Objective To study the effect of siRNA targeting survivin and tumor necrosis factor related apoptosis-inducing ligand on the proliferation and apoptosis of hepatocellular carcinoma ( HCC) cells. Methods siRNA eukaryotic expression vector was constructed and the effects of soluble tumor necrosis factor related apoptosis-inducing ligand ( sTRAIL) on HCC cells were observed. Results The recombinant plasmid Psilence ( + )-survivin was successfully constructed. Survivin mRNA expression inhibition ratio reached 73% by RT-PCR. Observed by MTT method sTRAIL failed to inhibit the survival rate of HepG2、HepG2/Silence( - ) cells at 12 h、24 h、48 h when compared to control groups. With the survivin gene being inhibited, the survival rate of HepG2/Silence( + ) cells(0. 518?0. 017) decreased in 12 h compared to control groups (0. 741?0. 005 ) and reached the lowest level in 48 h ( P

With patients' general situation, medication use, occurrence time of adverse drug reaction/event (ADR/ADE), clinical manifestations and prognosis as reference items, a retrospective study was made for 315 cases with ADR/ADE induced by Gastrodin in Chongqing from January 2008 to June 2014, in order to analyze the characteristics of ADR/ADE and provide reference for rational clinical medication. The results showed that among the 315 cases with ADR/ADE, 143 cases (45.4%) were males and 172 cases (54.6%) were females, most of them (74.9%) were aged above 45; 60 cases (19.0%) with ADE were caused by off-label indications and 66 cases (21.0%) with ADE were caused by over dosage; ADR/ADE cases induced by intravenous drip mainly happened within 30 min (85.5%), ADR/ADE cases induced by oral administration mainly happened within 2 h (74.4%), and all of ADR/ ADE cases induced by intramuscular injection happened within 10 min. Totally 593 ADR/ADE cases were reported, which were mainly damages in gastrointestinal system, skin and its adnexa; And 61.9% of ADR/ADE cases were newly reported. It is suggested that medical workers shall learn about the regularity and characteristics of ADR/ADE induced by gastrodin, apply it in clinic with standards, pay close attention to changes of patients' situations and attach importance to the monitoring of ADR/ADE, so as to enhance the safety of medication.
Adolescent ; Adult ; Aged ; Benzyl Alcohols ; administration & dosage ; adverse effects ; Child ; Child, Preschool ; China ; epidemiology ; Drug-Related Side Effects and Adverse Reactions ; epidemiology ; Drugs, Chinese Herbal ; administration & dosage ; adverse effects ; Female ; Gastrointestinal Tract ; drug effects ; Glucosides ; administration & dosage ; adverse effects ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Skin ; drug effects ; Young Adult

To improve ethanol production in Saccharomyces cerevisiae,an integration plasmid pUPGKAT with PGK promoter(phosphoglycerate kinase promoter),adh1 gene(the coding sequences of alcohol dehydrogenaseⅠ) and CYC1 terminator(Cytochrome c transcription terminator) was constructed.Firstly,a fusion fragment composed of PGK promoter and adh1 gene was generated by over lap extension PCR and ligated into pUG6 resulting in plasmid pUPGKA.Subsequently,CYC1 termi nator was amplified from pSH65 by PCR and ligated to the SpeⅠand SacⅡrestriction site of pUPGKA.To integrate PGK-adh1-CYC1 into S.cerevisiae genome,pUPGKAT was digested by TthⅢⅠand the lin-earized plasmid was used to transform S.cerevisiae YS2-△adh2(adh2 disrupted strain) by lithium acetate method.The yeast mutant YS2-△adh2-adh1 which had the adh1 gene placed under the PGK promoter and harbored the adh2 deletion was constructed.Anaerobic fermentation showed overexpression of adh1 by PGK promoter resulted in a 8.84% higher ethanol production compared to YS2-△adh2.

Red in vivo recombination is a new kind of genetic engineering technique based on homologous recombination. In this work, plasmid pKD46 which expresses Red recombination proteins is transferred into Escherichia coli strain DH5?.The kanamycine resistant gene is generated by PCR by using primers with homology to hisDCB gene of E.coli chromosome. Thus, the hisDCB gene was replaced with kanamycine resistant gene by the plasmid recombination system, then the resistant gene was eliminated by a helper plasmid encoding the FLP recombinase. At last, a E.coli histidine auxotroph which is sensitive to kanamycine was got. The results indicate that Red in vivo recombination is a convenient method to construct auxotrophs.

OBJECTIVETo observe the expression change of signal regulatory protein alpha1 (SIRPalpha1) in autoimmune hepatitis (AIH) and approach the relationship between SIRPalpha1 and the extent of inflammation.

METHODSImmunohistochemistry is used to detect the expression of SIRPalpha1 in the paraffin section preparations of 33 AIH and 10 normal hepatic tissue.

RESULTSSIRPalpha1 is positive or weakly positive expressed in AIH. The staining is localized in the cytoplasm of Kupffer cells in the hepatic sinusoid with focal distribution. It is negative in normal hepatic tissue. In light AIH, it is negative or weakly positive expressed with a 36.4 percent of the positive rate (4/11). The positive or strong positive expression is found in the moderate AIH with an 84.2 percent of the positive rate(16/19). There is statistical significance between both light AIH, moderate AIH and severe AIH (P less than 0.001) and moderate AIH and light AIH (P less than 0.001). There is no statistical significance between both light AIH and severe AIH (P = 0.145 ) and moderate AIH and severe AIH (P = 0.084).

CONCLUSIONSAs a negative regulatory factor, the expression of SIRPalpha1 in hepatic sinusoid Kupffer cells is some associated with the extent of AIH.

Adolescent ; Adult ; Aged ; Antigens, Differentiation ; metabolism ; Cell Communication ; Child ; Female ; Hepatitis, Autoimmune ; metabolism ; pathology ; Hepatocytes ; metabolism ; pathology ; Humans ; Kupffer Cells ; metabolism ; pathology ; Male ; Middle Aged ; Receptors, Immunologic ; metabolism ; Young Adult

PURPOSE: The purpose of this study was to investigate the influence of chronic virus-related liver disease severity on propofol requirements. MATERIALS AND METHODS: In this study, 48 male patients with chronic hepatitis B infection were divided into three groups according to Child-Turcotte-Pugh classification of liver function (groups A, B, and C with mild, moderate and severe liver disease, respectively). After intubation, propofol concentration was adjusted by +/-0.3 microg/mL increments to maintain bispectral index in the range of 40-60. Target propofol concentrations at anesthesia initiation, pre-intubation and pre-incision were recorded. RESULTS: The initial concentration used in group C was significantly lower than that used in group A or B (p<0.05), whereas no difference was observed between groups A and B. At pre-intubation, the actual required concentration of propofol increased significantly (3.2 microg/mL) in group A (p<0.05), which lead to significant differences between the groups (p<0.05). At pre-incision, the requirements for propofol decreased significantly in both groups A and B (3.0 microg/mL and 2.7 microg/mL, respectively) compared with those at pre-intubation (p<0.05), and were significantly different for all three groups (p<0.05), with group C demonstrating the lowest requirement (2.2 microg/mL). The required concentrations of propofol at pre-incision were similar to those at induction. CONCLUSION: In this study, propofol requirements administered by target-controlled infusion to maintain similar depths of hypnosis were shown to depend on the severity of chronic virus-related liver dysfunction. In other words, patients with the most severe liver dysfunction required the least amount of propofol.
Adult ; Anesthesia ; Anesthetics, Intravenous/*administration & dosage ; Chronic Disease ; Electroencephalography ; Hepatitis B, Chronic/complications/drug therapy/*surgery ; Humans ; Liver Diseases/*complications/surgery ; Male ; Middle Aged ; Piperidines/*administration & dosage ; Propofol/*administration & dosage ; Virus Diseases/*complications/surgery

OBJECTIVEThe aim of this study was to explore the possibility of creating a toxin, C-CPE-ETA', by fusing C-terminal high affinity binding domain of CPE (C-CPE) with a truncated form of Pseudomonas aeruginosa exotoxin A (ETA') and to examine whether C-CPE-ETA' could specifically target CLDN-3, 4 molecule and the targeted toxin was cytotoxic against CLDN-3,4-overexpressing ovarian cancer.

METHODSCLDN-3 and CLDN-4 expressions were analyzed at the mRNA level in three ovarian cancer cell lines and epithelial ovarian cancer tissues from 20 patients. After transforming an expression plasmid of C-CPE-ETA' into E. coli BL21 (DE3) plysS strain, the recombinant protein was purified using His-Bind resin chromatography column and analyzed by Western blot and Coomassie blue staining. The specific binding, proapoptotic and cytolytic activities were evaluated by flow cytometry, fluorescence microscopy with the JC-1 probe and MTT assay in CLDN-3,4-overexpressing ovarian cancer cells.

RESULTSQuantitive RT-PCR results showed there existed high levels of CLDN-3 and CLDN-4 in ovarian cancer cells, CAOV3, OVCAR3 and SKOV3. Moreover, high expressions of CLDN-3 and CLDN-4 were observed in 90.0% (18/20) and 60.0% (12/20) of ovarian cancer tissues, with an expression level 10-fold higher than that in the normal ovarian tissue. A 58 000 recombinant protein C-CPE-ETA' was demonstrated by Western blot and Coomassie blue staining. Purified and recombinant C-CPE-ETA' was bound with high affinity to CLDN-3,4-overexpressing ovarian cancer cells, CAOV3, OVCAR3 and SKOV3 cells. C-CPE-ETA' was strongly proapoptotic and cytotoxic towards the CLDN-3,4-overexpressing ovarian cancer cells. The concentration of IC(50) was 7.364 ng/ml for CAOV3 cells, 8.110 ng/ml for OVCAR3 cells and 22.340 ng/ml for SKOV3 cells, respectively. However, control CLDN-3,4-deficient cell line HUVEC was not susceptible to the recombinant C-CPE-ETA' at a concentration up to 10 µg/ml.

CONCLUSIONSThe C-CPE-ETA' protein exhibits remarkably specific cytotoxicity for CLDN-3,4-overexpressing ovarian cancer cells. Its therapeutic potential warrants further development for ovarian cancer molecular targeted therapy.

ADP Ribose Transferases ; metabolism ; physiology ; Apoptosis ; Bacterial Toxins ; metabolism ; Cell Line, Tumor ; Claudin-3 ; Claudin-4 ; Claudins ; genetics ; metabolism ; Enterotoxins ; metabolism ; physiology ; Exotoxins ; metabolism ; physiology ; Female ; Humans ; Immunotoxins ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Recombinant Fusion Proteins ; metabolism ; physiology ; Virulence Factors ; metabolism ; physiology

OBJECTIVETo investigate molecular mechanism of tanshinone II A inducing differentiation and apoptosis in acute promyelocytic leukemia NB4 cells.

METHODNB4 cells were cultured in vitro and treated with tanshinone II A and observed cellular morphology, cell category and the cellular proliferation. DNA microarray technique was used to analyze the gene expression profiles of NB4 cells induced by tanshinone II A.

RESULT92.8% of NB4 cells treated with 0.5 mg x L(-1) tanshinone II A were induced into mature neutrophils, in which myetocytes and melamyetocytes were 27.0%, banded and segmented neutrophits 68.2%. Cell growth were inhibited. cDNA microarray showed the enormously expressed 183 genes including 23 differentiation associated genes, and other interrelated genes.

CONCLUSIONTanshinone II A inducing differentiation in NB4 cells may be via regulation of many kinds of genes, especially differentiation associated genes expression. This partially explained the molecular mechanism of tanshinone II A inducing differentiation.

Cell Differentiation ; drug effects ; Cell Line, Tumor ; Diterpenes, Abietane ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; genetics ; metabolism ; Phenanthrenes ; pharmacology

OBJECTIVETo identify the possible mutation at possible sites in different mitochondrial genes that leads to hearing loss in a large Chinese pedigree.

METHODSBlood samples from a Hunan pedigree were obtained with informed consent. Genomic DNA was extracted from peripheral blood leukocytes using kit. The target fragments were amplified and detected by polymerase chain reaction (PCR) and directly sequencing respectively.

RESULTSThe result of direct sequencing revealed the A1555G mutation in 12S rRNA gene was inherited in this pedigree and no one has A7445G mutation or other mutations in its neighborhood region.

CONCLUSIONSequence analysis confirmed that the pedigree carries the A1555G mutation. With some members ever exposure of aminoglycoside antibiotics, mutation of A1555G may play a pivotal role in the pathogenesis of hearing loss in the large pedigree.

Base Sequence ; China ; DNA Mutational Analysis ; DNA, Mitochondrial ; chemistry ; genetics ; Family Health ; Female ; Hearing Loss ; genetics ; Humans ; Male ; Marriage ; Pedigree ; Polymerase Chain Reaction ; RNA, Ribosomal ; genetics ; RNA, Transfer, Ser ; genetics

OBJECTIVESTo establish DHPLC method in detecting mutations of mismatch repair genes, hMLH1 and hMSH2, and to identify germline mutations of hMLH1 and hMSH2 in HNPCC kindreds fulfilling Chinese HNPCC criteria.

METHODSFourteen peripheral blood DNA samples from 14 unrelated HNPCC probands fulfilling Chinese HNPCC criteria were obtained respectively. PCR amplified 35 exons of two main mismatch repair genes, hMLH1 and hMSH2. DHPLC followed by DNA sequencing was used to detect and confirm mutations.

RESULTSa total of 41 colorectal cancers and 19 extracolonic tumors were developed in 14 HNPCC kindreds, and gastric cancer was the most common extracolonic tumor type. Twelve single nucleotide changes were identified by DHPLC in 14 probands. Among them, three were missense mutations, one was a nonsense mutation. Other single nucleotide changes included five single nucleotide polymorphisms, two intron single nucleotide changes, one synonymous mutation. hMLH1 EXON19 CODON749 TAC-->TAG (Tyr-->X), hMSH2 EXON12 CODON629 CAA-->CGA (Gln-->Arg) and hMSH2 EXON15 CODON839 CAT-->CGT (His-->Arg) were new discovered mutations.

CONCLUSIONS(1) DHPLC was considered to be highly effective, convenient technique with consistent results for the mutation detection of hMLH1 and hMSH2 genes. (2) Valid mutations of hMLH1 and hMSH2 genes were identified in about one-third HNPCC kindreds fulfilling Chinese HNPCC criteria and missense mutation was the most common mutational types in this cohort of families.

Adaptor Proteins, Signal Transducing ; Asian Continental Ancestry Group ; genetics ; Base Pair Mismatch ; Carrier Proteins ; genetics ; Chromatography, High Pressure Liquid ; Codon, Nonsense ; Colorectal Neoplasms, Hereditary Nonpolyposis ; genetics ; DNA Mutational Analysis ; Female ; Genetic Testing ; Germ-Line Mutation ; Humans ; Male ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; genetics ; Mutation, Missense ; Nuclear Proteins ; genetics ; Pedigree