In this research a strain of isolated Pseudomonas alcaligenes which causes degradation of dexamethasone was acclimated further and its proteins of every position in the bacterium were separated by the osmotic shock method. The separated intracellular proteins which had the highest enzyme activity were extracted by the salting out with ammonium sulfate and were purified with the cation exchange chromatography and gel chromatography. The purified proteins which was active to cause degradation of dexamethasone had been detected were cut with enzyme and were analyzed with mass spectrometry. The results showed that the degradation rate to dexamethasone by acclimated Pseudomonas alcaligenes were increased from 23.63% to 52.84%. The degrading enzymes were located mainly in the intracellular of the bacteria and its molecular weight was about 41 kD. The specific activity of the purified degrading enzymes were achieved to 1.02 U x mg(-1). Its 5-peptide amino acid sequences were consistent with some sequences of the isovaleryl-CoA dehydrogenase. The protein enzyme may be a new kind degrading enzyme of steroidal compounds. Our experimental results provided new strategies for cleanup of dexamethasone in water environment with microbial bioremediation technique.
Amino Acid Sequence ; Bacterial Proteins ; chemistry ; Chromatography ; Dexamethasone ; chemistry ; Molecular Weight ; Pseudomonas alcaligenes ; enzymology

OBJECTIVE To design and construct eukaryocyte expression vector of SV40 virus large T antigen and induce its targeted expression in eukaryocyte.METHODS SV40 large T gene which excised intron was cloned by SOE(splicing by overlapping extension) and digested with restricted enzymes EcoR Ⅰ and BamH Ⅰ.By the same methods,we got the digested product of pEGFP-N1.After that,the two fragments were ligated to form SV40(TEGFP) by Ligation Kit,and sequenced by TaKaRa ABI Prism Terminator Cycle Sequence Kit.The reconstructed vector was transfected into primary cultured human fibroblast using a Lipofectin transfection method.At 48 h(after) transfection,the expression of SV40T was detected with PCR and RT-PCR using specific primer of T gene.(RESULTS) The restricted enzymes digested and sequencing results showed that SV40 large T gene had cloned into pEGFP-N1 vector successfully.The genome DNA and total RNA were isolated from the positive cells.With these samples,the specific 288 bp fragment was amplified using PCR and RT-PCR.CONCLUSIONS The recombinant plasmid SV40TEGFP will be a stable and valuable molecular tool for human eukaryocyte study.

Objective To isolate melanocytes from epidermis and identify their biological characters. Methods Taking TPA/bFGF/IBMX as basic supplements in medium DMEM/F12(1∶1), the cultured cells from the resected foreskin were purified with trypsin digestion and G418 selection, and the cellular structure and function were observed by Dopa-staining, melanin content assay, tyrosinase activity assay and immunohistochemical staining. Results Dopa-staining showed that the cultured melanocytes had melanin synthesis, melanin content assay and tyrosinase activity assay showed the tyrosinase function of the cells was normal, immunohistochemical staining demonstrated the cell differentiation was normal. Conclusion Melanocytes could be cultured by TPA/bFGF/IBMX and would maintain normal structure and biological function under such conditions.

To enable students to better grasp the basic skills of pathogenic biology and im-munology experimental teaching , and make full use of the characteristics of experimental teaching to train students' scientific quality and innovative consciousness , the reform of pathogenic biology and immunology experiment teaching was explored. Microbiology, Parasitology and Immunology experiment were integrated into an experimental course , and corresponding laboratory was set up to take an in-dependent experimental teaching. Through renewing experiment teaching idea, some measures were taken such as modularization and personalization of the teaching content, the establishment of a com-plete management system , writing a new experimental course to match the experiment , improving teaching methods and developing students' innovative experiments to improve their enthusiasm and in-terest for experimental class learning, thus enhancing their innovation ability.

Objective To investigate the immortalization of human melanocytes by transfection with SV40 T antigen ( SV40T ). Methods By using SofastTM,a gene transfection reagent, the reconstructed eukaryotic expression vector SV40T-pEGFP was stably transfected into cultured primary human melanocytes, then the positive cells were selected with G418. After the positive cells were expanded in culture, the expression of SV40T gene was detected by RT-PCR and PCR, and the protein expression of SV40T by Western blotting. Results The genome DNA and total RNA were isolated from the positive cell clones, and a 288 bp fragment, which was specific for the SV40T antigen gene, was amplified. The results of immunohis-tochemistry and Western blotting confirmed the expression of SV40T protein in transfected cells. Conclusion SV40T antigen gene can successfully induce the immortalization of human melanocytes.

Objective To evaluate the efficacy and safety of 308-nm excimer light in the treatment of stable vitiligo. Methods Thirty patients with stable vitiligo were enrolled in this clinical trial. All the subjects received the treatment with 308-nm excimer light on a twice-weekly schedule for 3 months. Results The repigmentation rate was 95.0%, 75.0% and 66.7% for lesions in the face and neck, trunk and limbs, with the treatment sessions averaging 10.22 ± 1.60, 19.10 ± 2.38, 37.74 ± 3.06, respectively, and accumulative irradiation dose averaging 7.50 ± 3.45, 10.60 ± 1.01, 18.56 ± 3.05 J/cm2 respectively. Significant differences were observed in the repigmentation rate and treatment sessions between the lesions in the face and neck, trunk and limbs (all P ＜ 0.05). No severe side effects were seen during the treatment. Conclusion 308-nm excimer light is effective and safe for the treatment of vitiligo.