OBJECTIVETo investigate the role of NADPH oxidase (Nox) in the oxidative stress injury of human dermal fibroblasts (HFbs).

METHODSAn oxidative stress injury model was established in HFbs by exposure to H(2)O(2). Normal HFbs and HFbs exposed to H(2)O(2) with and without pretreatment with NADPH oxidase inhibitor were tested for cell viability using MTT assay, and the intracellular reactive oxygen species (ROS) were determined with a DCFH-DA fluorescent probe. Western blotting was used to measure the protein expressions of membrane-bound subunit gp91phox of NADPH oxidase in the cells.

RESULTH(2)O(2) time- and concentration-dependently induced oxidative stress injury in the fibroblasts, causing a reduction of the cell viability to 40% after a 24-h exposure at 700 µmol/L (P<0.05) and an increase of ROS by 2 folds after a 2-h exposure at 700 µmol/L (P<0.05). Compared with the cells with oxidative stress injury, the cells with NADPH oxidase inhibitor pretreatment showed a 20% higher cell viability (P<0.05) and normal ROS level (P<0.05) following H(2)O(2) exposure. Western blotting demonstrated increased expression of gp91phox in the cells exposed to increasing H(2)O(2) concentrations, but gp91phox expression remained normal in cells pretreated with NADPH oxidase inhibitor.

CONCLUSIONH(2)O(2) can induce oxidative stress injury in the fibroblasts by affecting NADPH oxidase, especially its membrane-bound subunit gp91phox.

Cell Survival ; Cells, Cultured ; Fibroblasts ; cytology ; enzymology ; Humans ; Hydrogen Peroxide ; Membrane Glycoproteins ; metabolism ; NADPH Oxidase 2 ; NADPH Oxidases ; antagonists & inhibitors ; metabolism ; Oxidation-Reduction ; Oxidative Stress ; Reactive Oxygen Species ; metabolism

Trichomonas vaginalis induces proinflammation in cervicovaginal mucosal epithelium. To investigate the signaling pathways in TNF-alpha production in cervical mucosal epithelium after T. vaginalis infection, the phosphorylation of PI3K/AKT and MAPK pathways were evaluated in T. vaginalis-infected SiHa cells in the presence and absence of specific inhibitors. T. vaginalis increased TNF-alpha production in SiHa cells, in a parasite burden-dependent and incubation time-dependent manner. In T. vaginalis-infected SiHa cells, AKT, ERK1/2, p38 MAPK, and JNK were phosphorylated from 1 hr after infection; however, the phosphorylation patterns were different from each other. After pretreatment with inhibitors of the PI3K/AKT and MAPK pathways, TNF-alpha production was significantly decreased compared to the control; however, TNF-alpha reduction patterns were different depending on the type of PI3K/MAPK inhibitors. TNF-alpha production was reduced in a dose-dependent manner by treatment with wortmannin and PD98059, whereas it was increased by SP600125. These data suggested that PI3K/AKT and MAPK signaling pathways are important in regulation of TNF-alpha production in cervical mucosal epithelial SiHa cells. However, activation patterns of each pathway were different from the types of PI3K/MAPK pathways.
Cell Line ; Cervix Uteri/enzymology/metabolism/*parasitology ; Epithelial Cells/*enzymology/metabolism/parasitology ; Female ; Humans ; *MAP Kinase Signaling System ; Mucous Membrane/*enzymology/metabolism/parasitology ; Phosphatidylinositol 3-Kinases/genetics/*metabolism ; Proto-Oncogene Proteins c-akt/genetics/*metabolism ; Trichomonas Vaginitis/*enzymology/genetics/metabolism/parasitology ; Trichomonas vaginalis/*physiology ; Tumor Necrosis Factor-alpha/genetics/*metabolism

OBJECTIVETo investigate the expression of tripartite-motif protein 25 (TRIM25) and pyruvate kinase M2 (PKM2) protein in non-small cell lung cancer (NSCLC) and explore their role in the occurrence and progression of NSCLC.

METHODSThe expressions of TRIM25 and PKM2 protein were detected in 60 NSCLC specimens and 20 adjacent normal lung tissue (>5 cm from the lesions) with immunofluorescence histochemical method and in 10 fresh specimens of NSCLC with Western blotting. The results were analyzed in relation with the clinicopathological features of the patients.

RESULTSThe positivity rates of TRIM25 expression was 45% in the 60 lung carcinoma specimens, significantly higher than that in the 20 normal lung tissues (10%, P=0.005). TRIM25 protein was expressed in 28.6% of lung adenocarcinoma tissues and in 59.4% of squamous carcinoma tissues (P=0.017). TRIM25 protein expression was positively correlated with the TNM stages and lymph node metastasis of NSCLC (P<0.05). The expressions of PKM2 protein in 60 cases of lung carcinoma was 73.3%,while in 20 cases of normal lung tissues the expressions was 30%(P=0.001). The positivity rates of PKM2 expression differed significantly between lung adenocarcinoma and squamous carcinoma (57.1% vs 87.5%, P=0.008). An inverse correlation was noted between TRIM25 and PKM2 expressions (P=0.026).

CONCLUSIONTRIM25 and PKM2 protein may participate in the occurrence and progression of NSCLC, and their expressions are inversely correlated.

Adenocarcinoma ; enzymology ; Carcinoma, Non-Small-Cell Lung ; enzymology ; Carcinoma, Squamous Cell ; enzymology ; Carrier Proteins ; metabolism ; Humans ; Lung ; pathology ; Lung Neoplasms ; enzymology ; Lymphatic Metastasis ; Membrane Proteins ; metabolism ; Thyroid Hormones ; metabolism ; Transcription Factors ; metabolism ; Tripartite Motif Proteins ; Ubiquitin-Protein Ligases ; metabolism

OBJECTIVETo explore the effects of Panax Quinquefolium Saponin (PQS) on phosphatidylinositol 3-kinase/serine threonine kinase (PI3K/Akt) pathway of neonatal rat myocardial cells subjected to hypoxia.

METHODSNeonatal rat myocardial cells were cultured in vitro. After the myocardial cell injury was induced by hypoxia, the cells were randomized into 5 groups: the normal group, the model group, the positive control group (Ciclosporin A, 2 µ mol/L), the low-dose PQS group (PQSL, 25mg/L), and the high-dose PQS group (PQSH, 50 mg/L). Morphology and behavior of myocardial cells were observed under an inverted microscope. Apoptosis rate and lactate dehydrogenase (LDH) leakage rate of myocardial cells were determined by colorimetry. Mitochondrial transmembrane potential was assessed using a fluorexon laser. Phospho-glycogen synthase kinase (GSK)-3β and phospho-Akt as well as cytochrome C were determined by Western blot

RESULTSLDH leakage in the Ciclosporin A group, PQSH group and PQSL group reduced progressively compared with the model group (P<0.05). Akt and GSK-3β was strongly phosphorylated after treatment with Ciclosporin A and PQS compared with the model group (P<0.05, P<0.01). Compared with the model group (16.41±1.74; 35.28±6.30), both the integrated optical density of mitochondrial permeability transition pore (MPTP) and the mitochondrial transmembrane potential significantly increased in the PQSH group (42.74±2.12; 71.36±6.54) and the PQSL group (39.58±1.49; 66.99±5.45; P<0.05, P<0.01). However, the protein of cytochrome C outside the mitochondrion decreased in the PQSH group (273.66±14.61) and the PQSL group (259.62±17.31) compared with the model group (502.41±17.76; P<0.05).

CONCLUSIONThrough activation of the PI3K/Akt pathway and inhibition of the MPTP, PQS might protect the heart against ischemia injury and apoptosis of myocardial cells.

Animals ; Animals, Newborn ; Cell Hypoxia ; drug effects ; Cell Shape ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; L-Lactate Dehydrogenase ; metabolism ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; metabolism ; Myocytes, Cardiac ; cytology ; drug effects ; enzymology ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; drug effects ; Protein-Serine-Threonine Kinases ; metabolism ; Rats, Sprague-Dawley ; Saponins ; pharmacology ; Signal Transduction ; drug effects

OBJECTIVETo confirm the anticancer effect of total annonaceous acetogenins (TAAs) abstracted from Annona squamosa Linn. on human hepatocarcinoma.

METHODSThe inhibitory effect of TAAs was demonstrated in H22-bearing mice. The potency of TAAs was confirmed as its 50% inhibiting concentration (IC50) on Bel-7402 cell under Sulfur Rhodamine B staining. Both underlying mechanisms were explored as cellular apoptosis and cell cycle under flow cytometry. Mitochondrial and recipient apoptotic pathways were differentiated as mitochondrial membrane potential under flow cytometry and caspases activities under fluorescence analysis.

RESULTSThe inhibitory rate of TAAs in mice was 50.98% at 4 mg/kg dose. The IC50 of TAAs on Bel-7402 was 20.06 µg/mL (15.13-26.61µg/mL). Effective mechanisms of TAAs were confirmed as both of arresting cell cycle at G1 phase and inducing apoptosis dose- and time-dependently. Mitochondrial and recipient pathways involved in apoptotic actions of TAAs.

CONCLUSIONTAAs is effective for hepatocarcinoma, via inhibiting proliferation and inducing apoptosis.

Acetogenins ; chemistry ; pharmacology ; therapeutic use ; Animals ; Annona ; chemistry ; Antineoplastic Agents, Phytogenic ; chemistry ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; drug therapy ; enzymology ; pathology ; Caspases ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chromatography, High Pressure Liquid ; Dose-Response Relationship, Drug ; Humans ; Liver Neoplasms ; drug therapy ; enzymology ; pathology ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Mice ; Organ Specificity ; drug effects ; Spleen ; drug effects ; Thymus Gland ; drug effects ; Xenograft Model Antitumor Assays

Allosteric Regulation ; Carrier Proteins ; chemistry ; genetics ; metabolism ; Cell Proliferation ; Gene Expression ; Glycolysis ; genetics ; Humans ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Mutation ; Neoplasms ; enzymology ; genetics ; pathology ; Oxidative Phosphorylation ; Protein Conformation ; Protein Multimerization ; Protein Subunits ; chemistry ; genetics ; metabolism ; Thyroid Hormones ; chemistry ; genetics ; metabolism ; Tumor Cells, Cultured

OBJECTIVE: We detected expression of MMP9 to discuss its role in the occurrence and development of sinonasal squamous cell carcinoma. METHOD: The immunohistochemical staining, real-time PCR and Western blot were used to measure the expression of MMP9 in sinonasal squamous cell carcinoma tissues (Experimental group) and corresponding normal mucosa tissues (Control group). Relationship between MMP9 and the main clinical features of patients with sinonasal squamous cell carcinoma was analysed. RESULT: Positive expression rates of MMP9 in sinonasal squamous cell carcinoma tissues and corresponding normal mucosa tissues were 81. 25% and 18. 52% respectively. Positive expression rate of MMP9 was not significantly correlated with patient's age and gender (P>0. 05), but correlated with pathological type (P<0. 05). The expression of MMP9 mRNA in sinonasal squamous carcinoma tissues was 30. 66 times of tissues adjacent to carcinoma (P<0. 05). Western blot analysis also showed that the expression of MMP9 protein in squamous carcinoma tissues was significantly higher than tissues adjacent to carcinoma (P<. 05). CONCLUSION The expression of MMP9 was significantly higher in the sinonasal squamous cell carcinoma and correlated with the degree of differentiation. The results suggest that MMP9 may play a role in the occurrence and development of sinonasal squamous cell carcinoma and degree of malignancy from the protein and cellular and molecular level. The higher degree of malignancy, the stronger expression.
Carcinoma, Squamous Cell ; enzymology ; Head and Neck Neoplasms ; enzymology ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Mucous Membrane ; enzymology ; Paranasal Sinus Neoplasms ; enzymology ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Squamous Cell Carcinoma of Head and Neck

The tissue-specific and MeJA-induced transcriptional levels of BcUGT3 and BcUGT6 in Bupleurum chinense were analyzed in the present study. The transcriptional levels of BcUGT3 in root, leaf, flower and fruit were similar and they all were higher than those in stem. The transcriptional level of BcUGT6 was the highest in leaf and the lowest in flower among in all tested tissues. With non-treated adventitious roots as control, BcUGT6's transcriptional levels were elevated to nearly 2 folds for 2 h, 8 h, 24 h, 2 d and 4 d in MeJA-treated adventitious roots of B. chinense. It showed that the transcriptional level of BcUGT6 was slightly affected by MeJA. While, BcUGT3's transcriptional levels were gradually elevated, and till 4 d after MeJA treatment, the expression level was about 7 folds than that of non-treated control. Using pET-28a (+), the expressions of two genes was investigated. Induced by IPTG, the target proteins were expressed in E. coli and then purified. All the results obtained in the present study will be helpful for follow-up bio-function analysis of BcUGT3 and BcUGT6.
Acetates ; pharmacology ; Bupleurum ; cytology ; enzymology ; genetics ; Cell Membrane ; metabolism ; Cyclopentanes ; pharmacology ; Escherichia coli ; genetics ; Gene Expression ; Gene Expression Regulation, Plant ; drug effects ; Hexosyltransferases ; chemistry ; genetics ; isolation & purification ; metabolism ; Intracellular Space ; metabolism ; Oxylipins ; pharmacology ; Protein Sorting Signals ; Protein Structure, Secondary ; Protein Transport ; Sequence Analysis ; Transcription, Genetic ; drug effects

Chronic granulomatous disease (CGD) is a rare genetic disease, which is caused by defects in the NADPH oxidase complex (gp91phox, p22phox, p40phox, p47phox, and p67phox) of phagocytes. This defect results in impaired production of superoxide anions and other reactive oxygen species (ROS), which are necessary for killing bacterial and fungal microorganisms and leads to recurrent, life-threatening bacterial and fungal infections and granulomatous inflammation. The dihydrorhodamine (DHR) flow cytometry assay is a useful diagnostic tool for CGD that can detect absent or reduced NADPH oxidase activity in stimulated phagocytes. We report a patient with X-linked CGD carrying a novel mutation of the CYBB gene whose chimerism status following hematopoietic stem cell transplantation (HSCT) has been rapidly determined using the DHR assay. The level of DHR activity correlates well with short tandem repeat PCR analysis. Considering the advantages of this simple, rapid, and cost-effective procedure, serial measurement of DHR assay would facilitate the rapid determination of a patient's engraftment status, as a supplementary monitoring tool of chimerism status following HSCT.
Base Sequence ; *Chimerism ; DNA Mutational Analysis ; Flow Cytometry ; Granulomatous Disease, Chronic/*diagnosis/*enzymology/genetics/surgery ; *Hematopoietic Stem Cell Transplantation ; Homozygote ; Humans ; Infant, Newborn ; Male ; Membrane Glycoproteins/chemistry/*genetics ; Mutation ; NADPH Oxidase/chemistry/*genetics ; Polymerase Chain Reaction ; Rhodamines/chemistry/metabolism

To study the impact of the enterovirus 71(EV71) on the nuclear transport mechanism,The pGFP-NLS vector with nuclear location signal(NLS) was constructed, RD cells transfected by the pGFP-NLS vector were inoculated with the EV71 or cotransfected by EV71-2A vector. The results showed that GFP protein with NLS was expressed in the cytoplasm due to the inhibition of nuclear transport. In order to further study the mechanism of the EV71 to prevent nuclear transport,Nup62 was detected by Western blotting after RD cells were infected with EV71 or transfected by EV71-2A vector. The results showed that decreased expression of Nup62 could be detected after infection with EV71 and transfection by EV71-2A vector. This study demonstrates that the cleavage of Nup62 by EV71 2A protease may be the mechanism of nuclear transport inhibition.
Active Transport, Cell Nucleus ; Cell Line, Tumor ; Cell Nucleus ; metabolism ; Enterovirus A, Human ; enzymology ; genetics ; metabolism ; Enterovirus Infections ; virology ; Gene Expression Regulation, Viral ; Genetic Vectors ; Green Fluorescent Proteins ; metabolism ; Humans ; Membrane Glycoproteins ; metabolism ; Nuclear Localization Signals ; metabolism ; Nuclear Pore Complex Proteins ; metabolism ; Peptide Hydrolases ; metabolism ; Recombinant Fusion Proteins ; metabolism ; Transfection