Differentiation of Pc and P2 variants in class 1 integron by high-resolution melting analysis / 中华检验医学杂志

Objective To develop a simple high-resolution melting ( HRM) analysis method for differentiation of Pc and P2 variants in class 1 integron.Methods DNA fragments containing Pc and P2 variants were amplified from plasmids pACW ( PcW ) and pACWP2 ( PcW-P2 ) respectively , then these purified PCR products and P 2 promoters were analyed full-length amplicon by HRM .Eight DNA fragments containing different Pc promoters were amplified and site-specific mutated from plasmids pACS ( PcS ) , pACH2 ( PcH2 ) , pACH1 ( PcH1 ) , pACW ( PcW ) , genomic DNA of Klebsiellar pneumonia HS07-68 (PcWTGN-10)and HS05-1792(PcH2TGN-10)respectively.The purified PCR products and eight Pc variants were characterized by HRM analyses of an unlabeled probe and full-length amplicon.This assay was applied to the differentiate Pc and P2 variants in 109 class 1 integrons from 95 urine clinical Escherichia coli isolates in Huashan Hospital during 2004 -2007.The differentiation results were compared with that determined by direct sequencing .Results P2 promoter with a significant higher melting temperature ( Tm ) can be identified by HRM analysis clearly .P2 promoters were identified in 2 class 1 integrons and consistent with direct sequencing results .Eight Pc variants were classified into three groups: PcS, PcSTGN-10 , PcW, PcWTGN-10, PcH1, PcH1TGN-10.Using direct HRM analysis.PcH2, PcH2TGN-10 were classified into four groups:PcS, PcH1, PcH2, PcW, PcSTGN-10 , PcH1TGN-10 , PcH2TGN-10 , PcWTGN-10 according to the melting curves of the unlabeled probe .Combined the HRM analyses of the whole amplicon and unlabeled probe , the eight Pc variants can be differentiated from each other .Five different Pc variants, PcS, PcW, PcH1, PcH2TGN-10 and PcWTGN-10 , were identified and consistent with direct sequencing results .Conclusions This developed a simple Pc and P 2 variants differentiation method via simultaneous HRM analyses of an unlabeled probe and full-length amplicon .This method is cost-effective and accurate , could be used in differentiation of Pc and P2 variants of class 1 integrons in clinical isolates .

The clinical significance of nucleotide G1613A and C1653T mutations in the core promoter region of hepatitis B virus / 解放军医学杂志

Objective To evaluate the impact of hepatitis B virus (HBV) genome G1613A and C1653T mutations on disease progression, viral replication capacity, and transcription activity of HBV core promoter (CP). Methods A total of 258 patients were enrolled in the present study, including 65 patients with acute hepatitis B (AHB), 120 with chronic hepatitis B (CHB), and 73 with acute on chronic liver failure (ACLF). Serum HBV DNA was extracted from patients, and full-length HBV genome was amplified by PCR. The incidences of G1613A, C1653T and G1613A+C1653T in different groups were compared, and through functional experiments, the impact of mutants and wild-type virus on viral replication capacity and CP transcription activity was assessed. Results Genotype B, C and D were the three detected genotypes in 285 patients, with detection rates of 22.2%, 76.2% and 1.6%, respectively. The incidences of G1613A, C1653T and G1613A+C1653T mutations increased with the disease exacerbation, and they were 13.70%, 31.80% and 45.20% in AHB patients (P<0.01), 2.30%, 16.30% and 27.40% in CHB patients (P<0.01), and 2.29%, 12.07% and 23.29% in ACLF patients (P<0.05). Compare with wild-type strain, the G1613A mutant strain of HBV increased the viral replication capacity by 6%, reduced HBsAg level and core promoter activity by 15% and 16.2%, and reduced HBeAg to undetectable level; the C1653T mutant strain increased the viral replication capacity, HBsAg level, and core promoter activity by 10%, 55% and 17.1%, respectively, and the HBeAg level was comparable to that of wild-type strain; the G1613A+C1653T mutant strain increased viral replication capacity, HBsAg level and HBeAg level by 7%, 66% and 227%, respectively, while it had no influence on core promoter activity. Conclusion The G1613A and C1653T mutation in CP region may increase HBV replication capacity and alter CP activity and HBV antigens expression, the doublet mutation of G1613A+C1653T shows synergic effect on these changes, suggesting these mutations are associated with liver disease progression.

The association of DNMT3a, DNMT3b expression with the state of promoter methylation of ERαgene and ERαexpression in sporadic breast cancer / 天津医药

Objective To investigate the correlationship between DNMT3a, DNMT3b protein expressions and the state of promoter methylation of ERα gene and ERα protein expression in the development of sporadic breast cancer. Methods A total of 180 patients with sporadic breast cancer and 30 patients with breast fibroadenoma were included in this study. The expressions of DNMT3a and DNMT3b protein were detected by immunohistochemical method. The state of promoter methylation of ERα gene was detected by methylation specific PCR in 97 patients with sporadic breast cancer. Results There were no significant differences in positive expression rates of DNMT3a and DNMT3b protein between breast fibroadenoma and breast cancer. There were higher expression levels of DNMT3a and DNMT3b in breast cancer patient of Ⅲ～Ⅳstages than those of Ⅰ～Ⅱstages. The expression of DNMT3a was significantly higher in patients with lymph node metastasis than that of patients without lymph node metastasis (P＜0.05). Of 97 cases of breast cancer patients, ERα gene promoter methylation occurred in 39 cases (40.2%). The positive expression of DNMT3a protein was positively correlated with the ERα gene methylation (rS=0.250). The DNMT3a protein expression showed a significant influence to the overall survival (OS) in patients of breast cancer (P=0.035), no significant influence to the disease-free survival (DFS) (P=0.064). DNMT3b protein expression showed no significant influence to OS and DFS of patients with breast cancer (P=0.914 and 0.961). Conclusion The positive expressions of DNMT3a and DNMT3b are correlated with the invasion, metastasis and poor prognosis of sporadic breast cancer. DNMT3a was positively correlated with the state of ERα gene promoter methylation. The inhibition of DNMT3a and DNMT3b may have advantages in the prevention and treatment of sporadic breast cancer.

Association of genetic polymorphisms in the FGB promoter region with idiopathic deep venous thrombosis / 中华普通外科杂志

Objective To probe the association between possible single nucleotide polymorphism (SNP) in the FGB promoter region and idiopathic deep venous thrombosis.Methods A prospective analysis was performed in both IDVT group and control group (120 cases each) followed by a duplex examination using gene sequencing technique and restriction fragment length polymorphism (RFLP) in the promoter region of fibrinogen gene β.Possible SNPs in this region were detected arranged before HardyWeinberg equilibrium test and Linkage disequilibrium (LD) analyses.Ultimately,we compared the genotype frequencies between the two groups and undertook a multiple Logistic regression.Results Six kinds of SNPs were determined in the promoter region of β-fibrinogen gene:-148C/T,-249C/T,-455G/A,-854G/A,-993C/T and-1420G/A.A stronger linkage disequilibrium was confirmed between-993C/T and -455G/A (r2 =0.699) ;-993C/T and-148C/T (r2 =0.509) ;-455G/A and-148C/T (r2 =0.556).Statistical differences of genotype frequencies between two groups were observed in-148C/T,-249C/T,-455G/A and-1420G/A polymorphisms (all P ＜ 0.05).Conclusions The risk of IDVT was 4.579 times higher with every 1 g/L increase of fibrinogen concentration.Allele-148T,-455G and-1420A are IDVT risk factors.-993C/T may indirectly affect IDVT through linkage disequilibrium with-455G/A and-148C/T.

Building the Frequency Profile of the Core Promoter Element Patterns in the Three ChromHMM Promoter States at 200bp Intervals: A Statistical Perspective

Recently, the Encyclopedia of DNA Elements (ENCODE) Analysis Working Group converted data from ChIP-seq analyses from the Broad Histone track into 15 corresponding chromatic maps that label sequences with different kinds of histone modifications in promoter regions. Here, we publish a frequency profile of the three ChromHMM promoter states, at 200-bp intervals, with particular reference to the existence of sequence patterns of promoter elements, GC-richness, and transcription starting sites. Through detailed and diligent analysis of promoter regions, researchers will be able to uncover new and significant information about transcription initiation and gene function.

The targeting expression of IL-2 gene driven by alpha fetoprotein promoter and hepatitis B virus envelope in hepatic carcinoma cell / 肿瘤研究与临床

Objective To observe if IL-2 gene can express in hepatic carcinoma cells by double targeting of hepatitis B virus envelope (HBVE) and alpha fetoprotein (AFP) promoter.Methods HepG2 cells,L02 cells,and HepG2.2.15 cells were cultured in vitro.HBVE was obtained by PEG8000 concentration assay,and the acquired HBVE was used to pack recombintional gene.Human AFP promoter-IL-2 recombinational gene was obtained by PCR.Then HepG2 cells and L02 cells were transfected by transient transfection and the expression of IL-2 was detected by RT-PCR and Western blot.Results IL-2 was detected in HepG2 cells by RT-PCR and Western blot but not in L02 cells.Conclusion By using HBVE as a gene transporter,human AFP promoter-IL-2 recombinational gene can express in hepatic carcinoma cells,thereby it can increase the safety of exogenous gene transfection of hepatic carcinoma cells.

Mutation analysis of DJ-1 in patients with early-onset Parkinson's disease and relationship between the g.168_185del polymorphism and Parkinson's disease / 中华神经科杂志

Objective To evaluate the prevalence of the DJ-1 mutation in early-onset Parkinson's disease (EOPD) patients,and analyzed the association between the certain polymorphic marker g.168_185del in intron1 and Parkinson' s disease (PD).Methods We screened all 7 exons and exon-intron boundary regions of DJ-1 by PCR and direct nucleotide sequencing in 90 Chinese patients with EOPD.We also compared the allele and genotype frequencies of the g.168_185del polymorphism between EOPD patients and controls.Results We found no causative DJ-1 mutations in our cohort of Chinese EOPD patients.But we did identified 4 known polymorphic variants,including the g.168_185del in intron 1,g.5027G ＞ A (rs17523802),g.5065T ＞ C (rs226249),and g.5094C ＞ T (rs11121064) within exon 1.Del/Ins frequencies of the g.168_185 del polymorphism were 11.1％ (10/90)and 13.3％ (14/105) in EOPD group and normal group,respectively.Ins/Ins frequencies were 88.9％ (80/90) and 86.7％ (91/105),thex2 and P value of genotype frequency were 0.222 and 0.669 between EOPD patients and controls,respectively.The insert frequencies were 94.4％ (170/180)and 93.3％ (196/210) in EOPD patients and controls,the deletion frequencies were 5.6％ (10/180) and 6.7％ (14/210),thex2 and P value of allele frequency were 0.207 and 0.679 between EOPD patients and normal,respectively.Furthermore,the P value of genotype and allele frequencies were 0.736 and 0.744 between familial EOPD patients and controls,respectively;P values of genotype and allele frequencies were 0.847 and 0.852 between sporadic EOPD patients and control group,respectively.There was no statistical difference between groups.Conclusion Mutations in DJ-1 are uncommon in Chinese EOPD patients,and no association is observed between the DJ-1 intron 1 g.168_185del polymorphism and risk of PD.

Study on the correlation between methylation of APC genes and expression in pancreatic cancer / 中华检验医学杂志

Objective To investigate the effect of methylation of the APC gene on expression and the correlation with clinical data in pancreatic cancer.Methods Sixty postoperative tissue samples with pancreatic cancer were collected in the First Affiliated Hospital of Zhengzhou University from August 2010 to January 2011,20 benign pancreatic disease tissues were collected as control groups.APC promoter methylation and gene expression levels were detected by Methylation Specific PCR (MSP),Real Time PCR (RT-PCR) and Western blot in 60 pancreatic carcinoma,42 metastasis and 20 benign pancreatic disease tissues,then analyze the relation between methylation of the APC gene and the clinical data.Results APC promoter methlation was observed 48.53％,46.67％ and 1.16％ in pancreatic carcinoma,metastasis and benign pancreatic disease tissue,respectively.Methylation of APC in pancreatic carcinoma and metastasis increased significantly compared with control tissues (x2 =12.903,14.402; P ＜ 0.05).There were no statistically significant differences of APC expression in these tissues (P ＞ 0.05).There was a significant correlation between methylation of APC and clinicopathological stage (x2 =6.801,P ＜ 0.05),but no correlation with gender,age,tumor size,histological grade and metastasis (x2 =0.727,1.311,0.372,0.148,0.017 ; P ＞ 0.05).Conclusion The methylation of APC gene is closely related with pancreatic carcinoma inogenesis and the clinicopathological stage,but do not effect the expression of APC in tissues.

Correlation between polymorphism of rs5029924 in A20 promoter region and posttraumatic susceptibility to sepsis / 中华创伤杂志

Objective To investigate relationship of single nucleotide polymorphism (SNP)at rs5029924 locus in A20 promoter region and posttraumatic sepsis.Methods PCR-DNA sequencing was used to analyze different gene distribution at rs5029924 locus of 103 trauma patients with sepsis (Group A),120 trauma patients without sepsis (Group B) and 135 healthy peoples (control group).Relation of different genotypes at rs5029924 locus to sepsis susceptibility was analyzed.Peripheral blood cells of healthy peoples of different genotypes were stimulated using lipopolysaccharides (LPS) in vitro.Expression of A20 mRNA was measured by fluorescent quantitative PCR,expression of A20 protein by flow cytometry,and levels of TNF-α and IL-1β by ELISA method.Results Frequency of rs5029924 genotypes CC,CT and TT was respective 77.8％,20.0％ and 2.2％ in control group； 63.1％,34.0％ and 2.9％ in Group A； 83.3％,15.0％ and 1.7％ in Group B.Significantly lower frequency of CC genotype was observed in Group A when compared to Group B and control group (P ＜0.05),but no statistical differences were recorded between Group B and control group (P ＞ 0.05).CT/TT genotype increased risk coefficient of sepsis to 2.397-fold higher level when compared to CC genotype.Allele T increased prevalence of sepsis significantly as well (OR =2.056) when compared to allele C.After LPS treatment in vitro,CC genotype individuals revealed significantly higher levels of A20 mRNA and protein in peripheral blood leukocytes,but significantly lower levels of TNF-α and IL-1 β when compared to CT/TT genotype individuals.Conclusion Polymorphism of rs5029924 locus is associated with sepsis susceptibility and the reason may be that mutant genes affect promoter activity and down-regulate A20 expression,which fails to suppress inflammation.

Characteristics of polymorphism in promoter region of insulin-like growth factor-1 gene in 561 neonates / 中华围产医学杂志

Objective To investigate polymorphism in the promoter region of insulin-like growth factor-1(IGF-1) gene.Methods Five hundred and sixty-one neonates admitted to Peking University Third Hospital from June 1st,2010 to June 30th,2012 were recruited into the study.Gender,gestational age,birth weight and birth length were collected.Heel blood samples were collected on 3-5 days after birth.DNA was extracted to analyze the polymorphism in the promoter region of IGF-1 gene.Chi-square test,independent-sample t-test,analysis of variance and HardyWeinberg equilibrium were performed.Results Among the 561 neonates,413 were full term,and 148 were preterm; 92 were large for gestational age (LGA),433 were appropriate for gestional age (AGA),and 36 were small for gestional age (SGA).Seven different alleles and 23 genotypes in the promoter region of IGF-1 gene were identified in the population.The seven alleles were 188,190,192,194,196,198 and 200 bp respectively.The three most common genotypes were 190-192 bp,192-196 bp and 192-192 bp,whose frequencies were 23.2％ (130/561),15.0％ (84/561) and 12.8％(72/561).There were no significant differences of cytosine-adenosine (CA)19/CA19,CA19/CAno19and CAno19/CAno19 genotypes between full term and preterm infants [11.4％ (47/413) vs 16.9％(25/148),55.9％ (231/413) vs 50.7％ (75/148) and 32.7％ (135/413) vs 32.4％ (48/148)respectively,x2=2.96,1.21 and 0.00,all P＞0.05].There was no difference in the gestional age among infants with CA19/CA19,CA19/CAno19 and CAno19/CAno19 genotypes [(37.1±2.9),(37.6±3.1) and (37.4±3.1) weeks respectively,F=0.54,P=0.58].The frequency of CA19/CA19 in SGA neonates was higher than that in LGA and AGA neonates [25.0％ (9/36) vs 7.6％(7/92) and 12.9％ (56/433),x2 =7.01,P=0.03],but there were no differences in the frequency of CA19/CAno19 and CAno19/CAno19 among LGA,AGA and SGA neonates (CA19/CAno19:x2 =1.13,P=0.57; CAno19/CAno19:x2 =0.58,P=0.75).Conclusions Polymorphism exists in the promoter region of IGF-1 gene.The gestational age is not associated with the frequency of CA19 allele.

Construction of human surfactant protein B promoter luciferase reporter gene vectors and detection of their activities / 中国医师杂志

Objective To construct human surfactant protein B (SP-B) gene promoter luciferase reporter plasmids and detect their transcriptional activities in H441 cells.Methods (1)The fragment of SP-B promoter (-218/+ 435 bp) was acquired from human genome DNA by polymerase chain reaction (PCR) amplification and then was inserted into pGM-T vector by the T4 DNA ligase.The vector was transfected into TOP10 E.coli.The positive clone was identified by DNA sequencing.The identified target SP-B promoter sequence was cloned into pGL3-basic vector to construct the recombinant vector pGL3-basic-SP-B-promoter and was identified by enzyme digestion and sequencing; (2)The pGL3-basic-SP-B-promoter vector was converted into pGL4.17-SP-B-promoter vector through enzyme digestion.The identified recombinant vectors and control plasmid pRL-TK were transfected into H441 cells by lipofectamine 2000,and luciferase assays was performed using the dual-luciferase reporter assay system.Results The sequences of SP-B promoter in the recombinant luciferase reporter plasmids were consistent with the one published on Genebank.The firefly/renilla luciferase activity ratio of pGL3-basic/pGL4.17-SP-B-promoter vector (2.8 ± 1.1,66.5±3.8) was significantly higher than pGL3-Basic,pGL4.17 control vector (0.2 ±0.1,4.3 ±0.4) with statistical significance (t =4.182,27.419,P =0.000),respectively.The SP-B promoter activity of pGL4.17-SP-B-promoter vector was significantly higher than pGL3-basic-SP-B-promoter vector (t =27.712,P =0.000).Conclusions The pGL3-basic/pGL4.17-SP-B-promoter vectors are successfully constructed with SP-B promoter activity in H441 cells and pGL4.17-SP-B-promoter vector is the better choice for further study with higher luciferase activity.

Up-regulation effect of hepatitis B virus genome A1846T mutation on viral replication and core promoter activity / 解放军医学杂志

Objective:To evaluate the influence of hepatitis B virus (HBV) genome nucleotide A1846T mutation on the viral replication capacity and the transcription activity of HBV core promoter (CP) in vitro. Method A total of 385 patients with hepatitis B admitted to the 302 Hospital of PLA were enrolled in the study, including 116 with moderate chronic hepatitis B (CHB-M), 123 with severe chronic hepatitis B (CHB-S), and 146 with acute-on-chronic liver failure (ACLF). Serum HBV DNA was isolated and full-length HBV genome was amplified. The incidence of A1846T was analyzed. Full-length HBV genomes containing 1846T mutation were cloned into pGEM-T easy vector, and the counterpart wild-type 1846A plasmids were obtained by sitedirected mutagenesis. The full-length HBV genome was released from recombinant plasmid by BspQ /Sca digestion, and then transfected into HepG2 cells. Secreted HBsAg level and intracellular HBV core particles were measured 72 hours post-transfection to analyze the replication capacity (a 1.0-fold HBV genome model). 1846 mutant and wild-type full-length HBV genomes were extracted to amplify the fragment of HBV CP region, and the dual luciferase reporter of the pGL3-CP was constructed. The luciferase activity was detected 48 hours post-transfection. Result The incidence of A1846T mutation gradually increased with the severity of hepatitis B, reaching 31.03%, 42.27%, and 55.48% in CHB-M, CHB-S and ACLF patients respectively (P0.01). The replication capacity of 1846T mutants, level of secreted HBsAg, and transcriptional activity of CP promoter were increased by 320%, 28% and 85% respectively, compared with 1846A wild-type strains. While the more common double mutation A1762T/ G1764A in CP region was increased by 67%, 9% and 72% respectively, compared with its counterpart wild-type strains. A1846T had a greater influence on viral replication capacity in vitro. Conclusions A1846T mutation could significantly increase the replication capacity of hepatitis B virus, secretion of HBsAg and transcription activity of CP promoter, and cis-activate the downstream gene transcription. The finding indicates that HBV genome A1846T mutation might play a role in liver disease progression.

IL-10 gene promoter polymorphisms and leprosy in a Colombian population sample / Polimorfismos en el gen promotor de IL-10 en una muestra de pacientes colombianos con lepra

Introduction. Polymorphisms in promoters of genes code for cytokines that affect transcription levels. Several have been associated with leprosy patients that have functional and clinical implications. Objective. Polymorphisms in the promoter of the IL10 gene of leprosy patients will be compared frequencies in normal population. Materials and methods. SNPs (single nucleotide polymorphism) -1082 A/G (rs1800896), -819C/T (rs1800871), and -592A/C (rs1800872) were identified in 100 leprosy patients and in a control group of 100 volunteers from a leprosy endemic region of Colombia. Results. The genotypes C/C and C/T in the SNP -819 were associated together with leprosy (OR=4.34, p<0.001).Similarly, the genotypes C/C and C/A in the -592 SNP showed an association (OR=4.3, p<0.001). The haplotypes -819C-519C and -1082A-819C-592C showed significant association (OR=4.34, p<0.001 and OR=6.25, p<0.001) respectively. These haplotypes in homozygosis conditions were also associated with leprosy: -819C-519C/-819C-519C (OR=4.34, p<0.001), -1082A -819C-592C/-1082A -819C-592C (OR=1.90, p=0.04). The SNP -1082 was not associated with leprosy in this population. Conclusions. The haplotypes associated with leprosy, -1082A-819C-592C/-1082A-819C-592C, have been reported as low producers of IL-10. Functionally, the low production of IL-10 may have immune response consequences and clinical implications. Additional haplotypes of IL-10 have been reported as markers for leprosy susceptibility or resistance in other ethnic populations. This suggests that differences in distribution of diverse IL-10 gene polymorphisms among ethnic groups may indicate important gene-disease associations.

Introducción. Se han reportado polimorfismos en los genes promotores que codifican para citocinas y que afectan los niveles de transcripción, con implicaciones clínicas y funcionales en pacientes con lepra. Objetivo. Detectar los polimorfismos en el gen promotor de la interleucina 10 (IL-10), de los polimorfismos de un solo nucleótido (Single Nucleotide Polymorphisms, SNP) -1082 A/G (rs1800896), -819C/T (rs1800871) y -592A/C (rs1800872), en 100 pacientes con lepra y un grupo control de 100 voluntarios, de una región endémica de Colombia. Resultados. Los haplotipos -819C-519C y -1082A-819C-592C mostraron asociación significativa con lepra: OR=4,34, p=1 x 10-3, y OR=6,25, p=5 x 10-4, respectivamente. Estos haplotipos en condiciones de homocigoto, están también asociados con lepra: -819C-519C/-819C-519C (OR=4,34 p=1 x 10-3), -1082A -819C-592C/-1082A -819C-592C (OR=1,90 y p=0,04). El SNP -1082 no se encontró asociado con lepra en esta población. Los genotipos C/C y C/T en el SNP -819, se encontraron asociados a lepra (OR=4,34, p=1 x 10-3); de igual manera, los genotipos C/C y C/A en el SNP -592 mostraron asociación (OR=4,34, p=1 x 10-3). Conclusiones. El haplotipo que encontramos asociado con lepra, -1082A-819C-592C/-1082A-819C-592C, se ha relacionado con baja producción de IL-10. Funcionalmente, esta baja producción de IL-10 puede tener consecuencias en la respuesta inmunitaria, además de implicaciones clínicas. Se han reportado diferentes haplotipos de IL-10 como marcadores de vulnerabilidad y resistencia de lepra en otras poblaciones, lo cual sugiere que las diferencias en la distribución de diversos polimorfismos del gen de IL-10 entre grupos étnicos, es un factor importante al determinar la asociación entre enfermedad y genes.

Analysis of the status of DACH1 gene promoter methylation in endometrial carcinoma and its clinical significance / 中华妇产科杂志

ObjectiveTo analyze the status of DACH1 gene promoter methylation and explore its association with the expression of DACH1 gene promoter methylation and clinical significance of endometrium carcinoma(EC).Methods From February 2004 to August 2008,a total of 80 EC tissue samples with comprehensive surgical pathology staging were collected and used for this study.Twenty normal endometrium tissues in 2008 were abtained from the fractional curettage because of dysfunctional uterine bleeding as control.All samples were confirmed pathologically.Methylation specific PCR (MSP) was performed to detect the promoter methylation of DACH1 gene,and analyze its influence on the expression of DACH1 and the relationship between DACH1 promoter methylation and clinicopathological factors in EC.DACH1 protein expression was detected by western blot.Chi-square test and Pearson test were used for statistical analysis.ResultsThe rate of promoter methylation of DACH1 gene in the EC tissues was significantly higher than that in the normal endometrium issues (30％ vs.5％,P ＜ 0.05).There was an association between the expression of DACH1 and DACH1 gene promoter methylation ( r =- 0.30,P ＜ 0.01 ).There was statistical difference between the methylation of DACH1 and the pathological grade ( P ＜ 0.05 ) or histological type ( P ＜0.05).But DACH1 gene methylation was not related with the age,stage,myometrial invasion depth and lymphnode metastasis (P ＞ 0.05 ).Conclusions DACH1gene promoter methylaion could lead to a decrease or absence in the DACH1 expression in EC.The promoter methylation of DACH1 gene may induce the inhibition of DACH1 expression,which might be one of the mechanisms of DACH1 gene inactivation in human EC.

Effect of follicle stimulating hormone on the secretion of anti-mullerian hormone in the granulosa cells in patients with polycystic ovarian syndrome / 中华妇产科杂志

Objective To study the effect of follicle stimulating hormone(FSH)on the secretion of anti-mullerian hormone(AMH)in granulosa cells in patients with polycystic ovarian syndrome(PCOS).Methods From Aug.2008 to Dec.2009,33 patients with PCOS in Reproductive Medicine Research Center,Sixth Affiliated Hospital of Sun Yat-sen University were collected from 8-10 mm antral follicles and classified into the following three groups: FSH-unstimulated granulosa cells(unstimulated group,n =12),exogenous FSH-stimulated granulosa cells(exo-stimulated group,n =12)and internal FSH-stimulated granulosa cells(int-stimulated group,n =21).ELISA and real-time PCR were relatively used to measure protein and mRNA level of AMH.Luciferase reporting vector was constructed to detect the promoter activity of AMH.Results The levels of AMH secretion in PCOS granulosa cells were(11.4 ± 4.0)μg/L in unstimulated group,(7.9 ± 1.1)μg/L in exo-stimulated group and(5.6 ± 1.7)μg/L in int-stimulated group.Both the external and internal stimulation of FSH may suppress AMH secretion significantly(P ＜0.05).The mRNA level of AMH in PCOS granulosa cells were 2.5 ± 1.2 in unstimulated group,which were higher than 1.5 ± 0.5 in exo-stimulated group and 1.1 ± 0.7 in int-stimulated group significantly(P ＜0.05).The luciferase activity of AMH in PCOS granulosa cells were 11.5 ± 2.3 in unstimulated group,8.7 ± 2.4 in exo-stimulated group and 6.8 ± 2.4 in int-stimulated group.The luciferase activity of AMH in unstimulated group was significantly higher than those in exo-stimulated and int-stimulated groups(P ＜0.05).Conclusion FSH may inhibit the excessive secretion of AMH and stimulate follicle growth in PCOS granulosa cells by suppressing activity and expression of promoter.

Association between promoter polymorphisms of a disintegrin and metalloproteinase 10 gene and sporadic Alzheimer's disease / 中华神经科杂志

ObjectiveTo investigate the relationship between the polymorphisms of the promoter of a disintegrin and metalloproteinase 10(ADAM10) gene and sporadic Alzheimer's disease (SAD).Methods The promoter of ADAM10 gene in 10 controls and 10 SAD patients was sequenced.Three variations were found,then these variations in 298 SAD patients (SAD group) and 315 healthy controls (control group)were genotyped by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).ResultsThree polymorphisms were found in the promoter of ADAM10 gene: -279G/A (rs653765),- 630G/T( rs514049 ) and - 921GAGA/- ( rs33926666 ).For - 921GAGA/-,there were significant differences in genotype ( GAGA/GAGA:138 (46.3％ ),GAGA/-:155(52.0％),-/-:5(1.7％))and allele frequencies (GAGA:431 (73.6％),-:165 (27.7％) ) between SAD and control (genotype:x2 =34.130,P =0.000; allele:x2 =25.972,P =0.000). For - 279G/A,there were significant differences in genotype and allele frequencies between SAD and control in the subjects without ApoEε4 allele (genotype:x2 =8.734,P=0.013; allele:x2 =5.129,P=0.024). -279G and -921GAGA were relatively protective allele types for SAD,and they were not in linkage disequilibrium.ConclusionThe polymorphisms - 279G/A and - 921GAGA/- of ADAM10 are associated with SAD.Allele G or genotype G/G of -279G/A and the GAGA/GAGA genotype or the GAGA allele of -921GAGA/- might have a protective effect on SAD.

Preparation and primary application of antiserum to aminoacy1-3″-adenylyltransferase in integron / 中华检验医学杂志

ObjectiveTo prepare antiserum specific to aminoacy1-3″-adenylyltransferase [ AAD (3″) ],and to explore the application value of the prepared antiserum in detecting the expression levels of aadA2 gene that downstream of 8 different promoters (PcS,PcH2,PcH1,PcW,PcS-P2,PcH2-P2,PcH1P2 and PcW-P2 ) of variable regions in class 1 integron.MethodsaadA2 gene was amplified by polymerase chain reaction(PCR) and cloned into the expression plasmid pET19b.After inducing,the recombined aminoacy1-3″-adenylyltransferase[ AAD(3″)] with His-tag was expressed,purified and immunized rabbits to get anti- AAD(3″) specific serum.The prepared antiserum was used to detect the translation levels of aadA2 gene that downstream of different promoters of variable regions in class 1 integron by Western blotting (WB).Broth microdilution method was used to detect the minimal inhibitory concentrations (MIC) to streptomycin in Escherichia coli JM109 with aadA2 gene downstream of different promoters of variable regions.ResultsRecombined AAD (3″) expression plasmid pET19b-aadA2 was constructed successfully and was verified by sequence analysis.After transformed into E.coli BL21 ( DE3 ),a resoluble recombined AAD(3″) high expression strain was obtained.After fermentation,recombined AAD(3″) was purified and immunized rabbits.The anti- AAD(3″) specific serum was obtained with titer ＞ 1∶100 000.WB was used to detect the expression levels of AAD (3″),the translation product of aadA2 gene,that downstream of 8 different promoters of variable regions.The relative expression level of AAD (3″) that downstream of PcW was assumed to be 1,then the relative expression levels of AAD(3″),which all were detected 3 times independently,that downstream of PcS,PcH2,PcH1,PcS-P2,PcH2-P2,PcH1-P2 and PcW-P2 were 12.9±2.3,9.1±1.0,2.0±0.4,16.0±1.3,14.1 ±1.3,10.5±0.7 and 8.9 ±1.7 respective.Very different expression levels of AAD (3″) that downstream of different promoters of variable regions were obtained( F =32.421,P ＜ 0.01 ).The mean values of MIC,which all were detected 3 times independently,to streptomycin in E.coli JM109 with aadA2 gene downstream of PcS,PcH2,PcH1,PcW,PcS-P2,PcH2P2,PcH1-P2 and PcW-P2 were 256,256,64,128,32,128,4 and 64 mg/L respective.These results indicated the different expression levels of aadA2 gene that downstream of different promoters of variable regions can confer their host bacteria different resistance levels to streptomycin.Conclusions Resoluble recombined AAD(3″) is purified successfully and high titer anti- AAD(3″) specific antiserum is obtained from the immunized rabbits.This laid foundation for further investigation on the correlationship between the expressions of intI1 gene and the gene cassettes within variable regions.The expression levels of antibiotic gene cassettes that downstream of different promoters of variable regions are very different,so are the very different antibiotic resistance levels of the host bacteria.Therefore more attentions should be paid to the researches on the classification of promoters of variable regions when molecular epidemiology studies on the class 1 integrons in clinical isolates were conducted.

The effect of hepatitis B virus on the expression of kinesin family member 4 / 中华传染病杂志

Objective To investigate the differences of kinesin family member 4 (KIF4A) expression between hepatic carcinoma and adjacent tissue in patients with chronic hepatitis B virus (HBV) infection,and to understand the effect of HBV on the expression of KIF4A.Methods Reverse transcriptase-polymerase chain reaction and Western blot were used to measure the expression of KIF4A in hepatic carcinoma and adjacent tissues. HepG2 cells were co-transfected with KIF4A promoter containing the luciferase gene and HBV infectious clone pHBV1.3,and luciferase activity was measured.Expression of KIF4A in HepG2 cells was measured after tranfected with different doses of pHBV1.3.The student's t-test was used for statistic analysis.Results Expression of KIF4A was much higher in hepatic carcinoma than that in adjacent tissues.HBV enhanced KIF4 A gene promoter activity and the luciferase activities were increased as the concentration of pHBV1.3 increased ( 0,0.2,0.4,0.6 and0.8 μg/mL),which were (126.8± 13.4),(219.8±16.7),(387.6±21.5),(586.5 ± 228.9 ) and (657.6 ± 35.5 ) RUL/μg protein,respectively,while the luciferase activities were (123.6± 13.8),(131.8± 14.6),(129.7-13.5),(135.3± 13.4) and (127.1± 12.7) RUL/μg protein,respectively with different doses of control plasmids transfected,and statistical analysis showed significant difference between them (t=4.875,P=0.006).And HBV upregulated KIF4A mRNA and protein expressions in HepG2 cells in a concentration-dependent manner.Conclusion Expression of KIF4A is enhanced in hepatic carcinoma and HBV can upregulate KIF4A expression.

Association of CA repeats polymorphism in the promoter region of IGF-1 gene with metabolic syndrome / 中国医师杂志

Objective To explore the relationship between CA repeats polymorphism in the promoter region of IGF-1 gene and MS in the Han nationality.Methods 1047 subjects were recruited from general population of Dongcheng District in Beijing.MS was diagnosed based on the criteria for MS in 2005 by IDF.Genomic DNA was extracted by standard methods.PCR,Genescan,Genotyper and direct sequencing were conducted to screen CA repeats polymorphism in the promoter region of the human IGF-1 gene.Levels of plasma glucose,lipids,serum insulin and IGF-1 were determined.BMI and ISI were calculated.Results The prevalence of MS in (CA) 19 homozygote was lower than that in (CA) 19 heterozygote (9.1% vs 18.3%,x2 = 8.55,P ＜ 0.01) and without (CA) 19 (9.1% vs 24.0%,x2 = 18.05,P ＜ 0.01).The level of serum IGF-1 had differences among the three groups [ (114.0 ± 52.6) μg/L vs (136.6 ± 80.5) μg/L vs (129.2±49.1) μg/L,F =3.16,P ＜0.05],(CA)19 homozygote had lower serum IGF-1 than (CA)19heterozygote and without (CA) 19.BMI,WC,TG,FIns,2hIns and ISI had difference among the three groups (P ＜0.05).Conclusions (CA)19 repeats polymorphism in the promoter region of IGF-1 gene was significantly associated with MS in Han nationality.

Inactivation of DAPK1 gene by methylated oligonucleotides and its effect on the proliferation of leukemia cell line K562 / 白血病·淋巴瘤

Objective To inactivate Death-associated protein kinase 1 gene (DAPK1) by transfecting complementary methylated oligonucleotides and studies its effect on the proliferation of myelogenous leukemia cell line K562. Methods Methylated oligonucleotides complementary to DAPK1 gene promoter were transfected into K562 cells by Iipo2000. Methylation specific PCR (MSP) and Reverse transcription PCR (RT-PCR) were applied to detect DAPK1 gene promoter methylation status and its mRNA expressions respectively. MTT was used to detect the proliferation of K562 cells pre- and post- oligonucleotides transfection. Results DAPK1 gene promoter in non-treated and control groups were unmethylated with detectable mRNA expressions, but it became methylated with inhibited mRNA expressions after methylated oligonucleotide transfection. Proliferation in methylated oligonucleotide treatment group was significantly higher than that in non-treated and control groups. Conclusion Complementary methylated oligonucleotides could inactivate DAPK1 gene and inhibit its expression in K562 cells, which could promote its proliferation.