0.05),whereas the survival rate in each group of 160 and 320 ?mol/L was significantly higher than that in the control(P

Objective To establish a stable and feasible animal model of acute cerebral infarction,and to evaluate it with functional magnetic resonance imaging and pathology.Methods Twenty-five S-D rats were randomly divided into five groups,and there were 5 rats in each group.Rats in group A were sham-operated for control study.Unilateral middle cerebral artery occlusion(MCAO) was performed with improved thread in group B,C,D,E and were enrolled for MRI and MRS study at 3,6,12,24 hours after MCAO.All rats were examined by 1-hydrogen magnetic resonance spectroscopy(~1H MRS).Two or three rats in each group were sacrificed for 2,3,5-triphenyltetrazolium chloride(TTC) staining of the brain and two rats for pathological examination after MRS.Results Rats in group A showed no change in brain on ~1 H MRS or pathological study.~1H MRS of the rat brains after right MCAO showed a decrease of N-acetyl-aspartate(NAA),an increase of Lactate(Lac) in all groups.There was no significant change of Choline(Cho) and Creatine(Cr) peaks onrats in group B,C,D.The peaks of Cho and Cr were slightly dropped in group E.Conclusion The acute regional cerebral ischemic model in rats made by our approach is stable and reproducible,and it is suitable for evaluation and study with functional magnetic resonancespectroscopy accurately and sensitively.

OBJECTIVETo reveal the role of poly-ADP-ribosylation and DNA methylation in carcinogenic process induced induced by Cr (VI), and to discuss the relations between them.

METHODSThe pre-established Poly (ADP-ribose) glycohydrolase (PARG) deficient cells and 16HBE cells were treated with different concentrations of Cr (VI), and the changes of total genomic DNA methylation level in different groups were detected by methylation immunofluorescent detection, as well as the changes of the activity of methyltransferases. Moreover, RT-PCR and western blotting method were applied to analyze the changes of expression of DNMT1, DNMT3a, DNMT3b and MBD2, upon the protein level.

RESULTSAfter treated by Cr(VI) for 24 h, the healthy 16HBE cells showed a significant lower level of genomic DNA methylation; however, there was no significant changes (P > 0.05) found in PARG deficient cells by immunofluorescence assay. When the dose of Cr (VI) reached 5.0 µmol/L, the activity of methyltransferases in 16HBE cells and PARG deficient cells (49.33 ± 2.65, 80.05 ± 2.05) decreased by 20% and 50% comparing with contrast group (99.27 ± 1.10, 99.30 ± 0.60) . After treated by Cr (VI) for 24 h, the expression of mRNA and protein level among DNMT1, DNMT3a, DNMT3b and MBD2 decreased significantly in healthy 16HBE cells; and the expression of DNMT1 and DNMT3a decreased in PARG deficiency cells. The relevant expression levels of mRNA of DNMT1 were separately (0.99 ± 0.09), (0.79 ± 0.10), (0.59 ± 0.13) and (0.39 ± 0.02) (F = 247.17, P < 0.01), the expression levels of protein were separately (1.00 ± 0.03), (0.69 ± 0.15), (0.65 ± 0.10) and (0.55 ± 0.13) (F = 214.12, P < 0.01), the expression levels of DNMT3a mRNA were separately (1.00 ± 0.04) , (0.93 ± 0.11) , (0.79 ± 0.07) , (0.59 ± 0.05) (F = 498.16, P < 0.01) , and the expression levels of protein were separately (1.00 ± 0.14) , (0.97 ± 0.11) , (0.79 ± 0.17) , (0.57 ± 0.15) (F = 390.11, P < 0.01) when the dose of Cr (VI) at 0, 0.3, 1.2 and 5.0 µmol/L. However, there were no significant changes of expression found in DNMT3b and MBD2.

CONCLUSIONPoly-ADP-ribosylation could regulate the activity of DNMT3b and MBD2, protect cells against the DNA methylation alteration induced by Cr(VI) and maintain the global genomic DNA methylation level.

Cell Line ; Chromium ; toxicity ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; drug effects ; DNA-Binding Proteins ; metabolism ; Epithelial Cells ; metabolism ; Genome ; Humans ; Poly Adenosine Diphosphate Ribose ; metabolism ; RNA, Messenger ; genetics

OBJECTIVETo investigate the effect of poly-ADP-ribosylation in hexavalent chromium Cr(VI) induced cell damage.

METHODSThe study object, poly (ADP-ribose) glycohydrolase (PARG) deficient human bronchial epithelial cells (16HBE cells), was constructed previously by our research group. Normal 16HBE cells and PARG-deficient cells were treated with different doses of Cr (VI) for 24 h to compare the differences to Cr (VI) toxicity, meanwhile set up the solvent control group. On this basis, 5.0 µmol/L of Cr (VI) was selected as the exposure dose, after the exposure treatment, total proteins of both cells were extracted for two dimension fluorescence difference gel electrophoresis (2D-DIGE) separation, statistically significant differential protein spots were screened and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS/MS), and further validated by Western blot.

RESULTSAfter Cr (VI) treatment, the survival rate of PARG-deficient cells was higher than normal 16HBE cells. When the doses reached up to 5.0 µmol/L, the survival rate of 16HBE cells and PARG-deficient cells were respectively (59.67 ± 6.43)% and (82.00 ± 6.25)%, the difference between which was significant (t = -4.32, P < 0.05). 18 protein spots were selected and successfully identified after 2D-DIGE comparison of differential proteins between normal 16HBE cells and PARG-deficient cells before and after exposure. The function of those proteins was involved in the maintenance of cell shape, energy metabolism, DNA damage repair and regulation of gene expression. The differential expression of cofilin-1 was successfully validated by Western blot. The expression level of cofilin-1 in the 16HBE cells increased after Cr (VI) exposure with the relative expression quantity of 1.41 ± 0.04 in treated group and 1.00 ± 0.01 in control group, the difference of which was statistically significant (t = -18.00, P < 0.05), while the expression level in PARG-deficient cells had no statistically significant difference (t = -8.61, P > 0.05).

CONCLUSIONMost of the identified differential proteins are closely related to tumorigenesis, suggesting that poly-ADP-ribosylation reaction may resist the cytotoxicity of Cr(VI) by inhibiting Cr (VI) induced tumorigenesis, which provides important reference data to clarify the mechanisms of poly-ADP-ribosylation in Cr (VI) induced cell damage.

Bronchi ; Cell Transformation, Neoplastic ; genetics ; Chromium ; Cofilin 1 ; DNA Repair ; Epithelial Cells ; Glycoside Hydrolases ; deficiency ; physiology ; Humans ; Tandem Mass Spectrometry

Objective : To study the effect of hydroquinone on autophagy of human L-02 hepatocytes. Methods : L-02 cells were treated with different concentrations of hydroquinone(0,10,20,40 and 80 μmol/L) for 24 hours, the formation of autophagosomes was observed by transmission electron microscope;autophagosome formation was traced by mCheery-GFP-LC3B fusion protein. Western blot experiment was used to detect the expression of autophagy marker proteins LC3,Beclin-1,P62;immunofluorescence experiment was used to detect the subcellular localization and expression of autophagy proteins LC3,Beclin-1,P62. Results : Under the transmission electron microscope,it was observed that the autophagosomes and lysosomes of L-02 cells increased after the action of hydroquinone. Under the fluorescence microscope,the number of GFP-LC3B punctate yellow spots increased with the increase of hydroquinone dose,while the red flake spots decreased. Western blot results showed that hydroquinone increased the ratio of LC3Ⅱ/LC3Ⅰ protein in L-02 cells,and the difference between treatment groups and the control group was statistically significant(P<0. 05). Hydroquinone had no significant effect on Beclin-1 and P62 protein expression in L-02 cells(P>0. 05). The results of immunofluorescence experiments showed that LC3 and Beclin-1 were both expressed in the nucleus and cytoplasm of L-02 cells,hydroquinone had no effect on the subcellular localization of LC3 and Beclin-1 in L-02 cells,while 40 and 80 μmol/L hydroquinone could induce the translocation of P62 in L-02 cells from the cytoplasm to the nucleus. Conclusion Hydroquinone induces the increase of autophagosomes in L-02 cells,which may be related to the obstacles to autophagosome clearance.