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.

Objective To develop a simplified bisulfite genomie sequencing(BGS)method for DNA methylation marker scanning.Methods According to modified BGS protocol,the desalt DNA treated with bisulfite were directly used for bisulfite-PCR(BSP)without alkali treatmenL Complement of the bisulfite modification Wag accomplished by a prolonged pre-denaturation stage.After BSP,a second round PCR was performed with a pair of GC tagged primers to adjust the GC content of the amplieon for direct sequencing.To assess this improved protocol,promotor methylation of TNF-α gene in 3T3-L1 cell and androgen receptor(AR)gene in Hela cell was investigated.The real time BSP for Alu was also used to compare the sensitivity of the modified assay with traditional assay.Results Both the hypermethylated TNF-α promotor and hypomethylated AR promotor were successfully sequenced by improved BGS method,and the results were consistent with that of the traditional assay.The conversion rate reached 100%,while the conversion specificity was higher than 93.75%.The sensitivity of improved BGS method inereaged significantly(t=2.978 2,P＜0.05)and showed good reproducibility.Condusion The improved BGS method is simple and sensitive,facilitating more ambitious genomic methyhtion mapping studies.

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.

Salvia miltiorrhiza is a highly valued traditional chinese medicine for the treatment of atherosclerosis-related disorders in china, such as cardiovascular and cerebrovascular diseases in China. The wilt disease is serious in the culture of S. miltiorrhiza. Wilt disease cause biomass of plant shoots and roots is lessened, active components are decreased. To solve these problems, we research the pathogen causing wilt disease of S. miltiorrhiza. The suspected pathogen is identified by morphology and etiological test. The identification was further confirmed by alignment the sequences of internal transcribed spacer (ITS) amplified by PCR. Our result show the wilt disease of S. miltiorrhiza mostly occurred in July and August, which is hot and wetter. The wilt disease rate of S. miltiorrhiza continuous cropping for one year in S. miltiorrhiz stubble is 10%, but the wilt disease rate of S. miltiorrhiza continuous cropping for three years in S. miltiorrhiz stubble is 60%-70%. The root rot of S. miltiorrhiz caused by the wilt disease, so the wilt disease was mistaken for the rot root in production. Morphological characteristics show the pathogen is Fusarium oxysporum. The sequence of ITS wes determined and found by BLAST shared 99% identity to that of F. oxysporum f. sp. cucumerinum. So it comes to the conclusion that the causing agent of wilt disease on S. miltiorrhiza belongs to F. oxysporum.
DNA, Intergenic ; genetics ; Fusarium ; genetics ; isolation & purification ; physiology ; Plant Diseases ; microbiology ; Polymerase Chain Reaction ; Salvia miltiorrhiza ; microbiology ; Seasons

In order to demonstrate PTB bind to HPRE,reverse transcription,PCR-mediated detection,were used.HepG2.2.15 cell line and HBs-HPRE transient expression cells were adopted to identify PTB function in HBV life cycle.The results showed that PTB could directly bind to HPRE RNA.Functional analysis indicated that PTB could inhibit the expression of HBs antigen and this inhibition was in a dose-dependent manner in HepG2.2.15 cells.Higher expression of HBs in cells transfected pcDNA3-HBs-HPRE comparing with pcDNA3-HBs,and this high expression could also be inhibited by PTB.The data demonstrated that PTB inhibits HBs expression by interacting with HPRE.

Objective To investigate the feasibility of the adaptive cardio sequence prospective ECG-gated dual-source CT coronary angiography (CTCA).Methods Thirty one patients underwent prospective ECG-gated dual-source CTCA. The patients were divided into two groups: group A, heart rate ≤ 75 times / minutes, 70% of R-R interval was chose; group B, heart rate> 75 times / minutes, 40% of R-R interval was chose. Two experienced radiologists assessed the image quality of 15 segments of coronary arteries in a double blinded fashion in a four score scale. Student t test was used to compare the image quality between two groups. CT dose index(CTDIvol) and effective dose(ED) were also calculated for both groups. Results 437 segments of coronary arteries in 31 patients were included this analysis, 408 segments (93.4%) had assessable image quality, while 29 segments (6.6%) had non assessable image quality. Of 226 segments in group A, 12 segments (5.3%) had non assessable image quality; however, 17 segments (8.1%) of 211 segments in group B had non assessable image quality. The average score of image quality was 3.57±0.64 in 31 patients. There was no statistical difference for mean image quality of group A (3.65±0.39) and group B (3.41±0.43)( t=1.62,P=0.12 ) . There was a statistical difference for mean image quality for segment 10 on a per segment basis ( t = 3.8 ,P＜0.05). CTDIvol and ED were (18.88±5.04) mGy and (4.31±1.05) mSv, respectively.Conclusion Adaptive cardio sequence prospective ECG-gated dual source CT can acquire assessable image quality of coronary artery with lower radiation dose to the patients, which has the potential to be a routine CTCA technique.

OBJECTIVETo screen and identify proteins that interact with hepatitis C virus NS3 by means of T7-phage display system.

METHODSHepatitis C virus NS3 was expressed by prokaryotic expression and used as a selected molecule to biopan the T7 select human liver cDNA library; the selected positive clones were identified using DNA sequencing and analyzed with BLAST program in GenBank.

RESULTSAfter BLAST analysis in all the positive clones, the proteins which interacted with the hepatitis C virus NS3 were found to be serpin peptidase inhibitor, clade A, member 1 (SERPINA1) and cyclophilin-LC.

CONCLUSIONT7-phage display system is a convenient, rapid and effective method for screening interacting proteins. The proteins thus selected will provide an important means for studying the pathogenesis and carcinogenesis of HCV.

Cell Line ; Gene Library ; Hepacivirus ; metabolism ; Humans ; Peptide Library ; Protein Interaction Mapping ; methods ; Viral Fusion Proteins ; genetics ; isolation & purification ; metabolism ; Viral Nonstructural Proteins ; genetics ; metabolism

OBJECTIVETo explore the influence of arsenic pollution caused by coal-burning on methylation (promoter and exon 5) and mutation (exon 5) of human p53 gene, and to analyze the relationship between methylation, mutation and arsenism.

METHODSAccording to the diagnostic criteria of endemic arsenism, 112 patients with arsenism (including 38 mild cases, 43 moderate cases and 31 severe cases) were selected in the areas with endemic arsenism from Xingren, Guizhou province. Among the subjects, 43 cases were diagnosed by dermatopathological methods, and they were divided into non-cancerous group (24 cases) and cancerous group (19 cases). 90 controls were selected from the non-arsenic polluted areas. Under the principle of informed consent, blood samples were collected from individuals. The methylation of p53 gene in promoter region and exon 5 were detected by extinction enzyme-PCR, the mutation of p53 gene (exon 5) was detected by PCR-SSCP, PCR products cloning and sequencing technology.

RESULTSThe positive rates of methylation of p53 gene in promoter region were 13.16% (5/38), 27.91% (12/43) and 45.16% (14/31) respectively among mild, moderate and severe arsenism group, which were obviously higher than the rates in the control group (1.11% (1/90), χ² values were 8.679, 23.690, 41.199, respectively, both P values < 0.017). The positive rates of methylation of p53 gene were 25.00% (6/24) and 63.16% (12/19) in non-cancerous and cancerous group respectively, which were obviously higher than those in the control group (1.11% (1/90), χ² values were 18.762, 57.497, respectively, both P values < 0.025). The positive rates of methylation of p53 gene (exon 5) were 55.26% (21/38), 51.16% (22/43) and 48.39% (15/31) respectively among mild, moderate and severe arsenism group, which were obviously lower than the rates in the control group (88.88% (80/90), χ² values were 18.151, 23.168, 22.420, respectively, both P values < 0.017). The positive rates of methylation of p53 gene (exon 5) were 54.17% (13/24) and 42.11% (8/19) in non-cancerous and cancerous group respectively, which were obviously lower than those in the control group (88.88% (80/90), χ² values were 15.201, 22.075, respectively, both P values < 0.025). The mutation rates of p53 gene (exon 5) were respectively 5.26% (2/38), 16.28% (7/43) and 25.81% (8/31) among mild, moderate and severe arsenism group; while the results in moderate and severe arsenism group were obviously higher than in the control group (0.00%, χ² values were 15.465, 24.870, respectively, both P values < 0.017). The positive rate of mutation of p53 gene (exon 5) were respectively 16.67% (4/24) and 31.58% (6/19) in non-cancerous and cancerous group, which were obviously higher than it in the control group (0.00%, χ² values were 15.545, 30.077, both P values < 0.025). The hypermethylation of p53 gene in promoter region was related with the mutation of p53 gene (exon 5) (coefficient of association was 0.294, P value < 0.05); and the hypomethylation of p53 gene (exon 5) was related with the its mutation (coefficient of association was 0.410, P value < 0.05).

CONCLUSIONArsenic pollution caused by coal-burning can cause the hypermethylation of p53 gene in promoter region, hypomethylation and mutation of p53 gene (exon 5), and the changes of methylation of p53 gene are related with its mutation and might be one of the important etiological factors of arsenic pathogenicity or carcinogenesis.

Adult ; Arsenic Poisoning ; etiology ; genetics ; Case-Control Studies ; Coal ; adverse effects ; DNA Methylation ; Environmental Pollution ; adverse effects ; Female ; Genes, p53 ; Humans ; Male ; Middle Aged ; Mutation ; Promoter Regions, Genetic ; Tumor Suppressor Protein p53 ; genetics

BACKGROUNDMultiple epiphysis dysplasia (MED) is a common skeletal dysplasia with a significant locus heterogeneity. In the majority of clinically defined cases, mutations have been identified in the gene encoding cartilage algometric matrix protein (COMP).

METHODSFive patients were included in the study. Linkage analysis and mutation analysis of the COMP gene were conducted in the patients and their family members.

RESULTSWe have identified a novel mutation in axon 14 of COMP gene in the family.

CONCLUSIONSThis mutation produced a severe MED phenotype with marked short stature, early onset osteoarthritis, and remarkable radiographic changes. Our results extended the range of disease-causing mutations in COMP gene and contributed more information about relationship between mutations and phenotype.

Adolescent ; Asian Continental Ancestry Group ; Cartilage Oligomeric Matrix Protein ; genetics ; Female ; Humans ; Male ; Osteochondrodysplasias ; genetics ; Pedigree ; Point Mutation ; genetics