Animals ; Antibiotics, Antineoplastic ; pharmacology ; Blotting, Northern ; Cells, Cultured ; Dexamethasone ; pharmacology ; Doxorubicin ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation ; drug effects ; Glucocorticoids ; pharmacology ; Kidney Glomerulus ; cytology ; drug effects ; metabolism ; Mice ; RNA, Messenger ; drug effects ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

Fibrolase is a non-hemorrhagic zinc metalloproteinase isolated from southern copperhead snake venom (Agkistrodon contortrix contortrix) and is capable of degrading fibrin clots resulting from purified fibrinogen or from blood plasma. Alfimeprase, a truncated form of fibrolase, as a clinical agent was successfully completed PhaseII clinical trials.The cDNA of alfimeprase was amplified by recursive PCR, digested with BamHI and HindII, and cloned into pET43.1a, pMALp2X and pMALc2X vectors to generate fusions with NusA, MBP and sMBP(with signal peptide), respectively. Nus/alfimeprase was expressed in soluble form by co-expressing with chaperone FkpA and inducing with1mmol/L IPTG. The fusion protein accounted for about 25 % of total protein following cell lysis. Alfimeprase was successfully purifiesd by Ni-NTA affinity chromatography and cleaved by enterokinase. The results demonstrate the fibrinolytic activity of recombinant alfimeprase using fibrin plate assays and fibrinogen hydrolysis.

A two-step method for the purification of blocking-type anti-human CD80 monoclonal antibody 4E5 from mouse ascites was developed using anion exchange and gel filtration in combination. The ascites was first purified by anion exchange after centrifugation and filtration. The experimental parameters of sample loading and elution were optimized. The optimized loading condition was pH 8.0,50 mmol/L Tris-HCl and satisfactory results were obtained using a 0~0.5mol/L NaCl step elution. The fraction containing the protein of interest was directly loaded on gel filtration column and eluted using a 20 mmol/L phosphate buffer at pH 7.2. The purity of the obtained monoclonal antibody was up to 95% with a recovery of 61%. The purity of mAb could efficiently inhibit the growth of Daudi cells. The amplification of the method was also studied using a Bio-Scale Q5 column and the result was satisfied.

The oncogenic Epstein-Barr virus (EBV) -encoded latent membrane protein 1 (LMP1) enables this virus's long-term survival within the cells of immune system. Mean while, LMP1 also plays a critical role for the transformation of resting B cells by EBV. It initiates the activation of signalling pathways, such as NF-kappaB, mitogen-activated protein kinase (MAPK), and JAK/STAT cascade by adaptor proteins including the tumor necrosis factor (TNF) receptor associated factors (TRAFs) and the TNF receptor associated death domain protein (TRADD). It increases the expression of adhesion molecules LFA-1, ICAM-1, and costimulatory molecule B7-1 of B cells, and regulates the antibody and cytokine secreted by B cells. LMP1 and CD40 have many common properties in signal transduction. Both of them co-localize in lipid rafts for signal transduction. Considering its close relationship with CD40, the research on LMP1 has become a hot spot in the immunology field.
Animals ; B-Lymphocytes ; immunology ; CD40 Antigens ; genetics ; physiology ; Gene Expression Regulation, Viral ; Herpesvirus 4, Human ; genetics ; metabolism ; physiology ; Humans ; Signal Transduction ; Viral Matrix Proteins ; genetics ; physiology

OBJECTIVETo observe the effect of single herb pilose antler (PA) on the expression of Smad2 and Smad3 in the cartilage of osteoarthritis (OA) rats.

METHODSOne hundred 3-month old female healthy SD rats, (200 +/- 20) g, were recruited and routinely fed for 1 week. They were randomly divided into 5 groups, i.e., the low dose PA group, the high dose PA group, the normal saline control group, the model group, and the normal control group, 20 in each group. The model was prepared using classic Hulth method except the normal control group. After 6-week modeling, the model was confirmed successful by pathologic observation. PA at 0.021 g/100 g and 0.084 g/1 00 g was given by gastrogavage to rats in the low dose PA group and the high dose PA group respectively. Normal saline was administered to those in the normal saline control group. No treatment was given to rats in the normal control group and the model group. Bilateral knee cartilages were harvested at week 2,4, and 6. mRNA and protein expressions of Smad2 and Smad3 were detected by immunohistochemical assay, fluorescent quantitative PCR, and Western blot.

RESULTSOA model was successfully prepared by pathological observation. Results of immunohistochemical assay showed that Smad2 and Smad3 expressed extensively in the cartilage, and located inside the chondrocyte membrane. Compared with the model group, mRNA expression of Smad2 and Smad3 obviously increased in the low dose PA group and the high dose PA group at week 2, 4, and 6, showing statistical difference (P < 0.05). Compared with the same group at week 4 after gastrogavage, mRNA expression of Smad2 and Smad3 obviously decreased in the low dose PA group and the high dose PA group at week 6, showing statistical difference (P < 0.05). Compared with the model group, protein expression of Smad2 and Smad3 obviously increased in the chondrocytes of the low dose PA group and the high dose PA group at week 2 and 4, showing statistical difference (P < 0.01). Compared with the same group at week 2 after gastrogavage, protein expression of Smad2 and Smad3 obviously increased in the low dose PA group and the high dose PA group at week 4, showing statistical difference (P < 0.01). Compared with the same group at week 4 after gastrogavage, protein expression of Smad2 and Smad3 obviously decreased in the low dose PA group and the high dose PA group at week 6, showing statistical difference (P < 0.01).

CONCLUSIONS(1) The pilose antler could repair cartilages by regulating mRNA and protein expressions of Smad2 and Smad3. (2) Up-regulating mRNA and protein expressions of Smad2 and Smad3 might be one of important mechanisms for the pathogenesis of OA.

Animals ; Antlers ; chemistry ; Cartilage ; cytology ; metabolism ; Chondrocytes ; drug effects ; metabolism ; Female ; Medicine, Chinese Traditional ; Osteoarthritis ; drug therapy ; metabolism ; Rats ; Rats, Sprague-Dawley ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism

OBJECTIVETo observe the effects of indole-3-carbinol (I3C) on the outcome of the tumor as well as the changes of the anti-oxidative system in null mice grafted with nasopharyngeal carcinoma.

METHODS48 BALB/c null mice were divided by means of random number table into control group (0.5% sodium carboxyl methyl cellulose), low dosage (0.02 g/kg), middle dosage (0.1 g/kg) and high dosage (0.5 g/kg) of I3C. The mice were administered with different solutions by gavage for 10 days before CNE1 cells were inoculated subcutaneously into the back (near the armpit) of the nude mice, then the solutions were continually administered by gavage. The tumor volume was measured and the tumor inhibitory rate was calculated. The level of malondialdehyde (MDA), the activity of superoxide dismutases (SOD), the activity of glutathione peroxidase (GSHPx) and the expression of cleaved caspase-3 were determined on the 31th day of the study.

RESULTSI3C could reduce the tumor volume [the tumor volumes of the control group, the middle dosage group and the high dosage group were (4.13 +/- 0.53) x 10(-6) m(3), (3.14 +/- 0.71) x 10(-6) m(3), (2.72 +/- 0.29) x 10(-6) m(3)], as compared with the control, the shrinkage of tumor volume of the middle dosage group and the high dosage group were significant (the t valued at 0.990 and 1.510, P < 0.01). The tumor inhibitory rates of 3 groups were 3.8%, 20.5% and 34.9%, respectively. The contents of MDA in the tumor tissue tended to decrease [the values of control group, the low dosage group, the middle dosage group and the high dosage group were (31.29 +/- 2.51) x 10(-6) mol/L, (30.12 +/- 2.37) x 10(-6) mol/L, (23.32 +/- 1.93) x 10(-6) mol/L, (16.45 +/- 1.43) x 10(-6) mol/L] (F = 98.752, P < 0.01), and that of the high and the middle dosage group could obviously be reduced (t = 8.970, 14.840, P < 0.01) as compared with the control. The activity of SOD seemed to be elevated according to the increase of I3C dosage [the values were (387.24 +/- 23.16) x 10(3) U/L, (399.37 +/- 34.45) x 10(3) U/L, (431.63 +/- 31.24) x 10(3) U/L, (476.45 +/- 44.67) x 10(3) U/L] (F = 53.444, P < 0.01). When compared with the control, the SOD activity of the middle and the high dosage group be obviously increased (t = 44.390, 89.210, P < 0.01). I3C could also elevate the GSHPx activity [the GSHPx values of the four groups were (226.98 +/- 18.35) x 10(3) U/L, (234.65 +/- 15.59) x 10(3) U/L, (247.72 +/- 22.73) x 10(3) U/L, (300.37 +/- 26.02) x 10(3) U/L] (F = 25.916, P < 0.01). The GSHPx of the high dosage group was enhanced remarkable (t = 73.390, P < 0.01) as compared with the control. The expression of cleaved caspase-3 (relative molecular weight = 19 000 000) seemed to be elevated according to the increase of I3C dosage and the relative expression levels of which were 0.87 +/- 0.01, 0.97 +/- 0.01, 1.02 +/- 0.06 and 1.14 +/- 0.02 (F = 39.864, P < 0.01). When compared with the control, the elevation of this kind of cleaved caspase-3 was considered statistical significant (the t values were 0.100, 0.086 and 0.303, respectively, P < 0.05). When I3C dosage increased, the expression of cleaved caspase-3 (relative molecular weight = 17 000 000) seemed to increased too [the relative expression levels of which were 0.00 +/- 0.00, 0.05 +/- 0.02, 0.11 +/- 0.02, 0.20 +/- 0.02 (F = 56.629, P < 0.01)], and the increase of this kind of cleaved caspase-3 was esteemed significantly as compared with those of the control (the t valued at 0.046, 0.103 and 0.193, respectively, P < 0.05). Linear correlate analysis showed that the correlation coefficients between the shrinkage of tumor volume and the expression of the two kinds of cleaved caspase-3 protein was -0.732 (t = 3.404, P < 0.01) and -0.901 (t = 6.642, P < 0.01).

CONCLUSIONI3C could reduce the growth of tumor, the mechanism underlie it could be related to the decrease of the content of MDA as well as the elevated levels of SOD, GSHPx, and perhaps could be related to the apoptosis transduced by cleaved caspase-3.

Animals ; Cell Line, Tumor ; Female ; Glutathione Peroxidase ; metabolism ; Humans ; Indoles ; pharmacology ; Male ; Malondialdehyde ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Nasopharyngeal Neoplasms ; drug therapy ; metabolism ; Neoplasm Seeding ; Oxidation-Reduction ; Superoxide Dismutase ; metabolism

OBJECTIVE1H-NMR technology was carried out to investigate the chemical difference between 30 batches of Cibotium baronetz decoction pieces and look for new method for quality control of C. baronetz decoction pieces.

METHODSix hundreds MHz H-NMR spectroscopy and principle component analysis (PCA) were used to discriminate between 30 batches of commercially available cibotium samples based on multi-component metabolite profiles.

RESULTSaccharide is the principle component of C. baronetz decoction pieces, and steroid and triterpene were the discriminately chemical component. Protocatechuic acid, protocatechuic aldehyde, cibotiumbaroside A, cibotiumbaroside B and 4-O-caffeoyl-D-glucoside could be used as the marker for controlling the quality of commercial C. baronetz decoction pieces.

CONCLUSIONPattern-recognition techniques applied to proton nuclear magnetic resonance (1H-NMR) spectra of 80% methanol extraction of C. baronetz could correctly discriminate not only the quality, but also the chemical component for batches of commercial C. baronetz decoction pieces.

Benzaldehydes ; chemistry ; Caffeic Acids ; chemistry ; Catechols ; chemistry ; Drugs, Chinese Herbal ; chemistry ; standards ; Ferns ; chemistry ; Furans ; chemistry ; Glucose ; chemistry ; Glucosides ; chemistry ; Glycosides ; chemistry ; Hydroxybenzoates ; chemistry ; Magnetic Resonance Spectroscopy ; methods ; Maltose ; chemistry ; Quality Control ; Steroids ; chemistry ; Sucrose ; chemistry ; Triterpenes ; chemistry

Objective To investigate the clinical value of virtual touch tissues quantification in the evaluation of kidney stiffness in patients of diabetic nephropathy .Methods A total of 90 cases of diabetic nephropathy were divided into 3 groups:infinitesimal albuminuria ,microalbuminuria and massive proteinuria groups.And other 30 health subjects were taken as control group.The shears wave velocity ( Vs) which reflected the tissue elasticity was measured.The Vs values were compared among different groups.Results In all groups,the highest Vs was present in renal cortex .And compared with the renal cortex ,the Vs of the renal medulla and renal sinus have statistically significant differences [ the normal control group:( 3.65 ± 0.26)m/s,(2.72 ±0.35) m/s,(1.83 ±0.54) m/s,t =9.30,18.20,both P <0.05;Infinitesimal albuminuria group:(2.98 ±0.28)m/s,(2.47 ±0.33)m/s,(1.65 ±0.31)m/s,t=5.97,15.57,both P<0.05;microalbuminuria group:(2.55 ±0.22) m/s,(2.22 ±0.28) m/s,(1.54 ±0.21) m/s,t =3.86, 11.83,both P<0.05;massive proteinuria group:(1.99 ±0.28)m/s,(1.49 ±0.30)m/s,(1.01 ±0.39)m/s, t=5.85,11.48,both P<0.05].The renal cortex Vs of Infinitesimal albumonuria group ,microalbuminuria group and massive proteinuria group show a gradually decreasing trend .And the renal cortex Vs of microalbuminuria group and massive proteinuria group have statistically significant differences compared with the normal control group(t=11.79,17.79,both P<0.05).Conclusions Virtual touch tissues quantification technique can reflect the renal tissue elasticity .It will contribute to the assessment of renal function in patients with early diabetic nephropathy .

Reduced radiosensitivity of lung cancer cells represents a pivotal obstacle in clinical oncology. The hypoxia-inducible factor (HIF)-1α plays a crucial role in radiosensitivity, but the detailed mechanisms remain elusive. A relationship has been suggested to exist between hypoxia and autophagy recently. In the current study, we studied the effect of hypoxia-induced autophagy on radioresistance in lung cancer cell lines. A549 and H1299 cells were cultured under normoxia or hypoxia, followed by irradiation at dosage ranging from 0 to 8 Gy. Clonogenic assay was performed to calculate surviving fraction. EGFP-LC3 plasmid was stably transfected into cells to monitor autophagic processes. Western blotting was used to evaluate the protein expression levels of HIF-1α, c-Jun, phosphorylated c-Jun, Beclin 1, LC3 and p62. The mRNA levels of Beclin 1 were detected by qRT-PCR. We found that under hypoxia, both A549 and H1299 cells were radio-resistant compared with normoxia. Hypoxia-induced elevated HIF-1α protein expression preferentially triggered autophagy, accompanied by LC3 induction, EGFP-LC3 puncta and p62 degradation. In the meantime, HIF-1α increased downstream c-Jun phosphorylation, which in turn upregulated Beclin 1 mRNA and protein expression. The upregulation of Beclin 1 expression, instead of HIF-1α, could be blocked by SP600125 (a specific inhibitor of c-Jun NH2-terminal kinase), followed by suppression of autophagy. Under hypoxia, combined treatment of irradiation and chloroquine (a potent autophagy inhibitor) significantly decreased the survival potential of lung cancer cells in vitro and in vivo. In conclusion, hypoxia-induced autophagy through evaluating Beclin1 expression may be considered as a target to reverse the radioresistance in cancer cells.

Objective To evaluate the influence and significance of preoperative design of surgical approach on stem cell transplantation via stereotactic surgery. Methods Six patients with stroke in the basal ganglia region were selected. The transplantation target and transcranial approach point were designed by magnetic resonance examination before stem cell transplantation via stereotacfic surgery to guarantee that the line connecting the transplantation target and transcranial approach point could avoid the important functional areas, the ventricular system and the softening focus. Postoperative magnetic resonance examination was performed to observe whether the practical target and surgical approach coincided with the preoperative design or not. Results The practical transplantation target was coincided with the designed transplantation target, distributed around the softening focus without implanted cells in the softening focus. Surgical approach was coincided with the preoperative design and it successfully avoided the important brain functional area, ventricular system and softening focus.Conelnsion The preoperative design of surgical approach can not only ensure the cells being exactly transplanted into the reservation target and guarantee the curative effect, but also promise the surgical approach successfully avoiding the important brain functional area, ventricular system and softening focus and reduce the operative injury.