Objective To investigate the mechanisms by which nuclear factor-κB (NF-κB)translocates to nucleus of human glioblastoma U-87MG cells. Methods The levels of NF-κB translocating to nucleus in U-87MG after activation or inhibition ofphospholipase C-γ1 (PLCγ1) were first determined by electrophoretic mobility shift assay (EMSA), and then the levels of NF-κBtranslocating to nucleus in U-87MG after activation or inhibition of protein kinase-α (PKCα) were determined by EMSA. Results The level of NF- κB translocating to nucleus of U-87MG peaked 60min after EGF stimulation, which could be reversed by the pretreatment of PLCγ1 specific inhibitor U-73122. Moreover, PKC specific agonist phorbol myristate acetate (PMA) stimulation for 60 min promoted the nuclear translocation of NF-κB to the peak, which could be also effectively reversed by the pre-treatment of PKCα specific antagonist Ro 31-8220. Conclusions Epidermal growth factor can mediate the nuclear translocation of NF-κB in U-87MG through the signal transduction of PLCγ1-PKCα,thereby regulating the transcription of related invasion and metastasis genes.

Objective To investigate the adverse effect of different doses of high power microwave(HPM) irradiation on oxidative stress in the brain of Wistar rats in order to contribute to establishing an animal model to evaluate protective agents which will be used for protection against microwave radiation.Methods Eighty male Wistar rats were randomly divided into 16 groups according to factor analysis.The average power density was 0,10,30 and 100 mW/cm2 and the sampling time was 6 h,1,3 and 7 d .The duration of exposure was 6 minutes for each radiation group.After exposure, the rats were sacrificed at each sampling time.Colorimetric method was used to measure the content of malondialdehyde(MDA) and protein carbonyl, the activity of GSH-px, SOD and CAT.Results The content of MDA and protein carbonyl of each radiation group was increased with the radiation dose, but decreased with the sampling time prolonged.The activity of superoxide dismutast(SOD),glutathion peroxidase(GSH-px) and catalase(CAT) in each radiation group was decreased with the radiation dose increased, and with the sampling time prolonged, but increased later.Conclusion Microwave radiation can cause oxidative stress in rats brain, as shown by the oxidative damage of lipid and protein and the decrease in the activity of antioxidant enzymes.Besides, the effect also depends on the radiation dose and sampling time.

OBJECTIVETo study the effects of pentobarbital sodium in generation and modulation of rhythmical respiration in neonatal rats.

METHODSThe effects of pentobarbital sodium were examined on hypoglossal nerve (XII) rootlets and inspiratory neurons in the medullary preparations including the medial region of the nucleus retrofacialis, pre-Bötzinger complex and the dorsal respiratory group of neonatal rats aged 0-3 days. The electrical activity of XII nerve rootlets and inspiratory neurons were recorded. Different doses of pentobarbital sodium (20, 40, 60, 80 micromol/L) were added into modified Krebs solution to observe changes in the discharge activity of XII nerve and inspiratory neurons. Bicuculline was used to further investigate the mechanisms that pentobarbital sodium suppresses respiration.

RESULTSThe discharge activity inhibition of XII nerve was increased as pentobarbital sodium doses increased from 20 to 60 micromol/L, but no significant difference was observed between the doses of 60 and 80 micromol/L. Bicuculline can partly restore the rhythmical respiration discharge activity.

CONCLUSIONPentobarbital sodium can suppress respiration partly via GABAA receptors.

Adjuvants, Anesthesia ; pharmacology ; Animals ; Animals, Newborn ; Dose-Response Relationship, Drug ; Medulla Oblongata ; cytology ; drug effects ; physiology ; Neurons ; drug effects ; physiology ; Pentobarbital ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, GABA-A ; physiology ; Respiration ; drug effects ; Respiratory Center ; drug effects ; physiology

OBJECTIVETo explore the role of protein kinase D3 (PKD3) in the regulation of matrix metalloproteinases 7 (MMP-7) expression in prostate cancer cells.

METHODSPC-3 cells were either stimulated with 100 nmol/L PMA to activate PKD3 kinase activity, or transiently transfected with PKD3 siRNA, and the relative expression level of MMP-7 mRNA were analyzed by real-time PCR using 2(-delta delta Ct) method. MMP-7 mRNA levels were also analyzed and quantified in HEK293 cells with over-expression of wild-type PKD3, PKD3 knockdown (using PKD3 siRNA), or over-expression of wild-type PKD3 followed by PKD3 knockdown.

RESULTSMMP-7 mRNA expression in PC3 cells was significantly decreased after PMA-induced PKD3 kinase activation. In contrast, PKD3 knockdown by siRNA transfection markedly increased MMP-7 mRNA level (P<0.01). MMP-7 mRNA level in HEK293 cells was significantly decreased by PKD3 over-expression, whereas obviously increased by PKD3 knockdown. Down-regulation of MMP-7 mRNA level in HEK293 induced by PKD3 over-expression was rescued by PKD3 knockdown.

CONCLUSIONPKD3 may contribute to the malignant progression of prostate cancer cells through negative regulation of MMP-7 expression.

Cell Line, Tumor ; Down-Regulation ; Gene Knockdown Techniques ; Humans ; Male ; Matrix Metalloproteinase 7 ; metabolism ; Prostatic Neoplasms ; enzymology ; metabolism ; Protein Kinase C ; metabolism ; Signal Transduction

OBJECTIVETo evaluate the efficacy and optimal re-implantation time of two-stage revision for management of periprosthetic infection following hip arthroplasty.

METHODSWe retrospectively analyzed the clinical data of 15 patients (15 hip joints) undergoing two-stage ipsilateral total hip arthroplasty (THA) revision from January, 2006 to January, 2010. In the first stage, after surgical debridement and thorough removal of all the implants, a self-made Vancomycin-loaded cement spacer was implanted. The second stage operation was performed 3-6 months later for debridement and removal of the antibiotic-loaded spacer, followed by re-implantation of Vancomycin-loaded bone cement prosthesis in 9 cases and cementless prosthesis in 6 cases. The patients were followed up for 9-46 months (mean 25 months) after the operation.

RESULTSNo reinfection or prosthesis loosening/displacement was found in these cases after the operation. The Harris score increased from 40.3 before the operation to 54.0 after the first-stage operation, and to 88.2 at the last follow-up.

CONCLUSIONTwo-stage revision is effective for treatment of periprosthetic infection following hip arthroplasty, and 3-6 months can be the optimal interval between the two the first-stage and second-stage operation for re-implantation.

Adult ; Aged ; Arthroplasty, Replacement, Hip ; methods ; Female ; Hip Prosthesis ; Humans ; Male ; Middle Aged ; Prosthesis-Related Infections ; surgery ; Reoperation ; Retrospective Studies ; Treatment Outcome

OBJECTIVETo To establish a method of phosphoprotein affinity profiling for identifying phosphoproteomic differences between Thp-1 cells with or without lipopolysaccharide (LPS) stimulation, aiming to screen potential regulators involved in LPS pathway.

METHODSThp-1 cells were stimulated with 100 ng/ml PMA for 48 h to induce differentiation into mature macrophages, which, after culture for another 48 h in the absence of PMA, were either treated with 100 ng/ml LPS for 30 min or left untreated. After desalting procedure with ultrafiltration, the phosphoproteins enriched by phosphoprotein metal affinity column (PMAC) of both groups were run on 2-D electrophoresis to find the spots with different phosphorylation status. Finally, some of these spots were identified by mass spectrometry (MS) and subsequent bioinformatic analysis.

RESULTSCompared to untreated Thp-1 cells, LPS stimulated Thp-1 cells showed 29 spots with reproducible alterations on the 2-D map, including 8 representing up-regulated spots, 7 new spots, 10 down-regulated spots, and 4 absent spots. The newly emerged and absent protein spots were subjected to MS analysis, and 4 of them were identified to be involved in various cellular processes such as proteolysis, signal transduction and protein folding. Among these, phosphorylation of proteasome C2 subunit was dramatically up-regulated in LPS-stimulated cells, as was consistent with previous reports; the phosphorylation of Z-DNA-binding protein 1 has not been reported so far and needs further confirmation.

CONCLUSIONPhosphoprotein affinity profiling is an attractive method for screening novel regulators involved in LPS signaling pathways and can be widely used in systemic study of signal transduction.

Cell Line ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Lipopolysaccharides ; pharmacology ; Macrophages ; cytology ; drug effects ; metabolism ; Mass Spectrometry ; Phosphoproteins ; analysis ; metabolism ; Phosphorylation ; Proteomics ; methods ; Signal Transduction

Objective To evaluate the effect of long-term microwave radiation on the expression of N-methyl-D-aspartate receptor(NMDAR),brain derived neurotrophic factor(BDNF) and related molecules in signal pathways in the hippocampus of rats.Methods Fifty male Wistar rats were exposed to microwave radiation at an average power density of 0,5,10,20 and 30 mW/cm2for 6 min/time,3 times/week,and for 6 weeks,which were sacrificed and the hippocampus was quickly removed at 14 d and 28 d after exposure.The changes in NMDAR (NR1,NR2A,NR2B),postsynaptic density protein(PSD)-95,cortactin,BDNF and tyrosine kinase receptor B (TrkB) in hippocampal neurons of each group were detected by Western blotting and image analysis techniques.Results Compared with the control group,the expressions of related proteins did not change significantly after microwave irradiation of 5 mW/cm2 at each time point.After 20 mW/cm2 microwave radiation,the expression of NR1 was increased at 14 and 28 d (P ＜0.05),the expression of NR2A was increased at 28 d (P ＜ 0.05),but the expression of NR2B was decreased at 14 and 28 d (P ＜ 0.05).At a average power density of 30 mW/cm2,the expressions of NR1,NR2A and PSD-95 and the expression of NR2B were decreased at 14 and 28 d(P ＜0.05),and cortactin,BDNF and TrkB were increased at 14 d after irradiation (P ＜ 0.05).Conclusion The effect of different dosages of long-term microwave radiation on the proteins of NMDAR and its signal pathway related molecules is different.Microwave radiation may affect the NMDAR of postsynaptic information transmission through the BDNF-TrkB signaling pathways,which might play an important role in the impediment of learning and memory function caused by microwave radiation.

The aim of the present study is to investigate the differences in cardiac function, and the expression and activity of calcium regulatory proteins between rabbit systolic heart failure (SHF) and diastolic heart failure (DHF) models. New Zealand white rabbits were randomly divided into three groups: sham operation (SO) group, DHF group (receiving abdominal aortic constriction) and SHF group (receiving aortic valve destruction and abdominal aortic constriction). The cardiac function was detected by echocardiographic and hemodynamic assays. The mRNA expression levels of sarcoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a) and phospholamban (PLB) were evaluated by RT-PCR. The protein expression levels of SERCA2a, PLB, phosphoserine 16-PLB (pSer-16-PLB) and protein kinase A (PKA) were evaluated by Western blot, and the phosphorylation status of PLB was determined by the ratio of pSer-16-PLB protein level to that of PLB. The activity of SERCA2a was measured through inorganic phosphate. The activity of PKA was measured by gamma-(32)P ATP-binding assays. Compared with SO group, there were significantly increased ventricular wall thickness, raised left ventricular end diastolic pressure (LVEDP), reduced diastolic function in DHF group (P<0.05 or P<0.01), and significantly increased ventricular cavity size and LVEDP, reduced systolic function in SHF group (P<0.05 or P<0.01). The expression levels of SERCA2a in DHF and SHF groups were lower than that in SO group (P<0.05), while the expression and activity of PKA in DHF and SHF groups were higher than that in SO group (P<0.05 or P<0.01), and there was no significant difference between DHF and SHF groups. The expression levels of PLB and pSer-16-PLB as well as the phosphorylation status of PLB and activity of SERCA2a in SHF group were lower than those in DHF and SO groups respectively. Posing a contrast, the phosphorylation status of PLB and activity of SERCA2a in DHF group were higher than that in SO group (P<0.05). These results indicate that the SHF and DHF models were successfully established, and there are some differences in the expression and activity of calcium regulatory proteins between two models.
Animals ; Calcium-Binding Proteins ; metabolism ; Disease Models, Animal ; Heart Failure, Diastolic ; metabolism ; Heart Failure, Systolic ; metabolism ; Rabbits ; Sarcoplasmic Reticulum ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism

AIM To establish an HPLC method for the simultaneous content determination of phellodendrine chloride,berberine hydrochloride,jatrorrhizine hydrochloride,(R,S)-goitrine,astilbin,hesperidin and atractylenolide Ⅲ in Supplemented Ermiao Granules (Phellodendri chinensis Cortex,Atractylodis Rhizoma,Smilacis glabrae Rhizoma,etc.) and to establish fingerprints.METHODS The analysis of 70％ methanol extract of this drug was performed on a 35 ℃ thermostatic Amethyst C18-H column (4.6 mm ×250 mm,5 μm),with the mobile phase comprising of 0.05 mol/L potassium dihydrogen phosphate-acetonitrile flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelengths were set at 237,291 nm.RESULTS Seven constituents showed good linear relationships within their own ranges (r =1),whose average recoveries were 95.77％-110.36％ with the RS-Ds of 0.06％-2.94％.There were twenty common peaks in the fingerprints of fourteen batches of samples with the similarities of more than 0.990,five of which (5-hydroxymethylfurfural,phellodendrine,astilbin,hesperidin and berberine hydrochloride) were identified.CONCLUSION This stable,reliable and reproducible method can be used for the quality control of Supplemented Ermiao Granules.