ABSTRACT
Objective To investigate the relationship between the expression of GATA6 mRNA and the occu-rrence of congenital heart disease(CHD). Methods A total of 60 cases of CHD (CHD group)were diagnosed at Obstetrical Department,Rizhao City Maternal and Child Health Hospital,and 60 cases of normal pregnancy women (control group)were collected in March 2013 to May 2017. The expression levels of mRNA of GATA6 in the peripheral blood of pregnant women in 2 groups were detected by using real-time quantitative PCR (qRT-PCR)technique,and the relevant information of 2 groups of mothers before and during pregnancy was collected so as to investigate the rela-tionship between mRNA and GATA6 in CHD. Results There were no significant differences in age,body mass index (BMI),passive smoking,alcohol consumption,pregnancy medication,pregnancy-induced hypertension,high -density lipoproteincholesterol (HDL-C),total cholesterol (TC),triacylglycerol (TG)between pregnant women in the congenital heart disease group and the control group (all P > 0. 05). The CHD group showed a statistical difference significance in the rate of taking folic acid(81. 67％)and gestational diabetes incidence(8. 33％),serum level of LDL-C[(3. 59 ± 0. 46)mmol/ L]compared with the healthy control group [96. 67％,0,(3. 20 ± 0. 44)mmol/ L] (all P < 0. 05). The expression level of GATA6 mRNA in peripheral blood of patients with congenital heart disease (0. 014 ± 0. 005)was significantly lower than that of the control group (0. 129 ± 0. 031),and the difference was statis-tically significant (t = 28. 386,P = 0. 000). Logistic regression analysis showed that the level of in GATA6 mRNA ex-pression increased in the peripheral blood and folic acid intake were protective factors for CHD (all P < 0. 05)during pregnancy;while fetal gestational diabetes,elevated levels of LDL-C are risk factors for congenital heart disease (all P < 0. 05)fetus. Conclusion Down regulation of GATA6 mRNA expression in the maternal peripheral blood is one of the independent risk factors for fetal congenital heart disease.
ABSTRACT
Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-α scFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient.
Subject(s)
Animals , Humans , Mice , Antibody Affinity , Cells, Cultured , Cytidine Deaminase , Genetics , Metabolism , HEK293 Cells , Immunoglobulin Variable Region , Genetics , Allergy and Immunology , Mutation , Single-Chain Antibodies , Chemistry , Genetics , Allergy and Immunology , Somatic Hypermutation, Immunoglobulin , Genetics , Allergy and Immunology , Tumor Necrosis Factor-alpha , Allergy and ImmunologyABSTRACT
The Rad1 gene is evolutionarily conserved from yeast to human. The fission yeast Schizosaccharomyces pombe Rad1 ortholog promotes cell survival against DNA damage and is required for G(2)/M checkpoint activation. In this study, mouse embryonic stem (ES) cells with a targeted deletion of Mrad1, the mouse ortholog of this gene, were created to evaluate its function in mammalian cells. Mrad1 (-/-) ES cells were highly sensitive to ultraviolet-light (UV light), hydroxyurea (HU) and gamma rays, and were defective in G(2)/M as well as S/M checkpoints. These data indicate that Mrad1 is required for repairing DNA lesions induced by UV-light, HU and gamma rays, and for mediating G(2)/M and S/M checkpoint controls. We further demonstrated that Mrad1 plays an important role in homologous recombination repair (HRR) in ES cells, but a minor HRR role in differentiated mouse cells.