Objective To express and purify the killer immunoglobulin-like receptor KIR3DL1 extracelluar domain.Methods pUC57-KIR3DL1 was used as template,KIR3DL1 extracelluar domain was amplified by polymerase chain reaction(PCR)and cloned into pGEM-T vector with A-T cloning technique.After DNA sequence analysis,the target fragment was inserted into the prokaryotic expression vector pET28a-DsbA to construct the recombinant vector pET28a-DsbA /KIR3DL1.The recombinant plasmid was transformed into E.coli BL21(DE3),and induced with IPTG.Bacterial pellets were resuspended in 8M urea and centrifuged to remove the insoluble material.The crude extract was purified by passing over a Ni-NTA-agarose column.After the inclusion body flowing through the Ni-NTA-agarose affinity chromatography was refolded successfully,it was purified by Superdex75 gel filtration and the purification effects of the fusion protein were identified by SDS-PAGE and western blot.Result Some fusion proteins were expressed in the supernatant,and the others were expressed in the form of inclusion bodies.The purity of fusion protein was over 95% after purification under denaturing condition.Conclusion The highly efficient expression of KIR3DL1 extracelluar domain laid the foundation for the further studies on exploration of the mechanism of immunization recognition between KIR3DL1 and its ligand.

The expression of ZNF191 mRNA in 31 ovarian malignant tumors and 31 normal ovary tissues were investigated by reverse transcription-polymerase chain reaction (RT-PCR). It was found that expression level of ZNF191 mRNA in malignant ovarian tumors was much less than that in normal ovary tissues (P

Acquired Immunodeficiency Syndrome ; prevention & control ; Adult ; Burnout, Professional ; Chi-Square Distribution ; Female ; Humans ; Male ; Medical Staff ; Middle Aged ; Rural Population ; Surveys and Questionnaires ; Young Adult

Objective To explore the prognostic value of pediatric critical illness score(PCIS)and pediatric risk of mortality score(PRISMⅢ)and the accuracy for evaluating the state of children with acute respiratory distress syndrome(ARDS).Methods Seventy-one cases hospitalized children from 29 days to 14 years old of Hebei ARDS cooperation group were selected during the 13 months between 2005 and 2006.All cases were confirmed according to ARDS diagnostic standard.For prospective studies,the patients were scored simultaneously with PCIS and PRISMⅢ at different times:when the patients entered PICU,when the patients were in the worst situation in PICU,when the patients were diagnosed as ARDS and when ARDS was serious.The data were performed by using Logistic regression etc.Results Values of Logistic regression were P

Animals ; Coal Tar ; toxicity ; Comet Assay ; DNA Damage ; drug effects ; Mice ; NIH 3T3 Cells ; drug effects

Objective:To explore the way to separate and culture eutopic and ectopic endometrial glandular cells and their stromal cells,providing an in vitro cell model of endometriosis for study of its mechanism. Methods:Digestion,filtration and sedimentation were used to isolate and culture the eutopic and ectopic endometrial glandular cells and their stromal cells. The estrogen level was imatated to study the way of promoting cell growth. Morphological characters of eutopic and ectopic endometrial cells were examined using optical microscope. Results:The success rate of separation and culture of normal control endometrial glandular cells and its stromal cells was 91.7%(11/12);of eutopic endometrial glandular cells and its stromal cells of endometriosis was 93.8%(15/16);of etopic endometrial glandular cells and its stromal cells of endometriosis was 75.0%(12/16) . Conclusion:The cultured eutopic and ectopic endometrial cells is more like human body feature than the endometriosis model of animals. So the isolation and culture of eutopic and ectopic endometrial glandular cells and their stromal cells may serve as an in vitro experimental model.

Euphorbia kansui (EK) is a toxic herbal drug, and often used after vinegar-processing to reduce its toxicity. In present study, a 1H-NMR based metabonomic approach was used to evaluate the detoxification effect of vinegar-processed EK. The water extracts of EK and VEK were administered orally to male SD rats at doses of 9 g x kg(-1) x d(-1) for 1 week, respectively, and one more week observation was further conducted. The control group was orally given with saline. Histopathological studies of liver samples on the 8th and 15th day were conducted, and the metabolites of rat urine and liver were analysed by 1H-NMR. Histopathological studies of liver samples from EK and VEK treated rats showed no negative impacts. In metabonomic analyses of urines, changes of metabolites indicated liver damages, kidney lesions and imbalance of gut microbes in the second week. VEK-treated rats showed a quite lower toxicity compared with EK-treated ones. The present study revealed that the metabonomic approach might be helpful for the evaluation of toxicity of EK and detoxic effect of VEK.
Acetic Acid ; chemistry ; Animals ; Chemistry, Pharmaceutical ; Drugs, Chinese Herbal ; chemistry ; pharmacokinetics ; toxicity ; Euphorbia ; chemistry ; Inactivation, Metabolic ; Liver ; drug effects ; metabolism ; Magnetic Resonance Spectroscopy ; Male ; Metabolomics ; methods ; Rats ; Rats, Sprague-Dawley ; Urinalysis

Objective To investigate the thermal effects on tissue structures during microwave endometrial ablation(MEA)and seek a feasible method of endometrial thinning and a fitting mode of applicator radiating microwaves.Methods Operations were performed between the group of thorough uterine curettage and the group of early follicular phase in in vitro or in vivo uterus.The former was treated with MEA after thorough uterine curettage;while the latter was treated with MEA in the early follicular phase directly.The applicator radiating microwaves were moved in "Z" or "Z+W" shape inside uterine cavity.At the same time the serosal temperature was measured in the uterine fundus,tael cornua uteri,the posterior wall and the lower part of anterior wall.After operations the uterine specimens were stained by hematoxylin- eosin,and respiratory enzyme dehydronicotinamide adenine dinucleotide phosphate diaphorase(NADH-d) methods.The morphologic changes and the depth of tissue thermal damage were evaluated using an optical microscope and electron microscope.Results(1)Under the optical microscope the endometrial glands became distorted,the cell boundaries disappeared,the nucleoli turned condensed and were stained darker. A large number of acute inflammatory cells appeared in fibrous tissue.In the shallow muscle layer cells were arrayed thickly,nucleoli were solidified and condensed,and cellular plasm were concentrated.The endometrial and the superficial muscle layers were damaged and colorless with NADH-d staining.The scope of the tissue thermal damage was clearly seen.Under an electron microscope,some smooth muscle ceils, chromatin,karyotheca and cellular membranes were destroyed.The mitochondria were swollen,membranes were ruptured,and the crista disappeared.Many organelles were destroyed.The chromatin was lightly wrecked in the transitional area between putrescence and the normal smooth muscle tissue.Karyotheca and cellular plasm still existed,the mitochondria were highly edematous and the crista were disappeared,and the granular endoplasmic reticula were slightly expanded and degranulated.(2)The serosal temperature in in vitro uterus was significantly higher than that in in vivo uterus(P0.05).The injury depth of the "Z+ W" radiation group increased significantly than that of the " Z" radiation group(P

OBJECTIVETo investigate the antithrombotic effects and its molecular mechanisms of prazosin combined with anisodamine (Ani).

METHODSIsolated rat tail artery rings model was employed to evaluate the vasodilative effects of drugs, mice tail thrombosis model induced by carrageenan was used to study the antithrombotic effects and its molecular mechanisms of the drug composition.

RESULTSAmong alpha1-adrenoreceptor antagonists, prazosin(Pra) had the greatest relaxation rate, which was (82.6 +/- 8.9)%, and the EC50 value was 0.44 micromol/L. The drug composition of anisodamine and prazosin of different doses could decrease the length of the tail thrombosis from (24.6 +/- 4.6)mm to (6.9 +/- 2.7)mm, and the rate of thrombosis was decreased from 86.6% to 50.0%. The drug composition could prolong the prothrombin time (PT) distinctively, but it had no effect on the activated partial thromboplastin time (APTT). It also could restrain the decrease of serum levels of tissue plasminogen activator (t-PA) and 6- Keto -PGF1alpha as well as the increase of type-1 plasminogen activator inhibitor (PAI-1) and thromboxane B2 (TXB2) in the mice.

CONCLUSIONThe drug composition formed by anisodamine and prazosin has good effects of relaxing extremities tiny blood vessels and it can fight against thrombosis, its antithrombotic mechanisms may be related to the influence of the extrinsic coagulation pathway, inhibition of platelet activation functions and the promotion of fibrinolysis function.

Adrenergic alpha-1 Receptor Antagonists ; pharmacology ; Animals ; Disease Models, Animal ; In Vitro Techniques ; Male ; Prazosin ; pharmacology ; Rats ; Rats, Wistar ; Solanaceous Alkaloids ; pharmacology ; Thrombosis ; drug therapy

The coding sequence for the mature peptide of porcine lactoferrin (Plf) was synthesized according to the codon usage of lactobacillus, to establish optimized porcine lactoferrin Lactobacillus expression system. The gene was ligated into the Xho I/BamH I site of Lactobacillus expression vector pPG612.1 and the recombinant plasmid pPG612.1-plf was transformed individually into Lactobacillus casei ATCC393, Lactobacillus pentosus KLDS1.0413, Lactobacillus plantarum KLDS1.0344 or Lactobacillus paracasei KLDS1.0652 by electroporation. After induction with xylose, expression of the recombinant proteins was detected by Western blotting and confocal laser scanning microscopy. Secretion of recombinant Plf proteins from four recombinant species was determined quantitatively by ELISA. The antibacterial activities of recombinant proteins were measured by agar diffusion method. The result shows that Plf was correctly expressed in four species of recombinant lactobacillus, with molecular weight of about 73 kDa. The expression levels in recombinant Lactobacillus casei, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paracasei were 9.6 μg/mL, 10.8 μg/mL, 12.5 μg/mL and 9.9 μg/mL, respectively. Antimicrobial activity experiment shows that the recombinant proteins were active against E. coli, Staphylococcus aureus, Salmonella typhimurium, Listeria, Pasteurella. The recombinant Plf expressed by recombinant Lactobacillus plantarum showed the best antibacterial activity among all recombinant lactobacillus species. These data represent a basis for the development and application of porcine lactoferrin from recombinant lactobacillus.
Animals ; Anti-Bacterial Agents ; biosynthesis ; Lactobacillus ; metabolism ; Lactoferrin ; biosynthesis ; Recombinant Proteins ; biosynthesis ; Swine