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.

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.

ObjectiveTo investigate glutathione S-transferase GSTO 1 Glu155△Glu genetic polymorphism and risks of arsenic poisoning caused by coal-burning in Guizhou.Methods GSTO1 Glu155 △Glu gene polymorphism was analyzed by polymerase chain reaction-with confronting two-pair primers among one hundred and thirty arsenic poisoning patients and one hundred and thirty healthy controls.The results were verified by DNA sequencing.The association between different genotypes and arsenic poisoning was analyzed by unconditional Logistic regression model.ResultsThe results of Glu/Glu and Glu/△Glu genotype detected by this method were consistent with those of DNA sequencing.The frequencies of GSTO1 Glu/Glu genotype and Glu/△Glu genotype were 94.85％(92/97) and 5.15％(5/97) in the patients,99.15％(117/118) and 0.85％(1/118) in the controls,respectively.The difference was statistically significant(x2 =3.896,P ＜ 0.05).△Glu/△Glu genotype was not found in both patients and controls.After age and sex adjusting,GSTO1 Glu155 △Glu polymorphism was found to be a risk factor of arsenic poisoning [odds ratio (OR) =1.85,95％ confidence interval (CI):1.39 - 17.48].ConclusionsThe study finds that GSTO1 Glu 155 △ Glu polymorphism is associated with risk of arsenic poisoning.The relationship between them should be further studied through increasing sample size.

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.

OBJECTIVETo explore the role of Compound Danshen Injection (CDI) in regulating the expression of aquaporin 3 (AQP3) in human amnion epithelium cells (hAECs), and to study the relation between c-Jun N-terminal kinase (JNK) signal pathway and AQP3.

METHODShAECs were isolated and primarily cultured from term pregnancy with normal amniotic fluid volume and from term pregnancy with oligohydramnios, and then hAECs were further divided into four groups, i.e., the blank control group (A), the SP600125 group (B), the CDI group (C), and the SP600125 +CDI group (D). The cell viability was measured by cell counting kit-8 assay (CCK-8). The expression of total JNK, phosphorylated JNK, and AQP3 were determined by Western blot.

RESULTS(1) In hAECs with normal AFV or with oligohydramnios: There was no statistical difference in the cell viability or the expression of total JNK among the 4 groups (P > 0.05). But there was statistical difference in the expression of p-JNK (P < 0.05). Compared with A group, the expression of p-JNK was obviously down-regulated in B group, but obviously up-regulated in C group (P < 0.05). The expression of p-JNK was significantly lower in D group than in C group, but higher than that in A group or B group (P < 0.05).The AQP3 expression in the hAECs with normal amniotic fluid volume of C group and D group were higher than that in the A group (P < 0.05). However, there was no statistical difference in the AQP3 expression between C group and D group (P > 0.05). In hAECs with oligohydramnios, the expression of AQP3 obviously decreased in B group, but up-regulated in C group (both P < 0.05). The expression of AQP3 was lower in D group than in C group, but higher than in B group (P < 0.05).

CONCLUSIONCDI could regulate the AQP3 expression in hAECs with oligohydramnios via activating the JNK signal pathway.

Amnion ; cytology ; drug effects ; Aquaporin 3 ; metabolism ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Epithelial Cells ; drug effects ; metabolism ; Female ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; MAP Kinase Signaling System ; physiology

Objective To investigate DNA methylation in the promoter region,mRNA transcription and protein expression of glutathione-S-transferases-P1 (GSTP1) gene and their relation with arsenism.Methods In endemic coal-pollution-borne arsenism area,Jiaole village of Xinren county,Guizhou province,according to the diagnostic criteria of endemic arsenism(WS/T 211-2001),123 cases with endemic arsenism were selected and divided into three groups (mild arsenism group:42 cases,moderate arsenism group:41 cases and severe arsenism group:40 cases).Forty seven residents were selected as controls in a village about 12 km away from the endemic arsenism area.With the informed consent principle,peripheral blood of all respondents was collected in order to analyze DNA methylation and check mRNA.DNA methylation of GSTP1 gene promoter region in peripheral blood was assayed by PCR,and GSTP1 mRNA expression was assayed using real-time quantitative PCR.In addition,other cutaneous specimens originated from 53 cases with arsenism that accepted surgical treatment voluntarily were taken.Of these specimens,general pathological changes were 28 cases,precancerous 20 cases and cancerous 5 cases.Skin tissues of 15 cases of non-tumor surgery patients without abnormal pathological changes were as control group.GSTP1 protein expression in the skin tissue was detected using immunohistochemistry (IHC).Results Among different groups of arsenic poisoning,the positive rate of DNA methylation of GSTP1 gene was 28.57％(12/42) in the mild group,57.10％ (23/41) in the moderate group and 65.00％ (26/40) in the severe group.Compared with the control group (6.38％,3/47),the difference was statistically significant (x2 =7.792,26.000,33.412,all P ＜ 0.01).Among different groups of arsenic poisoning diagnosed by dermapathology,the positive rate of DNA methylation of GSTP1 gene was 21.43％(6/28) in the general pathological change group,50.00％(10/20) in the precancerous group and 80.00％(4/5) in the cancerous group.Compared with the control group(6.67％,1/15),the difference was statistically significant (x2 =3.562,7.468,10.756,all P ＜ 0.05).It showed that the positive rate of DNA methylation of GSTP1 gene increased with aggravation of the disease and dermatic lesion of arsenism (tendency x2 =38.239,x2 =13.659,all P ＜ 0.01).Compared with the control group(0.184 26),the expressions of GSTP1 mRNA in peripheral blood in moderate (0.087 77) and severe arsenic poisoning groups (0.056 93) were significantly reduced(all P ＜0.01),and that of severe group was significantly lower than that of the moderate group (P ＜ 0.01) ; compared with the control group(0.338 45) and the general lesion group(0.276 74),GSTP1 mRNA expression was significantly reduced in precancerous lesion group(0.104 81) and cancerous group(0.043 70),in which the cancerous group was significantly lower than that of the precancerous lesions.The difference of skin tissue GSTP1 protein expression rate between groups was statistically significant (x2 =20.948,P ＜ 0.05),in which the difference between the precancerous lesion group(65.00％,13/20),the cancer group (40.00％,2/5) and the control group(100.00％,15/15)was statistically significant (x2 =12.183,11.778,P ＜ 0.01).Spearman correlation analysis showed that the degree of skin lesion and the level of GSTP1 protein expression was negatively correlated (r =-0.520,P ＜ 0.05).Groups were divided according to DNA methylation of GSTP1 gene,and the mRNA and protein expression of GSTP1 in methylation group(0.038 40,57.14％) was significantly lower compared with that of unmethylated group(0.187 07,95.74％; Z =9.032,x2 =23.134,all P ＜ 0.01).Conclusions Arsenism may lead to DNA methylation of human GSTP1 gene promoter region,thereby inhibiting expression of mRNA and protein.GSTP1 gene plays an important role in arsenism or carcinogenic process.

Objective To determine the protein expression of P14ARF,MDM2 and mutant type P53 (P53mt) in skin specimens of coal-burning-type of endemic arseniasis patients and to reveal the molecular mechanism of the disease.Methods Sixty skin specimens from 60 endemic arseniasis patients including 35 of skin lesions patients,19 of precancerous lesion and 6 of skin cancer and 9 normal skin specimens from non-cancer patients were studied.Expression of P14~,MDM2 and P53mt was evaluated by immunohistochemistry using corresponding monoclonal antibodies.Results There was significant difference in the positive rates of P14ARF,MDM2 and P53mt among the 4 groups(x2 =9.39,6.21,20.64,all P ＜ 0.05).The positive rates of P14ARF in precancerous lesion and skin cancer specimens were 46.1％ (6/19) and 33.3％ (2/6),respectively,which were significantly lower than that of the normal skin specimens [88.9％(8/9),all P ＜ 0.05].Decreased expression of P14ARF was correlated with the development of dermopathy (P ＜ 0.05).The positive rates of MDM2 and P53mt in skin lesions,precancerous lesion and skin cancer specimens were 54.2％ ( 19/35 ),63.2％ (10/19),66.7％ (4/6) and 25.7％(9/35),73.7％(14/19),83.3％(5/6),respectively,which were significantly higher than those of the control (0,0,all P＜ 0.05).The expression of MDM2 and P53mt increased with the development of dermopathy(all P ＜ 0.05).Conclusions P53mt protein in skin tissue of coal-burning-type of endemic arseniasis patients is over expressed.Abnormal expression of P14ARF and MDM2 may be one of the reasons lead to abnormal cell cycle control disorders and may play a role in the development of endemic arseniasis.

AlM: To investigate the effect of endocapsular phacoemulsification cataract extraction and intraocular lens (lOL) implantation with a 1. 8mm or 3. 0mm clear corneal incision on total root mean square ( RMS ) value of the cornea, corneal astigmatism, spherical aberration, coma, trefoil and tear film.
METHODS:ln a prospective study, 156 age- related patients ( 196 eyes ) were randomly distributed into two groups. 1. 8mm-group comprised 94 eyes that had a silicone lOL inserted through a 1. 8mm sutureless clear corneal incision, while, 3. 0mm- group comprised 102 eyes through a 3. 0mm clear corneal incision. Postoperatively, the changes in the total RMS value of the cornea, corneal astigmatism, spherical aberration, coma, trefoil and tear film at 1wk, 1 and 3mo were determined respectively.
RESULTS:ln both groups, postoperatively at 1wk,there were statistically significant differences ( P<0. 05 ) in the total RMS value of the cornea, corneal astigmatism, spherical aberration, coma, trefoil and tear film, while, there were statistically minimal differences ( P< 0. 05 ) between 1. 8mm-group and 3. 0mm-group at 1mo, but were not statistically significantly different ( P > 0. 05 ) between two groups at 3mo postoperative.
CONCLUSlON:This study confirms that incision size has strong impact on the corneal higher-order aberrations, especially, 3. 0mm incision caused significant differences in the total RMS value of cornea, corneal astigmatism, spherical aberration, coma, trefoil and tear film compared with 1. 8mm micro-incision, therefore, micro-incision is very beneficial for clinical use in phacoemulsification.

Objective To study the effect of exogenous prostaglandin E 1 (PGE 1) on the superoxide dismutase(SOD) and nitric oxide(NO) levels in brain tissue of neonatal rats with hypoxic-ischemic brain damage(HIBD).Methods Sixty 7-day old newborn Wistar rats to establish HIBD models,intraperitoneally and subcutaneous injected PGE 1 and TMP,then the rats were killed after hypo- xia and ischemia for 48 hours.Take cerebral cortex of arteria carotis ligation side and made them into homogenate to detect SOD and NO levels in brain tissue.Results SOD level in HIBD group was lower,and NO level was higher than those of normal group(P

Fibrolase is a non-hemorrhagic zinc metalloproteinase isolated from southern copperhead snake venom (Agkistrodon contortrix contortrix) and is capable of degrading fibrin clots resulting from purified fibrinogen or from blood plasma. Alfimeprase, a truncated form of fibrolase, as a clinical agent was successfully completed PhaseII clinical trials.The cDNA of alfimeprase was amplified by recursive PCR, digested with BamHI and HindII, and cloned into pET43.1a, pMALp2X and pMALc2X vectors to generate fusions with NusA, MBP and sMBP(with signal peptide), respectively. Nus/alfimeprase was expressed in soluble form by co-expressing with chaperone FkpA and inducing with1mmol/L IPTG. The fusion protein accounted for about 25 % of total protein following cell lysis. Alfimeprase was successfully purifiesd by Ni-NTA affinity chromatography and cleaved by enterokinase. The results demonstrate the fibrinolytic activity of recombinant alfimeprase using fibrin plate assays and fibrinogen hydrolysis.