ObjectiveTo investigate the protective effect of all-trans retinoic acid (ATRA) on injury of human immortalized hepatocytes (HHL-5 cells ) induced by sodium arsenite and possible mechanisms.Methods After cultured for 48 h,HHL-5 cells were divided into four groups:normal group,ATRA group,sodium arsenite group and ATRA + sodium arsenite group.HHL-5 cell viability was tested by using cell proliferation experiment (WST).Superoxide dismutase(SOD),glutathione peroxidase(GSH-Px) activity,malondialdehyde(MDA) content,and aspartate aminottransferase (AST) activity in each group were determined by biochemical method.The microstructure of HHL-5 cells in each group was observed under transmission electron microscopy.ResultsHHL-5 cell viability(0.57 ± 0.02) of sodium arsenite group was compared with that of normal group(0.70 ± 0.01 ),the difference was statistically significant(P ＜ 0.05).Levels of SOD,GSH-Px,MDA and AST[ (153.84 ± 2.35),(0.08 ±0.02)U/mg Prot,(4.15 ± 0.50)nmol/mg Prot,(265.43 ± 4.62) × 103 U/L] of sodium arsenite group were compared with that of normal group[(237.41 ± 18.30),(0.93 ± 0.02)U/mg Prot,(2.26 ± 0.40)nmol/mg Prot,(177 ± 9.85) ×103 U/L],and the difference was statistically significant (all P ＜ 0.05).HHL-5 cell viability (0.65 ± 0.04) of ATRA + sodium arsenite group was compared with that of sodium arsenite group, and the difference was statistically significant (P ＜ 0.05).Levels of SOD,GSH-Px,MDA and AST[ (286.85 ± 3.39),(0.56 ± 0.09)U/mg Prot,(3.36 ± 0.37)nmol/mg Prot, (220.02 ± 1.07) × 103 U/L] of ATRA+ sodium arsenite group were compared with that of sodium arsenite group,the difference was statistically significant(all P ＜ 0.05).Compared with normal group and ATRA group,the surface microvilli of HHL-5 cells of sodium arsenite group decreased,double-membrane structure was unclear,vacuolar degeneration was seen in the cytoplasm,and glycogen was aggregated.The damage level of ATRA + sodium arsenite group was decreased.ConclusionsATRA plays a protective role through increasing intracellular antioxidant enzyme activity of HHL-5 cells,removal or reduction of oxygen free radicals produced by sodium arsenite.

Objective To observe the influences of different doses of sodium arsenite on mRNA transcription of keratinizing related and nuclear factor E2-related factor 2(Nrf2) genes in HaCaT cells.Methods Cell proliferation was evaluated by Cell Counting Kit-8(CCK-8) assay after the HaCaT cells were exposed to 0.00,3.13,6.25,12.50,25.00,50.00,75.00,100.00 μ mol/L sodium arsenite for 48 h,respectively.Based on the previous results of cell proliferation,0.00(control),6.25,12.50,and 25.00 μmol/L of sodium arsenite were selected to treat HaCaT cells for 48 h,respectively.The mRNA expression of keratin 1,keratin 10,involucrin,loricrin and Nrf2 were detected by real-time fluorescent quantitative PCR.ResultsCompared with the control group (100.05％),HaCaT cell proliferation rates(83.06％,51.04％,39.52％,24.51％,16.99％ and 9.04％) were significantly lower in 6.25,12.50,25.00,50.00,75.00 and 100.00 μ mol/L of sodium arsenite groups and the 50％ inhibiting concentration was 12.38 μmol/L.Compared with the control group( 1.06 ± 0.28,1.00 ± 0.12,1.00 ± 0.08),the mRNA expression of keratin 1,involucrin and loricrin (0.08 ± 0.04,0.13 ± 0.12,0.05 ± 0.03;0.47 ± 0.11,0.21 ± 0.09,0.10 ± 0.15; 0.50 ± 0.27,0.31 ± 0.10,0.57 ± 0.23) were significantly decreased(all P ＜ 0.05) in HaCaT cells treated with 6.25,12.50,25.00 μmol/L sodium arsenite,respectively.But keratin 10 mRNA expression showed a rise trend and the 6.25 μmoL/L sodium arsenite group (1.83 ± 0.45) was significantly higher than that of the control( 1.07 ± 0.14,P ＜ 0.05 ).The Nrf2 mRNA expressions of HaCaT cells in 12.50,25.00 μmol/L sodium arsenite groups(0.13 ± 0.07,0.69 ± 0.33) were significantly lower than that of the control ( 1.00 ± 0.09,all P ＜ 0.05 ).ConclusionsThe cellular proliferation and keratinization are decreased when HaCaT cells are exposed to sodium arsenite,which may be regulated by lowering Nrf2 mRNA transcription.

ObjectiveTo evaluate the role of Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway in attenuation of myocardial ischemia-reperfusion (I/R) injury by tetramethylpyrazine in rats.MethodsSixty-four healthy male Wistar rats weighing 250-300 g were randomly divided into 4 groups( n ＝ 16 each):sham operation group (group S),myocardial I/R group(group I/R),teramethylpyrazine group (group T) and AG,490( a JAK2 inhibitor) group (group AG).Myocardial I/R was induced by 30 min occlusion of left anterior desecending coronary artery (LAD) followed by 120 min reperfusion in groups I/R,T and A.In groups T and A teramethylpyrazine 20 mg/kg was injected iv 20 min before LAD occlusion.In group A AG490 3 tμg/g was injected iv at 5 min before reperfusion.Blood samples were then taken from inferior vena cava at 120 min of reperfusion for measurement of serum creatine phosphokinase (CK) and lactose dehydrogenase (LDH) activities.Myocardial infarct size was then measured and myocardial tissue was obtained for microscopic examination.ResultsSerum CK and LDH activities were significantly higher in group I/R than in group S.Pretreatment with tetramethylpyrazine significantly decreased myocardial infarct size and I/R-induced increase in serum CK and LDH activities and histologic damage.The protective effect of tetramethylpyrazine against myocardial 1/R injury was attenuated by postconditioning with AG490.ConclusionJAK2/STAT3 signaling pathway is involved in attenuation of myocardial I/R injury by tetramethyl pyrazine in rats.

By means of preparative HPTLC and column chromatography over silica gel and Sephadex LH-20, nine diterpenoids were isolated and purified from the whole plants of Scutellaria strigillosa. Based on the physico-chemical properties and spectral data, their structures were elucidated as: 6-O-acetyl-7-O-nicotinoylscutebarbatine G(1), 6-O-nicotinoyl-7-O-acetylscutebarbatine G(2), 6,7-di-O-nicotinoylscutebarbatine G(3), scutebarbatine K(4), scutebarbatine B(5), 6-O-acetylscutehenanine A(6), 6-O-nicotinoylbarba- tin A(7), 6,7-di-O-acetoxylbarbatin A(8), scutebarbatine F(9). Compound 1 is a new diterpenoid, and compounds 2-9 were isolated from Scutellaria strigillosa for the first time.
Diterpenes ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Scutellaria ; chemistry ; Spectrometry, Mass, Electrospray Ionization

Objective To investigate the effects of dominant-negative truncation mutant?NTCF4, lacking the N-terminal form of TCF4 gene,on biological characteristics of renal cancer cell line GRC-I and explore the molecular mechanisms.Methods GRC-I cell was transfected with pCDNA3-?NTCF4 eukary- otie expression plasmid,pCDNA3 empty vector to construct the stable cell line GRC-I/?NTCF4 and GRC-I/ Mock respectively.The morphological changes of stable cells were observed and the cells growth curve was detected through light microscope.The cellular proliferation activities were determined using the MTT assay. The protein expression of Wnt pathway downstream target gene C-Myc and Cox-2 was evaluated by immuno- cytoehemieal method and Western Blot analysis.Results After the dominant-negative?NTCF4 gene was permanently expressed,the GRC-I/?NTCF4 stable cells morphologically showed that appearance changed from circular to long-spindle shape,growth rate decreased with less karyosehisis found,malignant pheno- types reversed to normal renal tubular cells.MTT assay revealed that the proliferation activities of GRC-1/?NTCF4 cells were inhibited by 11.2%-35.5% compared with GRC-I cells (P＜0.05),while the GRC- I/Mock cells have no difference with the control cells.Immunocytochemical analysis and Western Blot showed that the C-Myc and Cox-2 protein expression level of GRC-I/?ANTCF4 cells were significantly sup- pressed in comparison with that of GRC-I/Mock and GRC-I cells.Conclusions The dominant-negative truncation mutant?NTCF4 could partially inhibit the growth of renal cancer cells and down-regulate the pro- tein expression of Wnt pathway target gene C-Myc and Cox-2.These findings provide a experimental founda- tion for applying cell signal therapy to renal cell cancer by blocking the Wnt signaling pathway.

Autophagy, an evolutionarily conserved process by which components of the cell are degraded in lysosomes, may facilitate survival of cancer cells under stress conditions. 8-Azaguanine (8-AG), an inhibitor of purine nucleotide biosynthesis, shows antineoplastic activity in multiple tumor cells. However, chemoresistance has restricted its development as an anticancer agent, and the mechanism of 8-AG resistance is not fully understood. We report here that 8-AG induces a protective autophagy to eliminate its cytotoxicity, and inhibition of autophagy increases cellular sensitivity of cancer cells to 8-AG treatment. Using HepG2 or SMMC-7721 hepatic cancer cell lines, we found that 8-AG inhibited cell viability and induced intrinsic apoptosis, accompanied by the up-regulation of the pro-apoptotic protein BimS, one of Bim (also known as BCL-2-like protein 11, BCL2L11) isoforms. Furthermore, 8-AG treatment enhanced the autophagy flux by promoting the dephosphorylation and activation of Unc-51-like autophagy activating kinase 1 (ULK1) via Akt/mTORC1 (mammalian target of rapamycin complex 1) signaling inhibition. Depletion of autophagy-related gene 7 (ATG7) markedly enhanced the level of BimS, and promoted cell death in response to 8-AG. 8-AG in combination with autophagy inhibitor chloroquine (CQ) or bafilomycin A1 (Baf A1) promoted the 8-AG-induced apoptosis in hepatic cancer cells. Altogether, these findings suggest that autophagy promotes chemoresistance of cancer cells for 8-AG, and blocking autophagy increases cellular sensitivity of cancer cells to 8-AG treatment.

OBJECTIVETo evaluate the expression of hypoxia inducible factor (HIF)-1alpha, 2alpha in sporadic clear cell renal cell carcinoma and their relationships to the mutations of von Hippel-Lindau (VHL) gene.

METHODSMutations of VHL gene, expression of HIF-1alpha and 2alpha were detected by polymerase chain reaction (PCR), direct DNA sequencing and immunohistochemistry in 77 cases of Chinese sporadic clear cell renal cell carcinoma (CCRCC). The stage was pT(1)N(0)M(0)in 55 patients (71%), pT(2)N(0)M(0) in 7 patients (9%), pT(3)N(0)M(0) in 14 patients (18%), and pT(4)N(0)M(0) in 1 patient (1%). The classification according to the tumor nuclear grading system showed 15 carcinomas (19%) of tumor nuclear grade 1, 56 (73%) of tumor nuclear grade 2 and 6 (8%) of tumor nuclear grade 3.

RESULTSNone of the VHL gene mutations were found in all the normal tissue specimens. VHL gene mutations were detected in 40 (52%) cases of CCRCC. The positive rate of HIF-2alpha (81%) was higher than that of HIF-1alpha (66%) (chi(2) = 23.310, P < 0.01); The positive rate of HIF-1alpha and HIF-2alpha in the cases of mutations (98% and 93% respectively) was higher than that of them in non-mutations (32% and 68% respectively) (chi(2) = 36.386, 7.617, P < 0.01); The correlation between HIF-1alpha and VHL gene mutations was closer than that between HIF-2alpha and VHL gene mutations (partial correlation coefficiency was 4.481 and 2.027 respectively, P < 0.01). The expression of HIF-1alpha and 2alpha in different pathological grade and stage of CCRCC showed no significant difference (P > 0.05).

CONCLUSIONSOur study suggests that VHL gene mutations are frequent in sporadic CCRCC, and the high expression of HIF-1alpha and 2alpha are found in the group of VHL mutations. However, we have not found significant correlation between the expression of HIF-1alpha and 2alpha and pathological grade and stage of CCRCC in our study.

Adult ; Aged ; Aged, 80 and over ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Carcinoma, Renal Cell ; genetics ; metabolism ; pathology ; Female ; Humans ; Hypoxia-Inducible Factor 1 ; metabolism ; Immunohistochemistry ; Kidney Neoplasms ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Mutation ; Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics

OBJECTIVETo explore the relationship between chronic prostatitis (CP) and prostatic calculus (PC).

METHODSWe used transperineal ultrasonography (TPUS) to detect PC in 500 normal volunteers and 491 CP patients, and divided them into a CP and a CP + PC group according to the ultrasonographic results. Then we analyzed the NIH-CPSI scores, duration of symptoms and white blood cell count in the expressed prostate secretion (ESP).

RESULTSPC was found in 19.8% of the normal controls, 5% (5/100), 12% (12/100), 19% (19/100), 27% (27/100) and 36% (36/100) in the 20-30 yr, 31-40 yr, 41-50 yr, 51-60 yr and > 60 yr groups, respectively. In comparison, PC was detected in 42.2% of the CP patients, 15.8% (12/76), 30.1% (69/215), 55.7% (59/109), 66.2% (43/65) and 82.8% (24/29) in the above five age groups, respectively, with statistically significant differences between the control and CP groups (P < 0.01). The CP and CP + PC groups showed significant differences in the duration of symptoms and white blood cell count in ESP (P < 0.01) but not in CPSI scores (P < 0.05).

CONCLUSIONThe incidence of PC is higher in CP patients than in healthy men, and it is associated with inflammation, aging and symptom duration, but not with CPSI scores.

Adult ; Aged ; Aged, 80 and over ; Bodily Secretions ; Calculi ; pathology ; Case-Control Studies ; Chronic Disease ; Humans ; Inflammation ; Leukocyte Count ; Male ; Middle Aged ; Prostate ; secretion ; Prostatic Diseases ; pathology ; Prostatitis ; pathology ; Young Adult

OBJECTIVETo study the genetic characterizations of VP1 region of Human enterovirus 71 (HEV71) isolated from clinical specimens of hand, foot and mouth disease (HFMD) patients in Beijing in 2008.

METHODS285 clinical samples were collected from HFMD patients in hospitals and day-care centers in Chaoyang district. They were performed by reverse transcription-polymerase chain reaction (RT-PCR) specific for HEV71. 10 HEV71 isolates were selected for entire VP1 coding gene amplification and sequencing.

RESULTS129 samples were RT-PCR positive, the positive rate is 45.26%. The homology of the nucleotide and the amino acid of the 10 strains were 94.6%-99.6% and 95.9%-100%. The phylogenetic tree revealed that 10 Beijing strains clustered within the C4a evolution branch of C4 subgenotype.

CONCLUSIONSRT-PCR played an important role in identifying HFMD outbreak in Beijing in 2008. The HEV71 strains were all belong to C4a evolution branch of C4 subgenotype with several transmission chains, and it showed that C4 subgenotype HEV71 spread in mainland China widely after 1998. The molecular epidemiology surveillance and the research of genetic characterizations of HEV71 should be strengthened in mainland China.

Capsid Proteins ; genetics ; China ; epidemiology ; Disease Outbreaks ; Enterovirus A, Human ; genetics ; isolation & purification ; Enterovirus Infections ; epidemiology ; genetics ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; methods

OBJECTIVETo construct and screen the suppression subtractive hybridization (SSH) library of human renal cell carcinoma (RCC).

METHODSPoly A(+) RNA was isolated from RCC lines 786-O (tester) and renal cell (RC) lines HK-2 (driver), respectively. SSH procedure was performed according to the protocol of the PCR-Select cDNA Subtraction Kit (Clontech), and PCR products were cloned into pT-Adv vector and transformed E. coli TOP10F'. All positive clones picked out were digested and some of which were sequenced.

RESULTSThe SSH library contained 362 clones with SSH cDNA fragments distributed mainly from 0.3 to 0.9 kb. Among 50 clones sequenced randomly, 2 represented unknown genes and the other 48 derived from 36 known genes.

CONCLUSIONThe quality of the SSH library of human RCC is reliable and its construction is the basis for further screening differentially expressed genes of RCC.

Adenocarcinoma, Clear Cell ; genetics ; Cell Line, Tumor ; Gene Library ; Humans ; Kidney Neoplasms ; genetics ; Nucleic Acid Hybridization ; methods