ABSTRACT
In order to provide rationale for selection of good germplasm in Rubus chingii, main effective medicinal ingredients of green fruit such as gallic acid, ellagic acid, kaempferol-3-rutinoside, astragalin and tiliroside were measured using UPLC for the samples collected from Chun'an county of Zhejiang province, and such parameters as soluble solid contents of ripe fruit of some samples were also measured to study variation among individuals and correlation. It has been found that there were differences among individuals in the contents of gallic acid, ellagic acid, kaempferol-3-rutinoside, astragalin and tiliroside, which ranged from 0.010 2%-0.027 4%, 0.089 5%-0.291 1%, 0.010 5%-0.114 8%, 0.005 8%-0.041 2% and 0.010 9%-0.086 3%, respectively, with a CV of 18.60%, 27.02%, 44.23%, 44.17% and 47.29%, respectively. Gallic acid was positively correlated with ellagic acid, but negatively with kaempferol-3-rutinoside and astragalin significantly. Significantly positive correlation existed between kaempferol-3-rutinoside, astragalin and linden glycoside as well as between ellagic acid and fruit shape index of ripe fruit and between linden glycoside and the content of soluble solids. 51.35% of the individuals had a content of soluble solids more than 15%. Therefore, abundant variations have been found among individuals in effective medicinal ingredients in R. chingii, which shows great potential for selection, but only do 7.61% of the individuals meet the requirement of Chinese pharmacopoeia in terms of the contents of effective medicinal ingredients. Therefore, selection could be first performed in terms of fruit shape index of ripe red fruit, followed by the contents of ellagic acid and kaempferol-3-rutinoside measured. The individuals, in which the contents of effective medicinal ingredients don't meet the requirement of Chinese pharmacopoeia, could be considered for the selection in terms of edible fresh fruit.
Subject(s)
Humans , Ellagic Acid , Fruit , Glycosides , Plant Extracts , RubusABSTRACT
<p><b>OBJECTIVE</b>To investigate the mechanisms that coagulation factor VIIa promotes proliferation and migration of a colon cancer cell line (SW620 cells) in vitro.</p><p><b>METHODS</b>The expression of interleukin 8 (IL-8), tissue factor (TF), caspase-7 and p-p38 MAPK in SW620 cells treated with factor VIIa or protease activated receptor 2 agonist (PAR2-AP) was measured by ELISA, Western-blotting and QT-PCR.</p><p><b>RESULTS</b>Factor VIIa and PAR2-AP induced IL-8 expression at both mRNA and protein levels, upregulated TF mRNA expression and TF activity, but down-regulated caspase-7 mRNA and p-p38 MAPK levels in SW620 cells. The effects of factor VIIa were not only blocked by anti-TF but also by anti-PAR2 antibodies.</p><p><b>CONCLUSION</b>Factor VIIa binds to TF on cell surface, forming a complex which activates PAR2, then provoking IL-8 and TF expression, and suppresses caspase-7 expression, thus promotes the tumor cell proliferation and migration. p38 MAPK may negatively regulate this process.</p>
Subject(s)
Humans , Caspase 7 , Genetics , Cell Line, Tumor , Cell Movement , Cell Proliferation , Colonic Neoplasms , Metabolism , Pathology , Factor VIIa , Pharmacology , Interleukin-8 , Genetics , Oligopeptides , Pharmacology , RNA, Messenger , Metabolism , Receptor, PAR-2 , Thromboplastin , Genetics , p38 Mitogen-Activated Protein Kinases , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To construct a novel kind of nonviral gene delivery vector based on polyethylenimine (PEI) conjugated with polypeptides derived from ligand FGF with high transfection efficiency and according to tumor targeting ability.</p><p><b>METHODS</b>The synthetic polypeptides CR16 for binding FGF receptors was conjugated to PEI and the characters of the polypeptides including DNA condensing and particle size were determined. Enhanced efficiency and the targeting specificity of the synthesized vector were investigated in vitro and in vivo.</p><p><b>RESULTS</b>The polypeptides were successfully coupled to PEI. The new vectors PEI-CR16 could efficiently condense pDNA into particles with around 200 nm diameter. The PEI-CR16/pDNA polyplexes showed significantly greater transgene activity than PEI/pDNA in FGF receptors positive tumor cells in vitro and in vivo gene transfer, while no difference was observed in FGF receptors negative tumor cells. The enhanced transfection efficiency of PEI-CR16 could be blocked by excess free polypeptides.</p><p><b>CONCLUSION</b>The synthesized vector could improve the efficiency of gene transfer and targeting specificity in FGF receptors positive cells. The vector had good prospect for use in cancer gene therapy.</p>
Subject(s)
Animals , Female , Humans , Male , Mice , Binding Sites , Carcinoma , Therapeutics , Cell Line, Tumor , Cell Survival , Dose-Response Relationship, Drug , Fibroblast Growth Factors , Metabolism , Gene Transfer Techniques , Genetic Vectors , Chemistry , Pharmacology , In Vitro Techniques , Ligands , Liver Neoplasms , Therapeutics , Mice, Inbred BALB C , Mice, Nude , Particle Size , Peptides , Chemistry , Metabolism , Pharmacology , Polyethyleneimine , Chemistry , Metabolism , Pharmacology , Prostatic Neoplasms , Therapeutics , Receptors, Fibroblast Growth Factor , Genetics , Metabolism , Structure-Activity Relationship , Surface Properties , Transfection , Transplantation, Heterologous , Xenograft Model Antitumor AssaysABSTRACT
Objective:To study the enhancing effect of bFGF-targeted antisense phosphorothioate oligonucleotide (APO)on the chemosensitivity of human laryngeal squamous carcinoma cell line Hep2 to Doxorubicin,5-Fluorouracil, and Cisplatin.Methods:bFGF-specific APO was designed,constructed and transfected into Hep2 cells with jetPEI (polyethyleneimine).Expression of bFGF mRNA was evaluated by semi-quantitative RT-PCR after transfection;immuno- cytochemical method was used to examine the expression of bEGF expression before and after transfection of Hep2;the in- duction of cell apoptosis was analyzed by flow cytometry;cell proliferation was then analYzed by MTT assay after treatment with bFGF-specific APO or chemotherapeutic drugs,or a combination of both.Results:bFGF-specific APO inhibited the growth of Hep2 cells in a dose-and time-dependent manner,with the peak inhibitory rate being 25.5%.The expression of bFGF mRNA and protein decreased by 52.0% and 41.1%,respectively.The apoptosis rate of Hep2 cells was 20.5% after transfection,bFGF-specifie APO reduced the 50% inhibitory concentration of Doxorubicin,5-Fluorouracil,and Cis- platin in Hep2 cells by 75.5%,83.5% and 65.4%,respectively.Conclusion:bFGF-specific APO can enhance the chemosensitivity of Hep2 cells,which paves a new way for potential biologic chemotherapy of laryngeal squamous carcino- ma.
ABSTRACT
Apx IV, a forth RTX toxin indentified in Actionbacillus pleuropneumoniae recently, is expressed by all A. pleuropneumoniae regardless the serotypes and inducible only in vivo toxin, so it is the optimal to develop species-specific and differentiated diagnostic assay. Here the 2445bp DNA fragment of apxIVA gene of A. pleuroneumoniae was amplified and fused in-frame to the downstream of the T7 promoter and 6 His Tag of the prokaryotic expression vector pET-28b. The construct was transformed into E. coli BL21(DE3). After induction by 1.0 mol/L IPTG, a recombinant protein about 90 kD in size, designed as ApxIVAN, was detected, which was present as inclusion bodies and reacted specifically with swine antisera to the APP-serotype-1 by dot-blot. An indirect ELISA (ApxIVA-ELISA) was developed using purified recombinant ApxIVAN from the inclusion bodies as described previously, which had excellent specificity to A. pleuroneunoniae. Using the ApxIVA-ELISA, the ApxIV antibodies were not detected in the inactivated APP bacterins vaccinated pigs, but were detected in A. pleuropneumoniae serotype 1, 2 and 7 infected pigs and mice. These results suggested that ApxIVA-ELISA can be used not only to detect all serotypes of APP, but also to differentiate the naturally infected and inactivated vaccine immunized pigs.
Subject(s)
Actinobacillus Infections , Diagnosis , Microbiology , Actinobacillus pleuropneumoniae , Genetics , Allergy and Immunology , Metabolism , Bacterial Proteins , Genetics , Allergy and Immunology , Metabolism , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Methods , Gene Expression , Genes, Bacterial , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , MetabolismABSTRACT
To produce the recombinant baculovirus transfer plasmid pFast-ORF2, the ORF2 gene of Porcine Circovirus type 2 (PCV2) was subcloned into baculovirus transfer vector (pFastBac(TM1) ) using Bac-to-Bac baculovirus expression system. E. coli DH10Bac (Gibco BRL) containing baculovirus shutter vector (bacmid) and helper vector was transformed with recombinant plasmid pFast-ORF2. Within E. coli DH10Bac, the ORF2 gene was transposed into the bacmid. The colonies of E. coli containing recombinant bacmid (Bac. ORF2) were collected by blue/white selection. The Bac. ORF2 was transfected into sf9 cells to yield AcNPV carrying the PCV2 ORF2 gene, referred to as Ac. ORF2. Expression of the ORF2 gene of PCV2 was confirmed by indirect immunofluorescent assay (IIFA), SDS-PAGE and Western-blotting. The expressed ORF2 gene product had a molecular mass of 28kD and could be recognized by the positive serum of PCV2. The results indicated the ORF2 gene was properly expressed in sf9 cell. It was noteworthy that many self-assembled virus-like particles (VLPs) were found in purified and phosphotungstic acid (PTA) stained PCV2 ORF2 protein by electron microscope. The particles were of similar morphology to the PCV2 virion and some self-assembled virus-like particles had darkly stained centers that made them appear to be empty capsids. Both PCV2 particles and self-assembled particles were approximately 17 nm in diameter.