Objective To construct eukaryotic expression vector of wild type E 4F1 and the mutant deleting amino acid region 32-81, and to detect the interaction between wild type or mutant E 4F1 and p53 and to study the effect of E4F1 on the expression level of p21.Methods Wild type and mutant sequences of E 4F1 were amplified from the mammary library using standard PCR and recombinant PCR .The sequences were cloned into pXJ 40-MYC vector to generate the MYC-E4F1 and MYC-E4F1(Δ32-81) recombinant plasmids that were transfected into 293T cells and identified by Western blotting . FLAG-p53 and MYC-E4F1 or MYC-E4F1(Δ32-81) were co-transfected into 293T cells and immunoprecipitation assay was performed to detect the interaction of wild type or mutant E 4F1 with p53.Wild type and mutant E4F1 expressing vec-tors were co-transfected into osteosarcoma U2OS cells and the expression of p21was detected.Results Recombinant plas-mids of MYC-E4F1 and MYC-E4F1(Δ32-81) were successfully constructed.Both wild type and mutant E4F1 interacted with p53.Deletion of amino acid region 32-81 of E4F1 increased the interaction .The expression level of p21 was in-creased by wild-type E4F1, but not by mutant E4F1.Conclusion The eukaryotic expression vector of wild type E4F1 and its deletion mutant is successfully constructed .Both of them interact with p53.Deletion of amino acid region 32-81 of E4F1 increases the interaction .This study contributes to further studies on the regulation and mechanism of E 4F1 on p53.

OBJECTIVETo investigate the effect of p53 binding site (+31/+60) of hsp90 beta gene on its transcriptional regulation.

METHODSThe binding site was first inserted into pBS-SK. After the plasmid annealing and elongation with mutagenic and selective primers, nuclease digestion and bacteria transformation was performed twice to select the positive mutated plasmid. Electrophoretic mobility shift assays (EMSA) was employed to detect the binding of hsp90 beta gene fragment containing mutated p53 binding site and Jurkat cell nuclear extract transfected by p53 expression vector.

RESULTSThe sequence analysis profile confirmed a successful mutation of two bases on the core sequence of the second half binding site. EMSA results showed the specific DNA-protein complex band disappeared after the mutation.

CONCLUSIONSThe core sequence of p53 binding site plays a key role in the trans binding of p53 to hsp90 beta gene.

Binding Sites ; HSP90 Heat-Shock Proteins ; genetics ; Humans ; Leukemia, T-Cell ; pathology ; Mutagenesis, Site-Directed ; Mutation ; Transcription, Genetic ; Transcriptional Activation ; Tumor Suppressor Protein p53 ; genetics ; metabolism

OBJECTIVETo construct the localization system involving anti-TfR monoclonal antibody (McAb) and AFP promoters and assess its effect on human hepatoma cell lines.

METHODSThe conjugate of anti-TfR McAb and polylysine (PLL) was made by SPDP and purified by molecular screen chromatography. DNA blocking test determined that the ratio of one pEBAF/tk to six Ab-PLL was the most suitable to couple them. The pEBAF/tk recombinant plasmid bearing HSV-TK gene was coupled to Ab-PLL by noncovalent bond. The pEBAF/tk was transferred into human hepatoma cell line HepG2, SMMC7721 and pulmonary cancer cell line A549 by receptor-mediated gene delivery (Ab-PLL-DNA) and liposome procedure. The growth inhibitory rates of HepG2, SMMC7721 and A549 cells were measured by MTT assay.

RESULTSThe inhibitory rates of HepG2/tk in 100 mg/L and 1 mg/L of GCV were 60.5% and 24.3%, respectively. The inhibitory rate of GCV to SMMC7721 was 23.2% in 3 days. The pulmonary cancer cell A549, A549/tk (Ab) and A549 /tk (lipo) could not be inhibited by the addition of GCV.

CONCLUSIONThe localization system employed in this paper has high specificity, effectiveness and safety for gene therapy. It would be a promising strategy for gene therapy.

Antibodies, Monoclonal ; therapeutic use ; Carcinoma, Hepatocellular ; therapy ; Cell Line, Tumor ; Ganciclovir ; therapeutic use ; Genetic Therapy ; Humans ; Liver Neoplasms ; therapy ; Receptors, Transferrin ; immunology ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; alpha-Fetoproteins ; genetics

OBJECTIVETo screen the severe acute respiratory syndromes (SARS) mimotopes with random phage peptide library and to investigate their immunogenicity.

METHODSUsing SARS sera as selective molecule, a 12 mer phage peptide library was biopanned and positive clones containing the mimic epitopes were selected. The immuno-characteriation of the epitopes were then investigated.

RESULTS2 positive clones that having specific affinity to SARS sera were obtained. The DNA sequencing data showed no homology between the sequences of the deduced amino acid of the two mimic antigen peptides and the sequence of SARS.

CONCLUSIONSARS mimotopes were obtained by phage peptide library screening. This method might provide a new approach for SARS therapy and vaccine development.

Animals ; Antigens, Viral ; Base Sequence ; Biomimetic Materials ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; genetics ; immunology ; Humans ; Peptide Library ; Sequence Analysis, DNA ; Severe Acute Respiratory Syndrome ; immunology

OBJECTIVETo determine the association of CD133 expression with the sensitivity to radiotherapy among rectal cancer patients.

METHODSThe clinical data of 32 rectal cancer patients was retrospectively collected for patients who received a short-term preoperative radiotherapy(5 Gy/d,×5 d) from 2008 to 2010. Pretreatment tumor biopsies were immunostained for CD133 expression. Rectal cancer regression grade (RCRG) was used to evaluate the sensitivity of the rectal cancer to preoperative radiotherapy. The correlation of CD133 expression and sensitivity to radiotherapy was analyzed.

RESULTSCD133 differentially expressed in rectal cancer tissue with 17 high expression and 15 low expression. The expression of CD133 was associated with the differentiation of rectal cancer with higher expression of CD133 among poorly differentiated rectal cancers(P<0.05). Among the CD133-high patients, two patients showed 1st RCRG, five patients showed 2nd RCRG and ten patients showed 3rd RCRG. For the CD133-low patients, there were five 1st RCRG, seven 2nd RCRG and three 3rd RCRG. There was a significant association between CD133 expression and sensitivity to radiotherapy (P=0.037). Multivariate logistic regression analysis showed that the expression level of CD133(P=0.027) and the differentiation of rectal cancer(P=0.046) were independent predictive factors for the sensitivity of rectal cancer to radiotherapy.

CONCLUSIONSCorrelation between CD133 expression and sensitivity to radiotherapy of rectal cancer may exist, which may be helpful in predicting the sensitivity of rectal cancer to preoperative radiotherapy.

AC133 Antigen ; Antigens, CD ; metabolism ; Biomarkers, Tumor ; metabolism ; Biopsy ; Combined Modality Therapy ; Glycoproteins ; metabolism ; Humans ; Peptides ; metabolism ; Rectal Neoplasms ; metabolism ; radiotherapy ; Retrospective Studies

Objective To explore the value of dual-phase contrast-enhancement multislice computed tomography (MSCT) in the assessment of acute myocardial infarction volume and perfusion in porcine models. Methods The distal left anterior descending coronary arteries of 5 pigs were balloon-occluded for 90 min and followed by reperfusion. MSCT was performed 1 min (early phase) and 5 min (delayed phase) after administration bolus of 100 mL of iodinated contrast material 30 min after reperfusion. On the same day, hearts were excised, sectioned in 8 mm short-axis slices, and stained with TTC. Infarction volume was defined as the sum of the hyper-enhanced area and surrounding hypo-enhanced area in all slices on delay enhanced phase of MSCT and the TTC-negative area on TTC staining slices. Infarction volume was expressed as percentage of total slice volume. Results Acute infarction detected by MSCT was characterized by early myocardial perfasion defects in the early phase of the contrast bolus (early defects) with surrounding residual defects and late enhancement observed in the late phase. Mean CT attenuation value of early defects was significantly different from CT attenuation value of remote myocardium [(213±55)HU vs (304±30)HU](P < 0.05), CT attenuation values of residual defects and late enhancement were also significantly different from those of remote myocardium [(360±75) HU vs (90±37) HU and (152±23) HU vs (190±37) HU, repectively](P < 0.01, P < 0.05). The mean infarction volume was (8.9± 1.0)% on MSCT and (9.2±1.4)% on TTC pathology images. The infarction volume assessed by MSCT compared well with TTC staining slices. Conclusion Acute reperfused myocardial infarction zone has specific enhancement pattens different to remote normal zone on dual phase MDCT, which is in good agreement with in vivo Trc pathology in the assessment of acute reperfused myocardial infarction shortly offer reperfusion.

The three links theory applied in trauma emergency care system refers to an integrated system with the three important components of trauma emergency care system, viz. prehospital trauma services, hospital trauma services and critical care services. The development of the trauma emergency care system should be guided by the three links theory so as to set up a practical and highly efficient system: a prompt operating and monitoring transportation system, a smooth and real-time information system, a rational and sustainable system of regulations and contingency plans, and a system for cultivating all-round trauma physicians.
China ; Delivery of Health Care, Integrated ; organization & administration ; Emergency Medical Service Communication Systems ; organization & administration ; Emergency Medical Services ; organization & administration ; Emergency Medicine ; organization & administration ; Emergency Service, Hospital ; organization & administration ; Humans ; Models, Organizational ; Practice Guidelines as Topic ; Transportation of Patients ; organization & administration ; Wounds and Injuries ; therapy

The aim of this study was to investigate the gene expression profiles of acute promyelocytic leukemia (APL) cell line NB4 treated with arsenic trioxide (As2O3) by using cDNA microarray. cDNA probes were prepared through reverse transcription from mRNA of NB4 cells treated with or without arsenic trioxide. The probes were labeled with Cy3 and Cy5 fluorescence dyes individually, hybridized with cDNA microarray representing 201 different human genes, and their fluorescent intensities were scanned. The genes were screened through the analysis of the difference in the gene expression profile. The results showed that after the treatment of arsenic trioxide (2 micromol/L), 6 genes were up-regulated, and 12 genes related to apoptosis and signal transduction were down-regulated. The p21, survivin, cdc2 and Wee1Hu genes may be related to the differentiation and/or apoptosis of NB4 cells induced by As2O3. It is concluded that p21, survivin, cdc2 and Wee1Hu may play an important role in the mechanism underling arsenic trioxide-mediated NB4 cell apoptosis.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; genetics ; Arsenicals ; pharmacology ; CDC2 Protein Kinase ; Cell Line, Tumor ; Cyclin B ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Cyclin-Dependent Kinases ; Gene Expression Regulation, Neoplastic ; Humans ; Inhibitor of Apoptosis Proteins ; Leukemia, Promyelocytic, Acute ; pathology ; Microtubule-Associated Proteins ; metabolism ; Oxides ; pharmacology

Objective To observe the initial mechanism investigation in immune escaping correlated T cell proportion of Lewis lung cancer by Fuzheng-Peiyuan formula. To provide a theoretical basis for the application of the Chinese medicine of strengthening the body resistance in clinics. Methods The healthy C57BL/6J mice were divided into six groups by the random number table: the Chinese medicine group, the Chinese medicine plus chemotherapy group, the chemotherapy group, the model group and the normal control group. There were 6 mice in each group. All the groups expect the normal control group were inoculated subcutaneously with Lewis lung cancer cell suspension 0.2 ml. The medicine given to the Chinese medicine group were orally Fuzheng-Peiyuan formula with 8.17 g/kg. The combination group was given intraperitoneal injection of cyclophosphamide 60 mg/kg and orally Fuzheng-Peiyuan formula 8.17 g/kg. The chemotherapy group received the injection of cyclophosphamide 60 mg/kg and intragastric administration of normal saline. The model group and normal control group were administered with saline. After continuous administration for 13 days, the expressions of CD4, Foxp3, CD8 and CD28 in spleen cells of tumor bearing mice were observed by flow cytometry. Results Compared with the model group, the chemotherapy group and the chemotherapy combined with Chinese traditional medicine group showed that tumor weight (1.76 ± 0.42 g, 1.40 ± 0.43 g vs. 4.37 ± 0.59 g) significantly decreased (P<0.01); and the CD4+ Foxp3+ cells of mouse spleen cells in the Chinese medicine group and Chinese medicine plus chemotherapy group (11.25% ± 1.69%, 9.30% ± 2.68% vs. 14.21% ± 1.50 %) significantly decreased (P<0.05 or P<0.01). Conclusions The result initial proved that the Chinese medicine could strengthen the body resistance, adjust the proportion of Treg and CTL in spleen T cell in tumor-bearing mouse.

To investigate the effect of arsenic trioxide (As(2)O(3)) or all-trans retinoic acid (ATRA) on the mRNA and protein expression of tissue factor (TF) and thrombomodulin (TM) and procoagulant activity (PCA) in NB4 cells. The NB4 cells were cultured in vitro and treated with As(2)O(3) or ATRA, expression of TF and TM antigen, and PCA change of treated NB4 cells were detected with ELISA, TF and TM mRNA transcription on the NB4 cells was assayed with reversed transcription polymerase chain reaction (RT-PCR). The results showed that 1 micromol/L As(2)O(3) and 1 micromol/L ATRA both gradually downregulated the expression of TF antigen and mRNA on NB4 cells, a human promyelocytic leukemia cell line, in time-dependent manner, as compared with control. The levels of TF antigen expression in AS(2)O(3) group were 13.3 +/- 1.8, 8.6 +/- 1.9, 10.8 +/- 1.5, 2.0 +/- 0.6 and 2.6 +/- 0.9 ng/10(7) respectively; while the levels of TF antigen expression in ATRA group were 12.4 +/- 1.1, 11.3 +/- 1.8, 5.7 +/- 1.7, 2.8 +/- 0.8 and 2.0 +/- 0.6 ng/10(7) at 24, 48, 72, 96 and 120 hours respectively (P<0.05). The procoagulant activity (PCA) of NB4 cells was decreased, blood coagulation times were 123.5 +/- 10.5, 156.3 +/- 11.6, 179.3 +/- 15.3, 248.9 +/- 20.1, 312.0 +/- 29.8 seconds in As(2)O(3) groups, respectively; 76.4 +/- 5.6, 146.8 +/- 10.9, 198.2 +/- 15.6, 265.8 +/- 20.6 and 363.8 +/- 31.9 seconds in ATRA groups respectively at 24, 48, 72, 96 and 120 hours (P<0.05). ATRA upregulated TM antigen expression on NB4 cells. It is concluded that the As(2)O(3) and ATRA decrease mRNA transcription of TF, downregulate expression of TF and reduce procoagulant activity in NB4 cells. The TM transcription and expression upregulated by ATRA may alleviate dysfunction of coagulation in APL.
Antineoplastic Agents ; pharmacology ; Arsenicals ; pharmacology ; Humans ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Oxides ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Thrombomodulin ; biosynthesis ; genetics ; Thromboplastin ; biosynthesis ; genetics ; metabolism ; Tretinoin ; pharmacology ; Tumor Cells, Cultured