Objective To investigate the DNA methylation feature and DNA methylation regulation to its transcription and expression of O6-methylguanine-DNA methyltransferase gene (MGMT) in NaAsO2-treated HaCaT cells. Methods HaCaT cells were treated 72 hours at intervals and repeatedly by 3.13, 6.25,12.50, and 25.00 μmol/L NaAsO2, MGMT gene promoter region was amplified in the transcription initiation site - 329 - + 93 region by bisulfate-sequencing polymerase chain reaction (BSP), the mRNA transcription and the protein expression of MGMT was detected by real-time quantitative PCR and Western blotting. NaAsO2-untreated HaCaT cell was set as a blank control, and human epidermal squamous carcinoma cell strain A431 was set as a positive control. Results Among the groups of HaCaT cells treated with 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, the positive rates of the DNA methylation of promoter region in MGMT gene were 0.63%(l/160), 6.25% (10/160), 10.63%( 17/160) and 18.75% (30/160), respectively, and methylated CpG sites were mainly located in - 249--146 region relative to transcription start site. There was no DNA methylation in the blank control. There were significant differences between the blank control and the NaAsO2-treated cells (x2 = 76.687, P< 0.05). Average levels of MGMT mRNA were 1.518 31 ± 0.180 54, 1.425 22 ± 0.180 39, 1.014 54 ± 0.096 79 and 0.887 72 ± 0.020 00, respectively among the groups of HaCaT cells treated with 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, compared with the blank control cells(1.198 29 ± 0.159 97), there were significant differences(F = 37.359, P < 0.05). Average levels of MGMT protein were 1.174 47 ± 0.064 75, 0.848 83 ± 0.057 01, 0.471 63 ± 0.023 34 and 0.240 34 ± 0.014 43, respectively among the groups of HaCaT cells treated with 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, compared with the blank control cells (1.066 19 ± 0.061 24), there were significant differences(F = 20.687, P < 0.05). Conclusions Arsenic can cause CpC island hypermethylation in the promoter region of MGMT gene, which results in inhibited MGMT mRNA transcription and protein expression. It might be one of the important mechanisms of arsenic-induced skin lesion.

0.05).Conclusions Immunologic function alteration of MPP of infants is showed mainly that Th1 immunologic response is raised,Th2 immunologic response is abated,the cellular factor are changed horizontally,but the immunology index and cellular factor are recovered quickly.There are no relations between MPP of infant and PHF-AH genotype,but there is a downward trend in activity of PHF-AH in acute stage.

Adrenoleukodystrophy ; genetics ; therapy ; Humans ; Lovastatin ; pharmacology

Animals ; Cadmium Radioisotopes ; toxicity ; Female ; Fetal Development ; drug effects ; Fetus ; Maternal-Fetal Exchange ; Pregnancy ; Rats ; Rats, Sprague-Dawley

Diagnostic Errors ; Epilepsy ; diagnosis ; Ethylene Dichlorides ; poisoning ; Humans ; Male ; Young Adult

Objective To investigate the effect of NaAsO2 on the binding levels of methyl CpG binding protein 2(MeCP2),DNA methyltransferase 1 (DNMT1) and histone deacetylase 1(HDAC1) to the hypermethylation promoter region of MGMT gene in HaCaT cells,in order to provide a basis to deepen the interpretation of the role of arsenic poisoning mechanism.Methods HaCaT cells were treated repeatedly and interval with different concentrations of NaAsO2(3.13,6.25,12.50,25.00 μnol/L,respectively) for 72 h.Untreated HaCaT was used as blank control group and human epidermal squamous carcinoma cell line(A431 cells) as positive control group.The binding levels to the two transcription regulatory regions(ChIP1,ChIP2) and to the coding region(ChIP3) of MGMT 8ene were detected by chromatin immuno-precipitation combined with quantitative PCR.Results The differences of binding levels of MeCP2,DNMT1 and HDAC1 to ChIP1 and ChIP2 in each group were significant (F=7.387,84.634,78.442 and 19.263,69.649,26.546,all P ＜ 0.05).The binding levels of MeCP2,DNMT1 and HDAC1 to ChIP1 and ChIP2 in each NaAsO2 exposed group[3.13 μmol/L NaAsO2 exposed group:(136.00 ±16.97)％,(145.00 ± 2.83)％,(88.50 ± 19.09)％ and (106.50 ± 37.48)％,(112.34 ± 8.73)％,(59.71 ± 8.49)％;6.25 μmol/L NaAsO2 exposed group:(130.00 ± 42.43)％,(154.50 ± 4.95)％,(101.00 ± 1.27)％ and (88.50 ±3.54)％,(134.32 ± 2.82)％,(102.75 ± 19.91)％ ; 12.50 μmol/L NaAsO2 exposed group:(141.50 ± 23.33)％,(161.50 ± 7.78)％,(125.00 ± 11.31)％ and (119.50 ± 24.75)％,(171.59 ± 3.54)％,(167.61 ± 10.61)％; 25.00μmol/L NaAsO2 exposed group:(134.50 ± 43.13)％,(472.50+ 50.20)％,(383.50 ± 30.41)％ and (180.09 ±12.73)％,(348.50 ± 27.58)％,(158.45 ± 12.02)％] were higher than that in the blank control group[(51.50 ±9.19)％,(82.00 ± 12.73)％,(25.03 ± 2.91)％ and (37.02 ± 4.24)％,(91.56 ± 26.16)％,(19.09 ± 2.90)％,all P ＜ 0.05].The differences of binding levels of MeCP2 to ChIP3 in each group were not significant(F =1.670,P ＞0.05),but the differences of binding levels of DNMT1 and HDAC1 to ChIP3 were significant (F =4.404,9.863,all P ＜ 0.05),and only the binding levels in the 25.00 μmol/L NaAsO2 exposed group [(615.85 ± 29.63)％,(306.09 ± 59.40)％] were higher than that in the blank control group[(99.70 ± 12.02)％,(92.45 ± 48.79)％,all P ＜ 0.05].Conclusions MeCP2 can bind to the methylated MGMT gene transcriptional regulatory regions which are induced by arsenic and leads to histone deacetylation by the recruitment of DNMT1 and HDAC1 and,meanwhile,DNMT1 can bind to the coding region of MGMT gene to recruit HDAC1 in a methyl DNA binding protein(MBD) independence manner and media MGMT gene silencing through the chromatin remodeling way,which might be the early molecular events of arsenic poisoning.

Objective To investigate the transcription and expression of DNA methyltransferase 1 (DNMT1) mRNA in endemic arsenism patients by burning coal usage,to probe its effects on the development and carcinogenesis of arsenism. Methods In 2008,68 arsenism patients(including 24 mild cases,28 moderate cases and 16 severe cases) were selected in the areas with endemic arsenism according to Standarding of Diagnosis for Endemic Arsenism from Xingren county,Guizhou province. Among the subjects,40 cases were diagnosed by pathological methods,and they were divided into general pathological changes(20),precancerous(14) and cancerous group(6). Tweleve kilometer away from the endemic arsenism area,23 controls were selected in Daguoduo village (non-arsenism exposure). Under the principle of informed consent,blood samples were collected from individuals. The mRNA expression of DNMTI was detected by real-time quantitative reverse transcription polymerase chain reaction(FQ-PCR). At the same time,skin tissue samples were collected from the voluntary surgical treatment patients with endemic arsenism (total 61 cases,including 34 general pathological changes cases,21 precancerous cases and 6 cancerous cases) and from the control(15 cases). DNMT1 protein was detected by immunohistochemical method.Results Average level of DNMT1 mRNA were 0.221 83±0.595 09,0.246 11±0.509 79 and 0.389 27±0.411 33 respectively among mild,moderate and severe arsenism group. DNMT1 mRNA level of mild and moderate group were obviously lower than the control group(0.695 95±0.463 98,all P < 0.01). The mRNA average level of DNMT1 were 0.320 64±0.547 46,0.313 09±0.529 13 and 0.159 07±0.342 56 individually among general pathological changes,precancerous and cancerous group,which were obviously lower than the control group(0.695 95±0.463 98,all P < 0.05). The expression rates of DNMT1 protein in skin were 88.24%(30/34),100%(21/21) and 100% (6/6) among general pathological changes,precancerous and cancerous group were higher than the control group [0(0/15),all P < 0.01],and the extent of expression gradually increased with the aggravation of skin damage(r,= 0.740,P < 0.01). Conclusions DNMT1 participated in the development of the arsenism. High expression of its protein was an early event during the process of the arsenism. DNMT1 may be the new target markers for early diagnosis and treatment of arsenism.

AIM:To observe the effects of Dickkopf-3 ( Dkk-3 ) in diabetic retinopathy ( DR ) circulating blood in patients with the expression level, the Dkk - 3 development changes in the diabetic retinopathy of significance in the diagnosis of early DR.
● METHODS: Eighty - five type 2 diabetic patients, included the non - proliferative diabetic retinopathy ( NPDR ) 23 patients, proliferative diabetic retinopathy, proliferative DR ( PDR ) in patients with 30 and non-diabetic retinopathy ( NDR ) with 32 cases. The same period of healthy physical examination was selected as control group ( 80 cases ) . Serum samples were collected, and the relative expression level of Dkk-3 was detected by enzyme - linked immunosorbent assay ( ELlSA) double antibody sandwich assay. The statistical differences were compared between groups.
●RESULTS: The plasma level of Dkk - 3 ( 430. 16 ± 198. 11pg/mL) in DR patients was significantly lower than that in healthy control group (627. 48±294. 45 pg/mL; P<0. 05 ) and NDR patients ( 601. 99 ± 194. 16 pg/mL; P<0. 05). While there was no significant difference in Dkk-3 level between NDR and healthy control group ( P =0. 729). The level of PDR in patients with Dkk-3 (396. 38± 185. 59 pg/mL) was lower than that of NPDR (538. 82 ± 187. 20 pg/mL;P=0. 002).
●CONCLUSION:The decrease of Dkk-3 level may be related to the occurrence and development of diabetic retinopathy, and there is a significant correlation with PDR. Circulating blood Dkk - 3 protein in diabetic retinopathy has a certain differential efficacy, it is likely to become diabetic retinopathy patients peripheral blood test indicators.

Background Misfolding of Myocilin protein plays an important role in the pathogenesis of primary open angle glaucoma (POAG).GRP78 can transfer the misfolded proteins out endocytoplasmic reticulum and keep the protein synthesis function of endoplasmic reticulum.However the relationship between GRP78 and Myocilin in the pathogenesis of POAG has not been elucidated.Objective This study was to investigate and analyze the coexpressions of GRP78 and Myolicin in trabecular meshwork cells (TMCs) of POAG.Methods The trabecular meshwork tissues were obtained during the surgery of POAG and healthy donor for the isolated and in vitro culture of TMCs,and the morphology of the cells was compared between POAG-derived TMCs and donor-derived TMCs.The expression of specific factors for TMCs were detected by immunochemistry.The cells were divided into normal control group,tunicamycin (Tm)-treated group and staurosporine (STS)-treated group,and the cells were cultured by regular medium,the 1 μ mol/L Tm medium or 0.1 μmol/L STS medium for 24 hours respectively.The co-expression of GRP78 and Myocilin in the cells were detected using immunofluorescence assay.This study was approved by Ethics Committee of Xi'an No.4 Hospital,and written informed consent was obtained from each patient before surgery.Results Primarily cultured cells showed the fiat star-like and irregular appearance with 3-5 processes,round nuclei,abundant cytoplasm and black engulf particles.The 3-7 generations of POAG-derived cells grew well,showing positive expressions of laminin (LN),fibronectin (FN),neuronal specific enolase (NSE) and Vimentin.Immunofluorescence assay showed positive expressions of GRP78 and Myocilin in the cells of the Tm group,STS group and normal control group,with the reddish and green fluorescence separately.Incomplete co-expression of GRP78 and Myocilin was seen in donor-derived TMCs,and complete co-expression of GRP78 and Myocilin was found in the POAG-derived TMCs in the normal control group.Complete co-expression of GRP78 and Myocilin was exhibited in both donor-and POAG-derived TMCs in the Tm group and STS group.In addition,accumulation of GRP78 protein around nucleus was seen in both donor-and POAG-derived TMCs in the Tm group.Conclusions GRP78 and Myocilin proteins are completely co-expressed in POAG-derived TMCs,inferring that GRP78 and Myocilin play an interaction role in the pathogenesis and development of POAG.GRP78 may play a cytoprotective role against the apoptosis of TMCs caused by endoplasmic reticulum stress.

Dextrocardia ; diagnosis ; Female ; Heart Septal Defects, Atrial ; diagnosis ; Heart Ventricles ; abnormalities ; Humans ; Infant, Newborn ; Truncus Arteriosus, Persistent ; diagnosis