OBJECTIVE: To prepare Nicotinamide gel and to establish its quality control method.METHODS: Nicotinamide gel was prepared using nicotinamide as main ingredient,and the content of nicotinamide was determined by UV-visible spectrophotometry.RESULTS: Preparation was colorless gel,and its identification and test were all in conformity with the related regulation stated in Chinese Pharmacopoeia(2010 edition).The linear range of nicotinamide was 6.24～16.64 ?g?mL-1(r=0.999 5) with an average recovery rate of 102.0%(RSD=0.33%,n=5).CONCLUSION: The preparation procedure is simple and feasible,and the quality of the preparation is stable and controllable.

AIM: To observe the effect of E2F decoy DNA on proliferation and apoptosis of androgen-independent prostate cancer cell line PC-3M.METHODS: E2F decoy DNA,ARE decoy DNA and control decoy DNA were transfected into PC-3M cells with lipofectamine,respectively.Their effects on cell proliferation were detected by MTT assay.The changes of cell morphology were observed by inverted phase contrast microscope.The cell apoptotic rate was determined by flow cytometry(FCM) analysis and chromosome DNA ladder was detected by DNA gel electrophoresis.The expression of c-Myc mRNA and cyclin D1 mRNA was detected by RT-PCR.The protein levels of c-Myc and cyclin D1 were detected by Western blotting.RESULTS: The growth of PC-3M cells was inhibited after transfection.The transfected PC-3M cells displayed typical apoptotic morphological changes.The apoptotic rate was 26.35% and DNA ladder was observed after transfection.The expression of c-Myc and cyclin D1 were inhibited.CONCLUSION: These results indicate that E2F decoy DNA induces apoptosis of androgen-independent prostate cancer cell lines PC-3M and inhibits cell proliferation via inhibiting expression of c-Myc and cyclin D1.

AIM: To study the basic mechanism of transcriptional regulation,NKX3.1 gene promoter was cloned and its promoter activities in prostate cancer cell lines and other cancer cell lines were tested.METHODS:(1.04 kb)-promoter fragment of NKX3.1 gene was obtained by PCR and cloned into pGL_3-basic and pEGFP-1 that are promoter-less reporter vectors to examine its promoter activity driving the reporter gene transcription.The promoter activity was determined by dual-luciferase reporter assay and the expression of GFP reporter observed under fluorescence microscope.RESULTS: The sequence of the cloned(1.04 kb) promoter proved to be correct by DNA sequencing.The dual-luciferase reporter assay(M1/M2) showed that the promoter activity in LNCaP cell transfected with pGL_3-(1.04 kb) promoter was about 1.5-fold higher than that of pGL_3-control transfection and 50-fold higher than that of pGL_3-basic transfection.To investigate the 1.04 kb-promoter activity in different tumor cell lines,the constructed pGL_3-(1.04 kb) promoter and pEGFP-1.04 kb promoter were transfected into several cell lines,respectively.The results showed that the activity of(1.04 kb) promoter in LNCaP was highest among the tested cell lines.Multiple consensus sequence elements have been identified within the(1.04 kb) fragment using TRANSFAC database.Further experiments will be done to determine their founctions.CONCLUSION: Cloned 1.04 kb fragment upstream of NKX3.1 gene presented a strong promoter activity and its activity was highest in LNCaP cell among the tested tumor cell lines.

AIM: To study the basic mechanism of transcriptional regulation, NKX3.1 gene promoter was cloned and its promoter activities in prostate cancer cell lines and other cancer cell lines were tested. METHODS: 1.04 kb - promoter fragment of NKX3. 1 gene was obtained by PCR and cloned into pGL3 - basic and pEGFP - 1 that are promoter - less reporter vectors to examine its promoter activity driving the reporter gene transcription. The promoter activity was determined by dual -luciferase reporter assay and the expression of GFP reporter observed under fluorescence micro scope. RESULTS: The sequence of the cloned 1.04 kb promoter proved to be correct by DNA sequencing. The dual - lu ciferase reporter assay (M1/M2) showed that the promoter activity in LNCaP cell transfected with pGL3 - 1.04 kb promoter was about 1.5 - fold higher than that of pGL3 - control transfection and 50 - fold higher than that of pGL3 - basic transfec tion. To investigate the 1.04 kb - promoter activity in different tumor cell lines, the constructed pGL3 - 1.04 kb promoter and pEGFP - 1.04 kb promoter were transfected into several cell lines, respectively. The results showed that the activity of 1.04 kb promoterin LNCaP was highest among the tested cell lines. Multiple consensus sequence elements have been iden tified within the 1.04 kb fragment using TRANSFAC database. Further experiments will be done to determine their founc tions. CONCLUSION: Cloned 1.04 kb fragment upstream of NKX3.1 gene presented a strong promoter activity and its activity was highest in LNCaP cell among the tested tumor cell lines.

AIM: To observe the effect of exogenous androgen responsive element decoy on the promoter of prostate specific antigen (PSA) and the growth of LNCaP cells for searching the possibility of gene therapy for prostate cancer. METHODS: Firstly, pGL3 - PSA luciferase expression vector containing 640bp - promoter fragment of PSA gene was constructed. Then, a 23 -mer phosphorothioated ARE decoy based on the deduced ARE sequence at the promoter region of PSA gene was synthesized. pGL3 - PSA and ARE decoy DNA were cotransfected into PC3 - M cell by lipofectamineTM 2000. Through detecting the activity of luciferase, the effect of ARE decoy on the promoter of PSA was studied. Then the ARE decoy DNA was transfected into LNCaP cells. The effect of decoy DNA on the proliferation of LNCaP cells was examined by using MTT assay. The effects of apoptosis were detected by phase contrast microscopy, DNA agrose gel electrophoresis and flow cytometry. Meanwhile, the nuclear extract was prepared from LNCaP cells and DNA - protein interactions were examined by electrophoretic mobility shift assay (EMSA). RESULTS: The reporter assay showed that the aetivity of luciferase was significantly reduced in the ARE decoy - transfected cells, bnt not in the cells transfected with the control decoy. EMSA demonstrated specific binding of the ARE decoy to androgen receptor. The growth of LNCaP was remarkably inhibited and apoptotic morphological changes as well as DNA fragmentation were observed in the ARE decoy- transfected cells. The rate of apoptosis was 22.4% detected by FCM. CONCLUSION: The ARE decoy is capable of inhibiting the promoter of PSA gene and inducing the apoptosis in prostate cancer cells. It may become a potential therapeutic tool for prostate cancers.

We wished to ascertain the prevalence as well as the genetic and antigenic variation of infectious bronchitis viruses (IBVs) circulating in the Guangxi Province of China in recent years. The S1 gene of 15 IBV field isolates during 2012-2013 underwent analyses in terms of the similarity of amino-acid sequences, creation of phylogenetic trees, recombination, and serologic identification. Similarities in amino-acid sequences among the 15 isolates of the S1 gene were 54.3%-99.6%, and 43.3%-99.3% among 15 isolates and reference strains. Compared with the vaccine strain H120, except for GX-YL130025, the other 14 isolates showed a lower similarity of amino-acid sequences of the S1 gene (65.1-81.4%). Phylogenetic analyses of the S1 gene suggested that 15 IBV isolates were classified into eight genotypes, with the predominant genotype being new-type II. Recombination analyses demonstrated that the S1 gene of the GX-NN130048 isolate originated from recombination events between vaccine strain 4/91 and a LX4-like isolate. Serotyping results suggested that seven serotypes prevailed during 2012-2013 in Guangxi Province, and that only one isolate was consistent with the vaccine strain H120 in serotype (which has been used widely in recent years). The serotype of recombinant isolate GX-NN130048 was different from those of its parent strains. These results suggested that not only the genotype, but also the serotype of IBV field isolates in Guangxi Province had distinct variations, and that increasing numbers of genotypes and serotypes are in circulation. We showed that recombination events can lead to the emergence of new serotypes. Our study provides new evidence for understanding of the molecular mechanisms of IBV variations, and the development of new vaccines against IBVs.
Animals ; Antibodies, Viral ; blood ; Chickens ; China ; Coronavirus Infections ; blood ; veterinary ; virology ; Genetic Variation ; Genotype ; Infectious bronchitis virus ; classification ; genetics ; immunology ; isolation & purification ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; blood ; virology ; Sequence Homology, Amino Acid ; Spike Glycoprotein, Coronavirus ; chemistry ; genetics ; immunology