ABSTRACT
The aim of the study was to investigate the anti-proliferation and apoptosis-inducing effects of S1, a novel tetrandrine derivative, in human gastric cancer BGC-823 cells and explore the possible mechanism of action. The anti-proliferative activity was determined by MTT assay; the induction of cell cycle arrest and apoptosis were detected by flow cytometry. Quantitative real time RT-PCR and Western blotting were used to evaluate the mRNA and protein expression levels in mitochondrial pathway. S1 significantly reduced cell viability and induced a G2/M phase arrest and apoptosis in dose- and time-dependent manner. Further studies showed that S1 increased mRNA and protein expression of Bax and the Bax/Bcl-2 ratio. Moreover, S1 decreased the protein expression of procaspase-9 and procaspase-3, suggesting that the induction of apoptosis may be related to the alteration of the ratio of Bax/Bcl-2 and the activation of caspases. These findings suggested that S1 merits further investigation as a novel therapeutic agent for the treatment of human gastric cancer.
Subject(s)
Humans , Antineoplastic Agents , Pharmacology , Apoptosis , Benzylisoquinolines , Chemistry , Pharmacology , Caspase 3 , Genetics , Metabolism , Caspase 9 , Genetics , Metabolism , Cell Cycle Checkpoints , Cell Line, Tumor , Cell Proliferation , Cell Survival , Proto-Oncogene Proteins c-bcl-2 , Genetics , Metabolism , Stomach Neoplasms , Drug Therapy , Genetics , bcl-2-Associated X Protein , Genetics , MetabolismABSTRACT
AIM@#A quantitative ELISA kit for the detection of human secreted CD306/LAIR-2 was developed using monoclonal and polyclonal antibodies which were raised against a highly purified recombinant human secreted CD306/LAIR-2.@*METHODS@#Anti-human secreted CD306/LAIR-2 monoclonal and polyclonal antibodies were raised by immunizing mouse or rabbit with recombinant human secreted CD306/LAIR-2. The monoclonal antibody was purified by protein G affinity, whereas the polyclonal antibody was purified by protein A affinity. The best match pair of antibodies were found and used to develop a double antibody sandwich ELISA kit for the detection of human secreted CD306/LAIR-2 in human samples.@*RESULTS@#A human secreted CD306/LAIR-2 ELISA kit was formulated with highly purified recombinant human secreted CD306/LAIR-2, highly specific monoclonal and polyclonal antibodies. This kit realized the quantitative measurement of recombinant human CD306/LAIR-2 and natural CD306/LAIR-2 in human serum samples.@*CONCLUSIONS@#The developed human secreted CD306/LAIR-2 ELISA kit is a reliable quantitation immunoassay kit.
Subject(s)
Animals , Humans , Mice , Rabbits , Antibodies, Monoclonal , Allergy and Immunology , Enzyme-Linked Immunosorbent Assay , Methods , Mice, Inbred BALB C , Receptors, Immunologic , Blood , Allergy and Immunology , Recombinant ProteinsABSTRACT
?-lactamase inhibitor research is popular for its potential on ?-lactam antibiotics resistant strain.A ?-lactamase binding peptide SIPIS04-01 was obtained by the yeast two-hybid system.In vitro assay showed that it can inhibit the ?-lactamase activity.In order to improve the expression level of the recombinant peptide,a two-copy expression plasmid pYG563 was constructed by random orientation tandem repeat method after codon modification,the two-copy plasmid was successfully expressed and the product was increased by 48.4% than that of one-copy plasmid.Purified peptide showed inhibitory activity against TEM-1 ?-lactamase in vitro and the inhhibitory constant Ki was measured.
ABSTRACT
TTI gene coding for Tsetse thrombin inhibitor was modified with E.coli bias codon and expressed in Escherichia coli with high efficiency.Recombinant protein was purified to more than 98% purity.Assay for enzyme activity determination was set up.The result showed that the fusion protein exhibited inhibiting activity for thrombin.Inhibitory rate of purified TTI was 73% when concentration of thrombin and substrate was 10U/ml and 250?mol/L respectively.Inhibition pattern was determined as competitive with Ki at 35?mol/L.
ABSTRACT
A novel gene,located between dnrX and drrB in the genome of daunorubicin-producing strain Streptomyces coeruleorubidus SIPI-1482,was cloned and named as dauW.The full sequence of dauW was submitted to GenBank(Accession No.EF523565).Blast result indicated that it showed high homology with dnrW in GenBank.The exact function of dauW is as yet unknown despite the possibility that it might belong to a family of FAD-dependent oxidoreductases on the basis of conserved domain analysis.dauW was cloned into expression plasmids pET-28a(+)and pET-32a(+),respectively,and was successfully expressed in E.coli DE3 after induction with IPTG.The preliminary results of the expression of dauW suggested that it might be involved in the self resistance in Streptomyces coeruleorubidus due to the increased resistance to daunorubicin in the E.coli host.
ABSTRACT
Vitreoscilla Hemoglobin(VHb), with the function of increasing the growth of and product yield by a heterologous host, has been widely use in the area of fermentation, environment protection, transgenic animal and plant, recombinant protein expression, etc. Fusion protein of VHb with other enzyme or protein can enhance activity and stability of the enzyme or isolation efficiency of the protein. The reconstitution of VHb will be helpful to obtain ‘novel’ proteins which have better activity.
ABSTRACT
To improve the growth enhancement activity of Vitreoscilla hemoglobin(VHb), Vitreoscilla hemoglobin gene(vgb) was mutated by error-prone PCR and then reconstituted by DNA shuffling. The shuffling library was constructed by inserting the shuffled genes into the downstream of vgb natural promoter and transforming them into E.coli DH5?. Mutated active VHb proteins were first screened in test tubes according to host cell pellets color and then in shake flasks according to host pellets wet weight .One active mutant protein, VHb′042506, was obtained after second screening. It could increased the host wet weight by 31.25% and 58.75% than that of the control which bearing natural VHb under microaerobic and extremely microaerobic conditions, respectively. Sequencing and alignment results showed that 11 nucleotides were mutated, thus resulted in 4 amino acids changes occurred in this mutant protein. CO difference spectrum test also indicated that it had higher specific absorption.
ABSTRACT
TDP-4-ketohexulose reductase, encoded by dnmV, is important in daunorubicin biosynthesis. To obtain a daunorubicin block mutant, double cross-over plasmid pYG817 was constructed by inserting apramycin resistant gene and amplified dnmV together with upstream dnmU into vector pUC18. dnmV was successfully disrupted after transformation of daunorubicin-producing strain SIPI-1482 by pYG817. Daunorubicin was absent from metabolites of the resulting transformant, and its biosynthesis can be reconstituted by introducing dnmV expression plasmid into the disruptant, although the yield is lower than wild-type SIPI-1482, according to HPLC analysis. This mutant can be a good candidate for production of anthracycline such as epi-daunorubicin by introducing exogenous gene into the host.