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Polycystic ovarian syndrome (PCOS) is an endocrine and metabolic dysfunction, highly prevalent in women in their reproductive years. Hyperandrogenism, oligo-ovulation, polycystic ovarian morphology are the main features of this syndrome. PCOS is a genetic disorder with a multifactorial etiology and has a strong link with environmental components. It is frequently associated with obesity and insulin resistance. Recently, epigenetic mechanisms have been involved in the pathogenesis of PCOS. Several studies showed that methylation in DNA and miRNAs is altered in women with PCOS in blood, serum, adipose tissue, granulose cells and theca. This evidence indicates that women with PCOS have a different epigenetic regulation, which might be triggered by an adverse intrauterine environment or by postnatal environmental elements such as diet and or obesity.
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Humans , Female , Polycystic Ovary Syndrome/genetics , Gene Expression Regulation, Neoplastic/genetics , DNA Methylation/genetics , MicroRNAs/genetics , Epigenesis, Genetic/geneticsABSTRACT
Objective To observe the expressions of efflux pump gene cluster adeAB in acinetobacter baumannii isolated from the burn patients and the expression changes of its upstream regulatory genes adeR and adeS and determine the influence of those genes on drug resistance of acinetobacter baumannii.Methods Nine drug-resistant strains and nine sensitive strains of acinetobacter baumannii isolated from the burn patients treated between June 2012 and March 2013 were used.After strain identification using 16SrDNA sequencing,acinetobacter baumannii standard strain ATCC19606 was employed as the control.mRNA expressions of efllux pump genes adeA and adeB and their upstream regulatory genes adeR and adeS were detected by real-time quantitative PCR.Results (1) adeA and adeB genes presented higher expressions in drug-resistant strains (3.71 ±0.95,76.16 ± 8.75) than in sensitive strains (0.92 ± 0.94,0.72 ± 0.78) (F =38.71,663.65 respectively,both P ＜ 0.05).(2) adeS and adeR genes showed higher expressions in drug resistant strains (18.02 ± 6.71,3.02 ± 2.69) than in sensitive strains (1.64±1.51,0.76±0.61) (F =51.04,5.57 respectively,bothP＜0.05).Conclusions The over-expression of efflux pump gene cluster adeAB induced by the expression alteration of its upstream regulatory genes adeR and adeS is closely associated with the drug resistance of acinetobacter baumannii in the burn patient.Besides,the regulatory genes may depend on adeS to sense the nosocomial environment condition,activate or inactivate adeR and hence regulate efflux pump expression.
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Objective To explore the role of H19 imprinting in etiology of pre-eclampsia. Methods Placentas of 24 women with pre-eclampsia (3 with mild pre-eclampsia and 21 with severe pre-eclampsia) and 50 healthy pregnant women at full term (control) were collected during selected cesarean delivery between August 2007 and March 2008. The statuses of H19 imprinting with placental tissues from normal pregnancy and patients with pre-eclampsia were identified upon polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The systolic and diastolic pressure were analyzed in H19 heterozygotic women. Results (1) There were 20 (40%) heterozygotes in 50 cases placenta tissues of the third trimesters, 11 (45%) heterozygotes in 24 cases placenta tissues of pre-eclampsia, There were no significant difference between two groups ( P > 0.05 ). (2) All 20 heterozygotes in placenta tissues of the third trimesters are exclusively monoallelically expressed, while 5 cases (45%) in 11 heterozygotes of pre-eclampsia are biallelically expressed (loss of imprinting, LOI). There were significant difference between two groups (P < 0. 01 ). (3) The values of systolic and diastolic pressure of patients with monoallelic expression of H19 were (171 ±9) mm Hg (1 nun Hg =0.133 kPa) and ( 104±8) mm Hg, the values of systolic and diastolic pressure with biallelic expression were ( 194±21 ) mm Hg and ( 124±18) mm Hg. There were significant difference between two groups (P<0.05 ). Conclusion LOI of H19 can be identified in pre-eclamptic placentas and is associated with maternal blood pressures, which implies the involvement of H19 gene LOI in the pathogenesis of pre-eclampsia and its potential relationship with the severity of the disease.
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To explore the relationship between HPRE mutations and noncytolytic anti-HBV infection, the objective eukaryotic expression vectors were constructed by molecular cloning and PCR-based site-directed mutagensis in vitro, and identification was performed using PCR and sequencing analysis. The results showed that eukaryotic expression vectors containing HPRE segment and mutating point were constructed successfully as confirmed by sequencing analysis. The activity of CAT gene obviously increased in the T to C mutation at nt 1504 of HPRE and no alteration in the C to T(G) at nt 1508. The mutation at nt 1508 of HPRE may escape the suppression role of IFN-?on HPRE. These results suggested that the mutation of HPRE might be affected the function of HPRE and influence the regulative function of IFN-? on HPRE, but not of 1FN-? nor of TNF-?.
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Objective To investigate the efficiency of antitumor immune responses induced by a controlled live dendritic cell(DC)vaccine Methods DC precursors were isolated from Fischer 344 rat bone marrow and cultured with granulocyte macrophage colony stimulating factor and interleukin 4 The rat ovarian tumor cell line NuTu 19 was genetically modified by retroviral mediated suicide gene(HSV 1 TK), and the positive clones were selected using G418 Live DC vaccine was then fused with DC and NuTu 19/TK cell by polyethylene glycol The characteristics of live DC vaccine were assayed with flow cytometry and confocal laser scanning microscopy The specific expression of HSV 1 TK gene in live DC vaccine was evaluated by RT PCR and western blot The sensitivity of live DC vaccine to ganciclovir (GCV) was evaluated by methylthiazoletetrazolium assay In vivo, rats vaccinated twice with live DC vaccine were compared to those vaccinated with killed DC vaccine, unfused DC and NuTu 19/TK cell or phosphate buffered saline Seven days following the last immunization, the rats were sacrificed to test the specific cytotoxic T lymphocyte (CTL) activity by lactate dehydrogenase release assay, or challenged with NuTu 19 and tumor incidence was observed Results The fusion efficiency was approximately (23?14) Live DC vaccine displayed an up regulated expression of major histocompatibility complex (MHC) IIOX6 (87 6?3 4)%, costimulatory molecule B 1 2 (71 1?9 3)%, integrin OX 62 (68 0?7 4)%, and adhesion ICAM 1 (77 1?2 0)%, and specifically expressed HSV 1 TK gene. Our data showed that spleen T lymphocytes from rats vaccinated with live vaccine displayed enhanced CTL aetivity (61 8?8 3)% contrast to that of rats vaccinated with killed vaccines (26 0?3 8)% ( P
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Objective To clone the transcriptional regulatory sequences (TRS) in human breast cancer related DF3 antigen, and to test the relationship between the activity of the TRS and the cell surface DF3 antigen. Methods Authors designed a pair of primers according to the registered 5′-flanking region of DF3 antigen. The 771 base pairs of DNA fragment were amplified from the genomic DNA of human MCF-7 breast carcinoma cells by PCR, and cloned to the pMD18-T vector. The results were tested by restrictive enzyme analysis and DNA sequencing. The DF3 TRS was cut by double enzyme: Mlu I、Hind III,and cloned to the pGL3 vector . The activity of the DF3 TRS was expressed by analyzing the relative luciferase activities. Results Restrictive endonuclease identification and DNA sequencing proved that the sequence authors got, was correct. The luciferase activity in MDA-MB-231 was hardly detected, whereas in MCF-7 the luciferase activity was about 200 times than in MDA-MB-231. Conclusions The DF3 TRS was cloned successfully. The DF3 activity has a distinct relationship with DF3 antigen. The study shows that DF3 TRS can be used in the gene therapy of breast carcinoma.
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AIM: To investigate effect of endotoxin on adrenomedulin(ADM) gene transcription in vascular endothelial cells. The role of p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway was also observed.METHODS: The effect of endotoxin in the presence or absence of p38 MAPK specific inhibitor SB203580 on the transcription of ADM in vascular endothelial cells ECV304 was evaluated by reverse transcription nest DNA polymerase chain reaction. RESULTS: Treatments of endotoxin (0.1, 1.0, 10, 50, 100 ?g/L) for 24 h increasea the ratios of ECV 304 ADM mRNA/? actin mRNA. The induction of ADM mRNA by endotoxin could be inhibited partially by 10 mmol/L SB203580.CONCLUSION: Endotoxin stimulated the transcription of ADM gene in vascular endothelial cells. The activation of p38 MAPK might be an important mechanism of ADM gene induced by endotoxin.
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Objective This study aims to investigate the role of Nef or Vpu of HIV 1 in the process of CD4 down regulation. Methods After transfection/infection, the cells that constitutively express Nef or Vpu were then properly prepared for indirect pre or post embedding immunocytochemistry and for further semiquantitative analyses. Results The number of CD4 molecules on the cell surface and in the cytoplasm of Vpu + cells was less than those in Vpu - cells. The number of CD4 molecules on the cell surface of Nef + Jurkat and HPBALL cells was less than that on the Nef - cell membranes. While CD4 molecules in the cytoplasm of Nef - Jurkat and HPBALL cells were less than those in the cytoplasm of Nef + cells. That Vpu partially co localized with Gag was analyzed by confocal microscopy; however, no CD4 Vpu complex was found in the cytoplasm. Furthermore, neither Nef nor Vpu shows effect on the incorporation of Gag into viral particles. Conclusions The results showed that CD4 Nef complexes formed at the coated pits of cell surfaces, with or without expressing Vpu. Formation of CD4 Nef complexes could be important for the enhancement of CD4 down regulation.
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Objective To investigate the gene expression of Chk1 gene in cerebrum after brain ischemia-reperfusion, trying to provide evidence to elucidate the molecular mechanism of brain injury.Methods Eighty-five male Wistar rats were divided into 3 groups.Group Ⅰ served as normal control.In group Ⅱ (non-ischemia group) animals underwent the whole experimental procedures except the occlusion of the bilateral vertebro-arteries and common carotid arteries.In group Ⅲ (ischemia-reperfusion group) animals were further divided into 3 subgroups according to the duration of ischemia: 10min, 30min and 60min.Each subgroup was again further divided based on the duration of reperfusion: 30min,2h and 6h.The cerebrum was immediately removed from rats after complete brain ischemia-reperfusion. The RNA was isolated and the reverse-transcription-polymerase chain reaction (RT-PCR) was carried out .The cDNA was analyzed by automatic system and the parameters were assessed to define the status of Chk1 mRNA expression in different ischemia and reperfusion groups.Results The quantity of Chk1 mRNA expression in the cerebral cortex of normal adult rat was about half of the glyceraldehyde-3-phosphate-dehydrogenase. When reperfused for 30min, 2h or 6h following 10min, 30 min cerebral ischemia and reperfused for 30min or 2h following 60 min cerebral ischemia, the expression of Chk1 mRNA was not significantly different from that in non-ischemia group.Only reperfused for 6h following 60 min cerebral ischemia, Chk1 mRNA expression decreased significantly.Conclusions The results indicate that Chk1 gene might be involved in molecular mechanism of cerebrum damage during complete global brain ischemia-reperfusion.
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AIM: To investigate the mechanism of AngRem104-mediated regulation of fibronectin gene in human mesangial cells. METHODS: A series of deleted FN promoter sequences was constructed, which fuse to a luciferase reporter gene, and then the potential active regions that respond to AngRem104 in the upstream regulatory sequence of human FN gene was screened by detecting the luciferase activity of promoter-reporter gene. RESULTS: The detection of relative luciferase activity revealed that there was no significant differences when the HMC were transfected with FN122-reporter gene together with sense AngRem104 construct, but the luciferase activity significantly increased when transfection of FN507-reporter gene construct together with sense AngRem104 construct. However, no increase in luciferase activity was observed when transfection of FN1280-reporter gene construct together with sense AngRem104 construct. The potential regulatory region responds to AngRem104 is in the upstream sequence (-122 to -507) of human FN gene. CONCLUSION: Our preliminary study provided the evidence that AngRem104 may mediate the transcription of the FN gene via the activation of its promoter.
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Gene transcriptional regulation research is one of the major challenges in the post-genome era. Bioinformatics has become more important with the rapid accumulation of complete genome sequences and the advances of computational methods and related databases. The current computational approaches in promoter prediction, transcription factor binding site identification, composite elements prediction, co-regulation of gene expression analysis and phylogenetic footprinting in the regulatory region analysis are discussed in this review.