We established an ELISPOT for bovine interferon-gamma (BoIFN-γ), and applied it in the diagnosis of bovine tuberculosis (bTB). Monoclonal antibodies that can bind with native BoIFN-γ were screened as the coating antibody and detecting antibody. After optimization of detecting conditions including coating antibody concentration, cell number, and detecting antibody concentration, the ELISPOT assay was established. Peripheral mononuclear cells (PBMCs) isolated from 30 cows were co-cultured with PPD, and detected with the ELISPOT assay. The optimal conditions of ELISPOT assay were 2.5 μg/mL coating antibody 2G5, 2.5 x 10(5) cells/well, and 1 μg/mL detecting antibody Bio-5E11. In these 30 cows tested both with the ELISPOT assay and the BOVIGAM kit, 11 cows were proved to be positive in ELISOPT assay with the sensitivity of 78.6%, and 12 cows were proved to be negative in ELISOPT assay with the specificity of 75%. The ELISPOT assay for BoIFN-γ could be used to detect bTB efficiently and it might be an alternative method for the diagnosis of bTB.
Animals ; Antibodies, Monoclonal ; Cattle ; Enzyme-Linked Immunospot Assay ; veterinary ; Female ; Interferon-gamma ; isolation & purification ; Sensitivity and Specificity ; Tuberculosis, Bovine ; diagnosis

Actins ; metabolism ; Adult ; Breast Neoplasms ; metabolism ; pathology ; surgery ; Calcium-Binding Proteins ; metabolism ; Cell Differentiation ; Desmin ; metabolism ; Diagnosis, Differential ; Female ; Fibroadenoma ; metabolism ; pathology ; surgery ; Hamartoma ; pathology ; Humans ; Hyperplasia ; Leiomyoma ; pathology ; Microfilament Proteins ; metabolism ; Muscle, Smooth ; pathology ; Phyllodes Tumor ; pathology

OBJECTIVE: To study and exploit Chinese medicine Astragali Radix, the molecular markers that relates to the phenotypic traits on medicinal components of Astragali Radix and would be detected. METHODS: The genetic diversity of 43 Astragali Radix samples was analyzed with ISSR molecular marker technique and then the population genetic structure was studied through 13 selected markers. The association analysis between ISSR markers and 4 phenotypic traits of medicinal components were performed with GLM (general linear model) programs in Tassel 2.1. Certain genetic diversity was discovered among the 43 Astragali Radix samples. RESULTS: The genetic distance varied between 0.050 6 and 0.743 8, with an average of 0.274 1. Moreover, the cultivated Astragali Radix from Ningxia and Gansu province closely related to the wild Astragali Radix collected from Liupanshan town in Ningxia. On the other hand, No. 340 had the farthest relationship with other Astragali Radix. The content of polysaccharide, total saponins, total flavonoids, and Astragaloside IV ranged between 7.693-27.840 mg•g-1, 7.167-17.579 mg•g-1, 2.212-6.164 mg•g-1 and 6.070-107.920 μg•g-1, respectively. Meanwhile, linear regression analysis indicated that there was a significant positive correlation between the content of the total saponins and that of flavonoids (r=0.650 5,P=2.3×10-6<0.01), while the content of astragaloside had no significant correlation with that of polysaccharide, total saponins and total flavonoids. The population genetic structural analysis showed that the 43 samples were divided into 4 subgroups. There were total of 34 locus in 13 ISSR markers significantly associated (P<0.01) with the content of polysaccharide,total saponins, flavonoids and astragaloside , and the rate of explanation on the phenotype of related marker ranged from 8.14% to 51.39%. Among the locus, 15 were related with astragaloside content at interpretation rates above 30%, 1 with polysaccharide content an interpretation rate reached as high as 51.39% with high threshold (P<1×10-5). CONCLUSION: These results would provide supporting evidence for identification and protection of germplasm resources as well as molecular marker-assisted breeding.

OBJECTIVESTo investigate whether antisense Bmi-1 plasmid could inhibit the proliferation of Jurkat cells.

METHODSThe antisense plasmid was constructed by PCR amplification of a 171 bp segment spanning Bmi-1 start codon and zinc finger structure and the PCR product was subsequently inserted reversely to plasmid pLNCX2. The final construct was confirmed through restriction enzyme digestion. G418 was added into the medium after the plasmid was successfully introduced into Jurkat cells by using lipofectin-mediated DNA transfection. The proliferation of Jurkat cells were determined by MTT and colony formation assays. Cell cycle was determined by flow cytometry. The p16 expression of Jurkat cells was studied by immunofluorescent histochemistry.

RESULTSThe growth rate of antisense Bmi-1 transfected Jurkat cells was significantly lower than that of the controls, and the colony forming capacity of the transfected cells decreased significantly (P < 0.01), the colony numbers being (90.7 +/- 9.07)/10(3) cells, (83.3 +/- 6.11)/10(3) cells and (56.0 +/- 5.56)/10(3) cells for control cells, empty plasmid transfected Jurkat cells and antisense Bmi-1 transfected Jurkat cells, respectively. The percentage of G, phase cells was increased and the p16 expression of antisense Bmi-1 transfected cells was significantly upregulated than that of control cells.

CONCLUSIONAntisense Bmi-1 can inhibit the growth and upregulate the expression of p16 of Jurkat cells in vitro.

Cell Cycle ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Jurkat Cells ; Nuclear Proteins ; genetics ; Oligonucleotides, Antisense ; genetics ; Plasmids ; genetics ; Polycomb Repressive Complex 1 ; Proto-Oncogene Proteins ; genetics ; Repressor Proteins ; genetics ; Transfection

Objective To explore the effect of traditional incision and drainage combined with vacuum sealing drainage (VSD)in treating patients with purulent exudation-staged genual septic arthritis. Methods The patients with purulent exuda-tion-staged genual septic arthritis were selected in the Zhengzhou Orthopedics Hospital from January 2010 to December 2015, and they were divided into control group and observation group. Fifty-four patients in the control group were treated with open operative drainage,while fifty patients in the observation group were treated with open operative drainage combined with VSD. The operation time,intraoperative bleeding volume,tourniquet application time,washing time,antibiotic use time,hospitalization time,therapeutic effect and relapse rate were compared between the two groups. Results There was no statistical difference in operation time and tourniquet application time and intraoperative bleeding volume between the two groups(t = - 0. 216,2. 518, 11. 320;P > 0. 05). Compared with the control group,the antibiotic use time and hospitalization time were shorter,the washing time was longer in the observation group(t = 2. 270,- 46. 310,2. 518;P < 0. 05). The occurrence rate of postoperative compli-cations in the control group and the observation group was 9. 3％(5 / 54)and 6. 0％(3 / 50),there was no statistical difference in the occurrence rate of postoperative complications between the two groups(χ2 = 0. 331,P > 0. 05). The fineness rate in the control group and the observation group was 25. 9％(14 / 54)and 22. 0％(11 / 50),there was no statistical difference in the fineness rate between the two groups(χ2 = 0. 219,P > 0. 05). The relapse rate in the control group and the observation group was 33. 3％(18 / 54)and 8. 0％(4 / 50),the relapse rate in the observation group was lower than that in the control group (χ2 =0. 331,P <0. 05). Conclusion Traitional incision and drainage combined with VSD has advantages of shortening hospital-ization and intravenous antibiotic time and decreasing relapse rate in treating purulent exudation-staged genual septic arthritis.

Carcinoma, Hepatocellular ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Humans ; Liver Neoplasms ; genetics ; metabolism ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Tumor Cells, Cultured ; beta Catenin ; biosynthesis ; genetics

The aim of the study was to look for the chemical constituents of the herb of Lysimachia foenum-graecum. The herb of Lysimachia foenum-graecum was extracted with 70% EtOH. The isolation and purification was performed with a combination of multi-column chromatography and the structure was determined by spectral analysis. The flavonoid compound was obtained and elucidated as kaempferol-7-O(4"-(E)-p-coumaroyl-)-alpha-L-rhmanopyranosyl)-3-O-beta-D-glucopyranosyl (1-->4)-alpha-L-rhmanopyranosyl (1-->2)-beta-D-glucopyranoside. It is a new flavonoid compound.
Flavonoids ; chemistry ; isolation & purification ; Glucosides ; chemistry ; isolation & purification ; Molecular Conformation ; Molecular Structure ; Plant Components, Aerial ; chemistry ; Plants, Medicinal ; chemistry ; Primulaceae ; chemistry

Adenocarcinoma ; metabolism ; pathology ; surgery ; Cardia ; Esophageal Neoplasms ; metabolism ; pathology ; surgery ; Esophagogastric Junction ; metabolism ; pathology ; surgery ; Humans ; Neoplasm Staging ; Receptor, ErbB-2 ; metabolism ; Sirtuin 1 ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; surgery ; Survival Rate

Bovine interferon-gamma (BoIFN-gamma) gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA of bovine spleen lymphocytes stimulated with ConA. The products of RT-PCR were cloned into pVAX1 vector, positive recombinant clone was identified by restriction enzyme digestion and sequencing. The recombinant plasmid pVAX1-BolFN-gamma was transfected into COS-7 cells mediated by lipofectine, indirect immunofluorescent assay analysis confirmed that rBoIFN-gamma was expressed in COS-7 cells. BoIFN-gamma gene (without signal peptide) was cloned into pET-30a(+) and pGEX-6p-1 vector, and transformed into the Escherichia coli cells. After optimizing the induction condition, SDS-PAGE analysis showed that the expression products were all found in soluble form and had a molecular weight of 23 kDa and 43 kDa respectively. BoLFN-gamma precursor gene (with signal peptide) was cloned into transfer vector pFastBac 1, and transformated into DH10Bac E. coli cells. By site-specific transposition, BoIFN-gamma gene was integrated into shuttle vector Bacmid, and transfected into the Sf9 insect cells mediated by lipofectine to produce recombinant baculovirus. Indirect immunofluorescent assay analysis confirmed that rBac-BoLFN-gamma was expressed successfully in Baculovirus vector system. The antiviral activities of rHis-BoIFN-gamma, rGST-BoIFN-gamma and rBac-BoIFN-gamma were up to 8.389 x10(7) U/mg, 6.554 x10(5) U/mg and 4.096 x 10(4) U/mL respectively, which were analyzed in MDBK/VSV system. A sandwich ELISA was established using monoclonal antibodies 3E6 and 5G4, which can detect BoIFN-gamma in quantity and provide a useful method for the clinical practice and research of BolFN-gamma.
Animals ; Antiviral Agents ; pharmacology ; Baculoviridae ; genetics ; metabolism ; COS Cells ; Cattle ; Cercopithecus aethiops ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Interferon-gamma ; biosynthesis ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Transfection