ABSTRACT
We cloned genes of four sentrin-specific protease (SENP), three small ubiquitin-like modifiers (SUMO), enhanced cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP) by two-step PCR. Then we constructed expression vector B28 for SENP and B13 for ECFP-SUMO-EYFP. The recombinant plasmids were transformed into Escherichia coli BL21 and expression was induced by isopropyl beta-D-thiogalactoside, then recombinant proteins were purified by Ni-NTA agarose column ion-exchange chromatography. The proteins were analyzed with SDS-PAGE and identified with Western blotting. Except that SENP3 catalytic domain (SENP3C) truncated in the C termini and SENP5C expressed in inclusion body, others were expressed as soluble proteins. SDS-PAGE analysis showed that the relative molecular mass of these fusion proteins were consistent with theoretical ones, and the specificity of the fusion proteins were confirmed with Western blotting. The fusion proteins of SENP and ECFP-SUMO-EYFP can be successfully expressed in prokaryotic expression system. It lays the foundation for the fluorescence resonance energy transfer analysis.
Subject(s)
Humans , Bacterial Proteins , Genetics , Catalytic Domain , Cysteine Endopeptidases , Genetics , Endopeptidases , Genetics , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Green Fluorescent Proteins , Genetics , Luminescent Proteins , Genetics , Recombinant Fusion Proteins , Genetics , SUMO-1 Protein , GeneticsABSTRACT
Objective To study the bystander effects and associated mechanisms through irradiated conditioned medium(1CM). Methods Natural kilhr(NK) cells were obtained from peripheral blood samples. ICM irradiated with different doses of 60Coγ-rays was used for culturing K562 cell strain. The degree of injury of K562cells by activated NK cells was observed, as well as the apoptosis frequency of K562 cell was investigated. Results Severe injury was induced in K562 cells cultured in ICM than the control (sham-irradiated) as shown by increased sensitivity to NK cells (P < 0.05). The apoptosis frequency of K562 cell was increased significantly compared with the control cells (P < 0.05). Conclusions The bystander effect induced by irradiation is existent. ICM can trigger the bystander effect on K562 cell strains.
ABSTRACT
Monoclonal antibodies (McAbs) against human liver DnaJ-like protein (HLJ1) was produced by using lymphocyte-hybridoma technique and then one method for the detection of HLJ1 antigen was established. Two hybridoma cell lines which stably secreted monoclonal antibodies against HLJ1 were generated and named for A4C7 and C4C8. Subtypes of the two McAbs were both IgG1, and the antibodies showed high titer and good specificity. Using the prepared monoclonal antibody, human embryonic liver tissues were examined by immunohistochemistry. The results indicated that HLJ1 located in the cytoplasm of the human embryonic liver cell. A double antibodies sandwich ELISA was established by using C4C8 and HRP labeled A4C7. This assay had good specificity, and the lowest detection limit was 7.5 ng/mL and the linear range was 7.5-750 ng/mL. In conclusion, an immunohistochemistry method and a sensitive sandwich ELISA were established for the detection of HLJ1 protein.
Subject(s)
Animals , Female , Humans , Mice , Antibodies, Monoclonal , Enzyme-Linked Immunosorbent Assay , Methods , HSP40 Heat-Shock Proteins , Blood , Allergy and Immunology , Hybridomas , Bodily Secretions , Immunohistochemistry , Mice, Inbred BALB CABSTRACT
The biophysical characteristics of heavy ions make them a rational source of radiation for use in radiotherapy of malignant tumours. Prior to radiotherapy treatment, a therapeutic regimen must be precisely defined, and during this stage information on individual patient radiosensitivity would be of very great medical value. There are various methods to predict radiosensitivity, but some shortfalls are difficult to avoid. The present study investigated the induction of chromatid breaks in five different cell lines, including one normal liver cell line (L02), exposed to carbon ions accelerated by the heavy ion research facility in Lanzhou (HIRFL), using chemically induced premature chromosome condensation (PCC). Previous studies have reported the number of chromatid breaks to be linearly related to the radiation dose, but the relationship between cell survival and chromatid breaks is not clear. The major result of the present study is that cellular radiosensitivity, as measured by D0, is linearly correlated with the frequency of chromatid breaks per Gy in these five cell lines. We propose that PCC may be applied to predict radiosensitivity of tumour cells exposed to heavy ions.
Subject(s)
Chromatids , Chromosome Breakage/radiation effects , Radiation Tolerance , Radiotherapy/adverse effects , Carbon/chemistry , Cell Line , Cytogenetic Analysis , HumansABSTRACT
Objective To explore the development trends in community nursing. Methods The current situation of community nursing in Shanghai was analyzed by means of questionnaire surveys and literature review. Subjects A total of 3 173 questionnaires were distributed to community nurses, hospital nurses, community residents, and hospitalized patients and 2 942 of them were retrieved, the rate of retrieval being 93.8%. Results The demand for community nursing was not satisfied, the number of community nurses was insufficient and their general quality was low. Conclusion The scope of services in community nursing needs to be expanded with demand as a guide, more community nurses need to be cultivated, and the quality of the nursing contingent needs to be enhanced.
ABSTRACT
Objective To find out the tendency of medical accident arbitration in 5 hospitals of Shanghai and the causes. Methods Cases involving medical accident arbitration that occurred in the 15 years running from 1985 to 1999 in 5 third tier hospitals of Shanghai were collected and a retrospective correlation analysis was conducted. Then 45 of the cases were selected by random sampling and a detailed analysis was made. Results ①During the 15 years the number of cases involving medical accident arbitration by class 1 commissions was 295, with the average number per year in the latter five years rising 51% as compared with the first ten years. ②There was no significant correlation between the number of cases involving arbitration and the number of cases judged to be accidents. ③There was no correlation, either, between the total volume of emergency outpatient service, which amounted to 68, 6 million visits, and the number of cases involving medical accident arbitration. ④The sample survey showed that the operating room and the emergency room were sections where medical accidents occurred most frequently. Conclusion In the past 15 years, the number of cases involving medical accident arbitration has been on the rise, yet there is no correlation between the rise and the number of cases judged to be accidents or the volume of medical service. The causes for the rise include both iatrogenic and non iatrogenic ones. The paper puts forward 6 countermeasures.
