To investigate the induction of apoptosis of mouse colonic adenocarcinoma CT26 cells by recombinant Clostridium difficile toxin B (rTcdB), CT26 cells were exposed to different concentrations of rTcd B. Inhibition of cell proliferation was measured by MTT assay. The activation of Caspase 3 was measured by colorimetric method. Cell morphological analysis and flow cytometry were performed to confirm cell apoptosis. rTcd B inhibited the proliferation of CT26 cells in a timeand dose-dependent manner. Caspase 3 activity in CT26 cells was elevated remarkably after rTcd B exposure for 6 h, 12 h, 18 h or 24 h, as compared with the control group. Morphological changes were observed by fluorescence microscopy. The exposure of rTcd B to CT26 cells induced a timeand dose-dependent apoptotic cell death as determined by flow cytometry analysis. The results showed that recombinant Clostridium difficile toxin B induced apoptosis of CT26 cells.

0.05).P300 and amplitudes of the mismatch negativity(MMN) of group A were lower than that of group B.PL of MMN was obviously prolonged.By comparing PL of group A with thar of group B,there was obvious difference(P

Objective To construct a eukaryofic expression vector for A PP695 gene carrying green fluorescence protein (GFP) gene. Methods RT-PCR was used to amplify the full-length APP695 cDNA. The PCR products and the eukaryotic expression vector pIRES2-EGFP were digested by restriction endonueleases, and the digested APP695 gene was inserted into the digested eukaryotic expression vector. The positive recombinants were identified by PCR analysis, Nhe Ⅰ and Hind Ⅲ digestion and sequence analysis. Results The 2088-bp DNA fragment was amplified by PCR from the plasmid pCB6, and an identical DNA fragment was also amplified from the recombinants. The products of double restriction enzyme digestion were A PP695 gene with a 5.3-kb DNA fragment. Sequence analysis confirmed successful insertion of A PP695 gene into pIRES2-EGFP vector. Conclusion The eukaryotie expression vector pIRES2/APP695-EGFP has been successfully constructed.

Aspirin ; therapeutic use ; Diagnosis, Differential ; Female ; Humans ; Immunoglobulins, Intravenous ; therapeutic use ; Infant ; Male ; Mucocutaneous Lymph Node Syndrome ; diagnosis

OBJECTIVETo study the genotype distribution of extended-spectrum beta-lactamases (ESBLs) in ESBLs-producing Escherichia coli (E. coli) isolates from posthepatitic cirrhosis' patients with bloodstream infection.

METHODSE. coli were isolated in bloodstream from patients with posthepatitic cirrhosis between January and December in 2011. The strains were identified by VITEK-II. The antibiol susceptibility tests were performed with K-B method. beta-lactamases genes were detected multi-PCR, PCR, sequence and blast.

RESULTSA total of 79 non-duplicate clinical isolates of E coli were consecutively collected from liver cirrhosis' patients with bloodstream infection. There were 20 isolates produced TEM-1 type beta-lactamases and 1 isolate produced SHV-1 typebeta-lactamases. 40 clinical isolates were detected to produce CTX-M type ESBLs, there were 20 CTX-M-1 group and 26 CTX-M-9 group, including 6 stains habouring both CTX-M-1 and CTX-M-9 group. Eight CTX-M genotypes were confirmed by sequencing of the PCR products, including CTX-M-3, CTX-M-14, CTX-M-15, CTX-M-24, CTX-M-28, CTX-M-31, CTX-M-65 and CTX-M-79.

CONCLUSIONCTX-M genotype ESBLs was the most popular extended-spectrum beta-lactamases in E. coli isolated from liver cirrhosis' patients with bloodstream infection. The CTX-M-14 is the dominant epidemic type.

Bacteremia ; microbiology ; Cross Infection ; microbiology ; Drug Resistance, Bacterial ; Escherichia coli ; drug effects ; enzymology ; genetics ; isolation & purification ; Escherichia coli Infections ; microbiology ; Escherichia coli Proteins ; genetics ; Genotype ; Hospitalization ; statistics & numerical data ; Humans ; Liver Cirrhosis ; therapy ; Microbial Sensitivity Tests ; beta-Lactamases ; genetics ; metabolism

Aim To investigate the influence of inhibi-tory nanocomposite on EC-9706 cells and the effect of nanocomposite on ESCCAL _ 1 LncRNA expression, siRNA-loaded nanocomposite being prepared as non-vi-rus delivery system Methods Mesoporous silica nano-particles were prepared by sol-gel method under room temperature and coated by cationic polymerpolyethylen-imine (PEI)on the surface to stay positive charge, which could facilitate its combination with negatively charged ESCCAL _ 1 siRNA. The size and surface charge of nanocomposite were determined by laser par-ticle analyzer and TEM. The inhibitory rate of nanopar-ticles on EC-9706 cells was detected by MTT methods. Entrapment efficiency was determined by agarose gel e-lectrophoresis. The uptake-siRNA was detected by flu-orescence microscope. The expression of ESCCAL _1 LncRNA was detected by RT-PCR. Results The MSNP appeared to have a high dispensability and hom-ogeneous size by particle size analyzer and transmission electron microscopy(TEM). The formed nanoparticles had a surface mesoporous diameter of 3 ~ 5 nm. The proliferation of ESCCAL_1 was inhibited significantlly (P < 0. 05),and the 72h inhibitory rate was (54. 93 ± 2. 6)%;the siRNA loading could be effectively up-taken by EC-9706 cells;ESCCAL_1 silencing efficien-cy was 69. 5% . Conclusions The tumor targeting nanocomposite with high encapsulation efficiency is prepared. The proliferation of esophageal cancer EC-9706 cells can be effectively inhibited by anocompos-ite-mediated siRNA,and the expression of ESCCAL_1 is effectively silenced in EC-9706 cells. The nanocom-posite is an efficient gene delivery system and may have potential application in gene therapy.

OBJECTIVETo explore a technology for diagnosing VHL mutations from a single cell and provide experimental evidences for the feasibility of applying technology in detecting genetic mutations from a single cell.

METHODSAfter whole genome amplification (WGA) based on multiple displacement amplication (MDA) for a single cell, we did regular PCR following sequencing and detected the genotypes using the real time PCR based on TaqMan probes. We detected VHL mutations by the different terminal fluorescent changing.

RESULTSThe rate of amplification for single cell based on MDA was 90.91%. The rate of contamination was 0. After sequencing, the allele drop out (ADO) rate of heterozygotes was 26.67%(8/30); combined with the different terminal fluorescent changing, the rate of ADO of heterozygotes was 16.67%.

CONCLUSIONWGA based on MDA for a single cell followed by regular PCR with sequencing and real time PCR can specially and accurately detect the VHL genotypes of single cells.

Base Sequence ; DNA Mutational Analysis ; Female ; Genotype ; Humans ; Lymphocytes ; metabolism ; pathology ; Middle Aged ; Molecular Sequence Data ; Mutation ; Polymerase Chain Reaction ; Preimplantation Diagnosis ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics ; von Hippel-Lindau Disease ; blood ; genetics

OBJECTIVESTo study the effects of trichostatin A (TSA) on protein-protein interaction between HBx and histone deacetylase protein 1 (HDAC1).

METHODSBoth HBx and HDAC1 expressing vectors were constructed by the method of routine molecular cloning. The expression of HBx and HDAC1 were observed by Western blot assay. The protein-protein interaction was tested between HBx and HDAC1 by GST pull-down in vitro as well as co-immunoprecipitation in vivo.

RESULTSBoth HBx and HDAC1 expressing vectors were successfully constructed. Protein-protein interaction between HBx and HDAC1 existed both in vitro and in vivo. TSA, an inhibitor of HDAC1, had no effect on the interaction between HBx and HDAC1.

CONCLUSIONSHBx interacts with HDAC1 in vivo and in vitro in a non- TSA dependent way.

Histone Deacetylase 1 ; metabolism ; Humans ; Hydroxamic Acids ; metabolism ; Immunoprecipitation ; Plasmids ; Protein Interaction Mapping ; Trans-Activators ; metabolism

Acquired Immunodeficiency Syndrome ; epidemiology ; virology ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; China ; epidemiology ; Female ; Humans ; Male ; Middle Aged ; Young Adult

This study was aimed to investigate the effects of rituximab (RTX), a chimeric human anti-CD20 monoclonal antibody, on lymphoma cell injury induced by X ray irradiation. The human Burkitt EBV-infected and moderate radioresistance lymphoma cells (Namalwa) were used in the this study. Cytotoxicity of rituximab combined with X ray irradiation on Namalwa cells was measured by sulforhodamine B (SRB)-staining; the apoptosis of Namalwa cells was detected by flow cytometry with FITC-Annexin V/PI double staining; the morphologic changes of cells were observed under transmission electron microscope (TEM) and the change of intracellular free calcium level ([Ca(2+)]i) in response to irradiation and rituximab was determined by means of the fluorescent dye fluo-3 and confocal microscopy. The results showed that the growth inhibition in Namalwa cells exposed to irradiation was enhanced by treatment with rituximab. Compared with irradiation alone, rituximab combined with irradiation significantly induced the cell apoptosis and a sustained rise of intracellular free calcium ([Ca(2+)]i) level in Namalwa cells; the serial apoptotic appearances of cells could be observed under TEM. It is concluded that rituximab can enhance the sensitivity of lymphoma cells on X ray irradiation as to induce cell more apoptosis, in this process the intracellular free calcium ([Ca(2+)]i), as an intracellular signaling molecule probably plays an important role.
Antibodies, Monoclonal, Murine-Derived ; immunology ; pharmacology ; Antigens, CD20 ; immunology ; Apoptosis ; Calcium ; analysis ; Cell Line, Tumor ; Humans ; Lymphoma ; drug therapy ; metabolism ; pathology ; Radiation Tolerance ; drug effects ; Rituximab