: (1) Elevated levels of TNF-alpha in plasma from mice of immunologically- mediated aplastic anemia. (2) Induced production of TNF from spleen cells were much lower in exprimental groups than that in normal control group. (3) Decreased TNF-? gene expression present in spleen cell in AA group compared with normal control group.

pPIC9 was used as a template and ?-factor gene of Saccharomyces cerevisiae was amplified by PCR.The gene was cloned in the intracellular expression vector pYES2/CT for Saccharomyces cerevisiae and the secreting expression vector named pYES2/CT/?-factor(pYC?)was constructed.The gene of mannase(man)from recombinant vector of pKLAC1-man(pKLman) was cut by restriction enzymes and linked with pYC?.This recombinant vector pYC?-man was used to determine the secretory ability and stability of pYC?.The excellent secretory ability of pYC? was proved by two experiments.One showed that INVSc1/pYC?-man clones formed the clear rings around the clones on the medium contained trypan blue,while INVSc1/pYC? clones had no rings.Further analysis of mannase activity of extracellular supernatant and intracellular extracts showed that both extracellular and intracellular mannase activities of INVSc1/pYC? were not detected,while INVSc1/pYC?-man had evident extracellular mannase activities and no intracellular mannase activities.The stability of pYC? was also very good proved by continuous cultivation for about 150h.

The expression cDNA library of A. tabescens was constructed by SMART technique, which useλTriplEx2 as a vector. The titer and the percentage of the constructed library were about 1.0 × 106pfu/mland 98.3% respectively, and the titer and the capacity of the amplified library were about 3.1 × 108pfu/mland 4.2 × 1010. The library was used to provide expressed sequence tags (ESTs). 147 Expressed SequenceTaqs (ESTs) were gained from 176 clones, which were selected randomly and sequenced at the 5'end. Thesequences were submitted to the EMBL database. Blasting the sequences in the GenBank, 43 of them werefound that they have significant similarity with data in GenBank. EST AJ620046 was has significantsimilarity with the arabinosidase of Bacteroides thetaiotaomicron. Using SMART-RACE a full-length cDNA ofAJ620046 was successfully obtained. In order to initially characterize the biochemical properties ofAJ620046, the ORF of AJ620046 named AF was cloned and expressed in Pichia Pastoris yeast.Recombinant pHIL-S1-AF constructed by inserting AF into pHIL-S1 was transformed into Pichia PastorisGS115. Preliminary experiments indicated that AJ620046 was expressed as a 32 kDa protein in recombinantyeast.

A novel biosensor for aflatoxin B1 detecting has been reported. The biosensor electrode for AFB1 detecting was assembled by immobilized aflatoxin-oxidoreductase using open-ended multi-walled carbon nanotubes as matrix. Its linear range was between 0.16μM and 3.2μM. And if the specific anti-aflatoxin B1 antibody and aflatoxin oxidoreductase were both immobilized on the electrode with Multi-Walled carbon nanotubes, the detection limit of the modified electrode could be 16 nM with a 10 times improved sensitivity. The aflatoxin enzyme biosensor assembled this way strode one step forward its practical application.

Objective To evaluate the value of intraduetal ultrasonography (IDUS) in diagnosing biliary and pancreatic disorders. Methods The findings by endoscopic retrograde cholangiopancreatography (ERCP) and IDUS from 19 patients with suspected biliary and pancreatic disorders from July 2006 to August 2007 in our hospital were analyzed retrospectively. Results Of the 19 patients, 17 had obstructive jaundice (including 6 eases of cholangiocarcinoma, 2 pancreatic adenocareinoma, 2 gallbladder carcinoma, 2 chole-docholithiasis with bile duct stricture, 2 autoimmune pancreatitis, 1 papillary adenocarcinoma, 1 papillary adenoma, and 1 sclerosing cholangitis) and 2 intraduetal papillary mueinous tumor (IPMT). The diagnosis was confirmed by surgery and pathological findings in 11 patients. The diagnostic accuracy of ERCP and IDUS was 73. 7% (14/19) and 84. 2% (16/19), respectively, and that of ERCP combined with IDUS was 89. 5% (17/19). The sensitivity and specificity of ERCP to differentiate benign bile duct strictures from ma-lignant ones were 100. 0% (11/11) and 83.3% (5/6), respectively; and those of IDUS were 100. 0%(11/11) and 100. 0% (6/6), respectively. The sensitivity and specificity of ERCP in diagnosing cholan-gioeareinoma were 83.3% (5/6) and 60% (3/5), respectively; and those of IDUS were 100. 0% (6/6) and 40. 0% (2/5), respectively. Conclusion Combination of ERCP with IDUS can improve the diagnostic accuracy of pancreaticobiliary disorders. Additionally, IDUS shows higher sensitivity and specificity in differ-entiation between benign and malignant bile duct strictures, but it is still difficult to identify the etiologic factors of malignant bile duet strictures by IDUS.

Objective Tyrphostin AG1024(3-Bromo-5-t-butyl-4-hydroxybenzylidenemalonitrile) is a specific insulin like growth factor type Ⅰ receptor tyrosine kinase blocker,this study is to investigate the effect of AG1024 on the proliferation and apoptosis of hepatocellular carcinoma cell lines.Methods Treated with AG1024 on vailed concentrations(0～40 μmol/L),human hepatocellular carcinoma cel lines HepG2 and SMMC-7721 were observed for morphological and molecular biology changes,the effect of AG1024 on the cell lines proliferation invasion ability as well as apoptosis was evaluated. Results MTT showed that AG1024 dose-dependently inhibited the proliferation of hepatocellular carcinoma cells,flow cytometry suggested that AG1024 significantly promoted cell lines apoptosis,the cell invasion assay indieated that AG1024 significantly inhibited cell's invasion ability.RT-PCR showed over-expression of IGF-IR in liver cancer cells.and AG1024 dose-dependently increasedtheexpressionofcytochreme C. According totheresultsof Western, blotting,the phosphor-ERK and procaspase-3 were down-regulated while the total ERK remained unchanged. Conclusion AG1024 as a specific IGF-IR blocker blocks the downstream signaling cascade and thus inhibits the proliferation of hepatocellular carcinoma cells and induces cell's apoptosis.

AIM To develop an HPLC method for the simultaneous content determination of berberine hydrochloride,jatrorrhizine hydrochloride,palmatine hydrochloride,kaempferol,luteolin and apigenin in Qingre Zhili Pills (Berberidis Radix and Portulacae Herba).METHODS The analysis of methanol extract of this drug was performed on a 35 ℃ thermostatic Agilent TC-C18 column (4.6 mm × 250 mm,5 μm),with the mobile phase comprising of acetonitrile-0.02％ potassium dihydrogen phosphate flowing at 0.9 mL/min in a gradient elution manner,and the detection wavelengths were set at 265 nm and 360 nm.RESULTS Six constituents showed good linear relationships within their own ranges (r≥0.999 3),whose average recoveries were 97.16％-99.90％ with the RSDs of 0.62％-1.48％.CONCLUSION This simple method can be used for the rapid quality control of Qingre Zhili Pills.

Objective To profile the intraspecific type of Trichophyton mentagrophytes clinically isolated from different anatomical sites of patients, and to compare the performance of different target sites for the identification of Trichophyton mentagrophytes complex strains. Methods A total of 48 Trichophyton mentagrophytes strains, which were clinically isolated from Department of Dermatology, Wuhan No. 1 Hospital in the latest 3 years, were included in this study. The phenotypes of these Trichophyton mentagrophytes isolates were primarily determined by morphological observation and the urease test. PCR was performed to amplify the nuclear ribosomal internal transcribed spacer(ITS) region and the D1?D2 domains of the large?subunit ribosomal DNA(28S rDNA)followed by DNA sequencing. Then, Clustal X2 software and MEGA 6.0 software were used to analyze the ITS and D1?D2 sequences and to build phylogenetic trees by the maximum?likelihood method (bootstrap = 2000). Results As the ITS sequence?based phylogenetic tree showed, the probability that the 48 isolates were grouped into the Trichophyton interdigitale clade reached 92%. However, Trichophyton interdigitale could not be effectively differentiated from Trichophyton quinckeanum by the D1?D2 sequence?based phylogenetic tree. In addition, Trichophyton interdigitale showed various appearances, including woolen type, downy type, cream type, powdery type and granular type. Conclusions Trichophyton mentagrophytes can be identified to the species level based on the sequence of ITS region, which shows higher efficiency in identifying Trichophyton mentagrophytes complex than the D1?D2 domains. Morphological characteristics can not serve as the basis for intraspecific typing of Trichophyton mentagrophytes.

Objective To observe the clinical effects of irbesartan combined with ambrette capsule for treatment of mild-to-moderate proteinuria in type 2 diabetic nephropathy. Methods Foutty-six patients with diabetic nephropathy( DN),who meet the diagnostic criteria for diabetes and DN diagnostic criteria which WHO promulgated in 1999 years,were randomly divided into control group(n =23)and treatment group(n =23) . Patients in control group were administrated conventional hypoglycemic drugs,lipid,calcium antagonists step-down and diet control,while in treatment group were administrated irbesartan(75 mg,1 times/d)combined with ambrette capsule(5 pills,3 times/d)on this basis. Both the treatment duration was 8 weeks. Urine protein,blood urea nitrogen( BUN),serum creatinine( SCr)and C reactive protein( CRP)were measured respectively before treatment and 8 weeks after treatment. Results After 8 weeks treatment,the total effective rate in treatment group was 87. 0%(20/23),higher than that of control group(52. 2%(12/23);χ2 =6. 571,P﹤0. 05). The CRP had decreased significantly in treatment group( from( 11. 7 ± 0. 9 ) mg/L to( 5. 8 ± 0. 3 ) mg/L ) which compared with control group(from(10. 1 ± 0. 6)mg/Lto(9. 8 ± 0. 4)mg/L;P﹤0. 05). But BUN and SCr had no significant difference compared with pre-treatment( P ﹥ 0. 05 ). Conclusion Irbesartan combine with ambrette capsule can significantly decrease the urine protein and regulate on the micro-inflammatory state. It is worthy of popularization and application.

Objective To determine the dose-response relationship of 0.2％ ropivacaine for ultrasoundguided stellate ganglion block (SGB).Methods Seventy-five ASA physical status [or Ⅱ patients with migraine,aged 23-55 yr,with body mass index of 22-28 kg/m2,scheduled for elective ultrasound-guided SGB,were randomly divided into R1-5 groups (n =15 each) using a random number table.In R1,R2,R3,R4 and R5 groups,the patients underwent ultrasound-guided SGB with 0.2％ ropivacaine 1,2,3,4 and 5 ml,respectively.A successful SGB block was confirmed by the onset of ptosis (Horner syndrome) on the injected side.Probit analysis was used to calculate the effective dose of 0.2 ％ ropivacaine in 50 ％ and 95 ％ of the patients (ED50 and ED95) and 95％ confidence interval (95％ CI).Results The ED50 of 0.2％ ropivacaine for ultrasound-guided SGB was 2.2 ml (95％CI 1.9-2.5 ml) and ED95 was 3.2 ml (95％CI 2.8-4.1 ml).Conclusion The ED50 and ED95 of 0.2％ ropivacaine for ultrasound-guided SGB are 2.2 and 3.2 ml,respectively.