Animals ; Cells, Cultured ; Chondrocytes ; Interleukin-1beta ; NF-kappa B ; Osteoarthritis/drug therapy* ; Rats ; Tumor Necrosis Factor-alpha

Polymerase Chain Reaction (PCR)in recent years is a new disease detection methods basedon molecular biology level.It has the advantage of sensitive,specific,trace,simple,fast,but in the operation due to multiple factors,quality control is not easy to control.Especially in the first stage of quality control,it engages alot of operations and is weakest stage such needs most concern.It is necessary and important to have a good quality control;it not only improve the reliability and accu-racy of PCR testing,treatment of molecular biology,the professional level of work staff,but also reduce patient disputes and increase technology access,acceptance of the PCR laboratory.In this paper,the author from the factors which influence the first stage of PCR:whether correctly fill PCR test application form,patient preparation,handling,transportation,storage of good sample,unqualified specimen rejection,the requirement of laboratory technicians,calibration and maintenance on the in-struments;quality check on reagents,consumables quality inspection,standard operating procedures (SOP)writing,and la-boratory safety.Hope this paper can do a good reference for other same profession.

OBJECTIVETo investigate the effects of Propyl Gallate (PrG) on cellular adhesion between human To investigate the effects of Propyl Gallate (PrG) on cellular adhesion between human umbilical vein endothelial cells (HUVEC) and polymorphonuclear leukocytes (PMN) as well as the expression umbilical vein endothelial cells (HUVEC) and polymorphonuclear leukocytes (PMN) as well as the expression of intercellular adhesion molecule-1 (ICAM-1, CD54) and E-selectin (CD62E) on the VEC surface. of intercellular adhesion molecule-1 (ICAM-1, CD54) and E-selectin (CD62E) on the VEC surface.

METHODSA human VEC inflammation model was induced by tumor necrosis factor alpha (TNF-alpha). VECs were pre- A human VEC inflammation model was induced by tumor necrosis factor alpha (TNF-alpha). VECs were preincubated with varying concentrations of PrG (0.001-5 mmol/L) or 1 per thousand DMSO (v:v) or 10 mmol/L acetylsalicylic incubated with varying concentrations of PrG (0.001-5 mmol/L) or 1 per thousand DMSO (v:v) or 10 mmol/L acetylsalicylic acid (ASA) for 1 h, and then were stimulated with 10 ng/mL TNF-alpha for 6 h. Rose bengal vital staining method acid (ASA) for 1 h, and then were stimulated with 10 ng/mL TNF-alpha for 6 h. Rose bengal vital staining method was used to measure the adherence rate of PMN to VEC, while flow cytometry was used to determine the was used to measure the adherence rate of PMN to VEC, while flow cytometry was used to determine the expression of CD54 and CD62E on the VEC surface. expression of CD54 and CD62E on the VEC surface.

RESULTSAfter 6 h of incubation with TNF-alpha, the adherence After 6 h of incubation with TNF-alpha, the adherence of PMN to HUVECs as well as the percentage of fluorescence-positive cells and mean fluorescence intensity of PMN to HUVECs as well as the percentage of fluorescence-positive cells and mean fluorescence intensity (MFI) of surface CD54 and CD62E in HUVECs increased significantly ( (MFI) of surface CD54 and CD62E in HUVECs increased significantly (P<0.01). Pre-treatment of HUVECs with <0.01). Pre-treatment of HUVECs with PrG (0.1-5 mmol/L) significantly suppressed the adherence of PMN to VECs induced by TNF-alpha (PrG (0.1-5 mmol/L) significantly suppressed the adherence of PMN to VECs induced by TNF-alpha (P<0.05). PrG <0.05). PrG (1-5 mmol/L) inhibited the VEC surface expression of CD62E and CD54 in a dose-dependent way ( (1-5 mmol/L) inhibited the VEC surface expression of CD62E and CD54 in a dose-dependent way (P<0.05). PrG <0.05). PrG at lower concentrations (0.001-0.1 mmol/L) showed no effect on CD54 expression, while it showed a slightly at lower concentrations (0.001-0.1 mmol/L) showed no effect on CD54 expression, while it showed a slightly increasing trend in CD62E expression (increasing trend in CD62E expression (P>0.05). ASA at 10 mmol/L had no obvious effect on the positive rate of >0.05). ASA at 10 mmol/L had no obvious effect on the positive rate of CD62E and CD54. CD62E and CD54.

CONCLUSIONSHigh concentrations of PrG (0.1-5 mmol/L) exert its inhibitory effect on cellular High concentrations of PrG (0.1-5 mmol/L) exert its inhibitory effect on cellular adherence of PMN to HUVECs, and its mechanism may be related to inhibiting surface expression of CD54 and adherence of PMN to HUVECs, and its mechanism may be related to inhibiting surface expression of CD54 and CD62E in HUVECs. Its action concentration was lower than that of ASA. CD62E in HUVECs. Its action concentration was lower than that of ASA.

Cell Adhesion ; drug effects ; Endothelial Cells ; cytology ; drug effects ; Flow Cytometry ; Fluorescein-5-isothiocyanate ; metabolism ; Fluorescence ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Neutrophils ; cytology ; drug effects ; Propyl Gallate ; chemistry ; pharmacology ; Staining and Labeling ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo investigate the effects of propyl gallate (PrG) on the thrombus formation time and the coagulation/fibrinolysis system in an experimental carotid artery thrombosis model in rats.

METHODSFifty SD rats were randomly divided into 5 groups (10 animals/group): the normal group (normal saline 2 mL/kg), the model group (normal saline, 2 mL/kg), the heparin control group (1,250 IU/kg), the low dose PrG group (30 mg/kg), and the high dose PrG group (60 mg/kg). Thirty minutes after intravenous injection of saline or the corresponding drugs, a carotid artery thrombus was induced by continuous electric stimulation in all rats except for those in the normal group. The duration from the initiation of the electric stimulation to the sudden drop in carotid temperature was recorded as the thrombus formation time. Levels of plasma tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) were determined by ELISA.

RESULTSPrG (30 and 60 mg/kg) can prolong the thrombus formation time, but the effect was obviously weaker than that of heparin (P<0.05, P<0.01). Compared with the model group, PrG (30 and 60 mg/kg) elevated the plasma activity of t-PA (both P<0.05) and showed an increasing tendency in elevating the ratio of t-PA/PAI-1 (P>0.05), while it had no significant effect on the level of PAI-1.

CONCLUSIONPrG has a certain antithrombotic effect and can slightly regulate the imbalance of the t-PA /PAI-1 ratio.

Animals ; Blood Coagulation ; drug effects ; Carotid Artery Thrombosis ; drug therapy ; Female ; Fibrinolysis ; drug effects ; Male ; Plasminogen Activator Inhibitor 1 ; blood ; Propyl Gallate ; pharmacology ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Tissue Plasminogen Activator ; blood

The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2 strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50 degrees C was 8.5 and the optimum temperature at pH 8.5 was 55 degrees C. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT), mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO) stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.
Bacillus subtilis ; classification ; enzymology ; Enzyme Activation ; Enzyme Stability ; Peptide Hydrolases ; chemistry ; isolation & purification ; Species Specificity ; Substrate Specificity

cDNA for Insulin-like growth factor binding protein 3 was cloned and constructed a prokaryotic expression vector--pET-DsBA-IGFBP3. The construct was transformed into E. coli BL21 (DE3)plysS. The induced fusion protein (D-IGFBP3) was expressed successfully in soluble form. We obtained D-IGFBP3 the purify of which is over 95% after purification by His affinity chromatography. The product was identified by Western-blot. The cell assay showed that the obtained fusion protein can inhibit the growth of MCF-7 and bind with IGF-I in vitro.
Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chromatography, Affinity ; Dose-Response Relationship, Drug ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Gene Expression ; Humans ; Insulin-Like Growth Factor Binding Protein 3 ; genetics ; metabolism ; pharmacology ; Insulin-Like Growth Factor I ; metabolism ; Protein Binding ; Recombinant Proteins ; isolation & purification ; metabolism ; pharmacology ; Solubility

OBJECTIVETo study on the effect and mechanism of curcumin on inhibiting injury induced by free radical in pulmonary fibrosis.

METHODOne hundred and forty-four male SD rats were randomly divided into 6 groups (24 rats in each group). Rats in the model control group, positive medicine group, and high, moderate and low curcumin groups were injected with a single dose of bleomycin by trachea, and rats in sham-model control group with same volume normal saline. One day after the injection, curcumin solution of different dosages (200,100,50 mg x kg(-1) x d(-1)) was respectively given to rats in the high, moderate and low curcumin group by daily gastrogavage, while equal volume of normal saline was given to those in the sham-model control group and model control group, and an equal volume of prednisone (0.56 mg x kg(-1) x d(-1)) was saline was given to those in positive medicine control group. On the 7, 14, 28 days, the contents of GSH-Px, SOD, MDA and iNOS in pulmonary tissues of different groups were measured.

RESULTCurcumin can raise the content of SOD and GSH-Px and lessen the level of MDA and iNOS.

CONCLUSIONCurcumin can regulate the level of free radical in the body of rats with pulmonary fibrosis and lessen the oxidative injury of pulmonary tissues caused by free radical, in the body of rats with pulmonary fibrosis. The mechanisms of curcumin on idiopathic pulmonary fibrosis lie in adjusting the level of free radical and inhibiting the injury of lung tissue induced by free radical.

Animals ; Antioxidants ; isolation & purification ; pharmacology ; Bleomycin ; Curcuma ; chemistry ; Curcumin ; isolation & purification ; pharmacology ; Free Radicals ; metabolism ; Glutathione Peroxidase ; metabolism ; Lung ; drug effects ; metabolism ; pathology ; Male ; Malondialdehyde ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Plants, Medicinal ; chemistry ; Pulmonary Fibrosis ; chemically induced ; metabolism ; prevention & control ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism

Objective: To investigate jugular bulb venous oxyg en partial pressure(PjO2)， hemoglobin saturation (SjO2) and the arterial t o jugular bulb venous oxygen content difference(AjDO2) during anesthesia with desflurane and isoflurane in patients with brain tumor. Methods: Fifty-six patients with brain tumor were randomized into desflur ane or isoflurane for maintaining anesthesia. PjO2, SjO2 and AjDO2 in pati ents were measured during normoventilation, hyperventilation and hypoventilation . Results: During normoventilation， SjO2 and PjO2 in desflu rane group was significantly higer than those in isoflurane group（P<0.05 or P<0.01）, and AjDO2 in desflurane group was significantly lower than that in isoflurane group（P<0.05）.Except that PjO2 in desflurane group was si gnificantly higer than that in isoflurane group during hyperventilation （P< 0.01）, there were no differences in SjO2, PjO2 or AjDO2 between the 2 g roups during hyperventilation or hypoventilation. While anesthesia with desflura ne and isoflurane, there was a positive correlation between PaCO2 and SjO2. Conclusion: At the same anesthetic effect concentration, desflur ane can significantly increase SjO2 and PjO2 in comparison to isoflurane un der normoventilation, suggesting that desflurane may have stronger effect of rel axing cerebral vessel than isoflurane.

Ectopic Cushing’s syndrome caused by pheochromocytoma is rare. We reported a 15-year-old female patient who was admitted to hospital with typical Cushing’s syndrome. She had not started menstruation. Her plasma adrenocorticotropic hormone (ACTH) and 24-hour urinary free cortisol levels were extremely high. Gonadal and progestational hormone levels were also abnormal. Abdominal computed tomography scans and enhanced scans revealed multiple irregular tumors in the right adrenal. Pelvic echogram showed an infantile uterus, while the ovaries were at an immature stage of development. Retroperitoneal laparoscopic right adrenalectomy was performed without intraoperative complications. Histology and immunohistochemistry of the tumor were consistent with pheochromocytoma. Retroperitoneal laparoscopic adrenalectomy is a safe procedure with satisfactory outcomes and allows for rapid recovery.
Adolescent ; Adrenal Gland Neoplasms ; diagnosis ; secretion ; therapy ; Adrenocorticotropic Hormone ; secretion ; Female ; Humans ; Pheochromocytoma ; diagnosis ; secretion ; therapy

OBJECTIVETo screen low molecular weight protein biomarkers relevant to portal vein tumor thrombi (PVTT) in serum of hepatocellular carcinoma (HCC) patients.

METHODSSerum samples were obtained from 12 healthy volunteers, 12 HCC patients without PVTT and 12 HCC patients with PVTT. Using two-dimensional gel electrophoresis (2-DE) in which the second dimension was 16% SDS-PAGE, serum protein images of the 3 groups were analyzed by ImageMaster software. The differential protein spots were further identified by MALDI-TOF MS/MS.

RESULTSComparing the results using 12.5% SDS-PAGE gel, there were more protein bands (between 3 x 10(3) and 20 x 10(3)) and low molecular weight (MW) protein spots (less than 20 x 10(3)) were clearly shown in the 16% SDS-PAGE gel. Fifteen differential protein spots representing 5 proteins were found in the 3 groups by inter-class comparison and they were then identified. Compared with those in the healthy group, apolipoprotein A-I, lipoprotein CIII, transthyretin and DNA topoisomerase II were all down regulated in HCC groups and haptoglobin-2 was over expressed. All 5 proteins decreased more in the PVTT group than in the non-PVTT group.

CONCLUSIONThe expression of low MW serum protein obviously changes in the beginning and in the progressive stage of HCC, and differentially expressed low MW proteins might be potential biomarkers in an early prognostic prediction and surveillance in the treatment for HCC and PVTT.

Adult ; Blood Proteins ; analysis ; Carcinoma, Hepatocellular ; blood ; pathology ; Electrophoresis, Gel, Two-Dimensional ; methods ; Female ; Humans ; Liver Neoplasms ; pathology ; Male ; Middle Aged ; Neoplastic Cells, Circulating ; pathology ; Portal Vein ; pathology ; Proteome ; analysis