OBJECTIVETo detect the expression changes of the demethylase TETs (Ten-eleven translocation enzymes) in human embryonic kidney cell (HEK293) exposed to high dose cadmium chloride (CdCl2), and to investigate the regulation effects of TETs on global genomic methylation.

METHODSHEK293 cells were exposed to CdCl2 for 24 h, 48 h and 72 h, the survival rate was tested by CCK-8 (cell counting kit-8) method, and the cell morphology was observed. The levels of TETs mRNA and protein were detected by fluorescence quantitative PCR and Western blot, respectively. The genomic DNA methylation level was detectedby pyro sequencing assay.

RESULTSCdCl2 had toxic effects on HEK293 cells, and the half inhibitory concentration (IC50) was 1.78 µmol/L. After exposure of CdCl2 for 24 h, 48 h and 72 h, the morphology of HEK293 cells was altered, and the high dose group (2.0 µmol/L) showed vacuolar changes and fuzzy appearance. The level of TET1 mRNA in groups of 0.0, 0.5, 1.0, 2.0 µmol/L were 0.23 ± 0.13, 0.48 ± 0.12, 0.59 ± 0.16 and 0.95 ± 0.39, respectively (F = 182.89, P = 0.002); The level of TET2 mRNA in groups of 0.0, 0.5, 1.0, 2.0 µmol/L were 0.23 ± 0.12, 0.32 ± 0.02,0.31 ± 0.10 and 0.34 ± 0.07, respectively (F = 27.94, P < 0.001); The level of TET3 mRNA in groups of 0.0, 0.5, 1.0, 2.0 µmol/L were 0.26 ± 0.10, 0.27 ± 0.11, 0.25 ± 0.11 and 0.28 ± 0.09, respectively (F = 1.76, P = 0.036). The interaction effect existed between exposure time and doses of TET1 mRNA, TET2 mRNA and TET3 mRNA (F values were 32.94, 23.04 and 13.78, respectively; P values were < 0.001, 0.041 and < 0.001, respectively). Western blot showed that in different exposure time and dose, the protein expression levels of TETs had the similar trend as mRNA levels. In 24 h (55.01 ± 3.62)%, 48 h (48.31 ± 8.99)%, 72 h (48.76 ± 6.60)%, the DNA methylation had significant differences (F = 18.50, P < 0.001); In groups of 0.0 µmol/L (55.29 ± 2.83)%, 0.5 µmol/L (55.35 ± 3.11)%, 1.0 µmol/L (48.58 ± 6.40)% and 2.0 µmol/L (43.56 ± 7.89)%, the differences of DNA methylation had significant differences (F = 7.03, P = 0.048); the effect of interaction was also existed (F = 2.73, P = 0.043).

CONCLUSIONIn the short term exposure to CdCl2, the levels of TETs mRNA and protein showed a trend of increase according to the exposure time and dose, and the methylation level of whole genomic DNA was also altered. The demethylase TETs may play a role in regulating the genomic methylation level of HEK293 exposed to cadmium.

Cadmium Chloride ; toxicity ; DNA Methylation ; Dioxygenases ; genetics ; Epithelial Cells ; drug effects ; HEK293 Cells ; Humans ; RNA, Messenger

Objective: This study aimed to investigate the clinical and prognostic significance of TET2 single nucleotide polymorphism I1762V in patients with acute myeloid leukemia (AML) . Methods: The high-throughput sequencing method was used to sequence 58 hematological tumor-related genes in bone marrow samples from 413 patients with AML. TET2 I1762V and other somatic mutations were annotated and compared with patients' clinical information and prognosis. Results: I1762V was found in 154 patients with AML, which was significantly different from the general population in NyuWa Chinese Population Variant Database (χ(2)=72.4, P<0.001) . I1762V was not related to sex, age, and karyotype of patients with AML (P>0.05) . Patients with I1762V had a significantly higher proportion of NPM1 and KIT gene mutations than others (P<0.001) . NPM1 and KIT mutations were mutually exclusive. The survival analysis results revealed that the overall survival (OS) and progression-free survival (PFS) of patients with AML with I1762V were significantly greater than those of wild-type patients (HR=0.57, P=0.030; HR=0.55, P=0.020) , whereas the OS and PFS in patients with AML with DNMT3A mutation (with or without I1762V mutation) were lower than those of wild-type patients (HR=1.79, P=0.030; HR=1.74, P=0.040) . Conclusion: TET2 SNP I1762V has been linked to AML. I1762V is a prognostic factor of patients with AML, which can be used to guide the treatment and evaluate the prognosis of AML.
DNA-Binding Proteins/genetics* ; Dioxygenases/genetics* ; Humans ; Leukemia, Myeloid, Acute/genetics* ; Mutation ; Nuclear Proteins/genetics* ; Polymorphism, Single Nucleotide ; Prognosis

Mean hemoglobin (Hb) concentration of about 3 500 subjects derived from 17 studies of Himalayan highlanders (Tibetans, Sherpas, and Ladakhis) was compared with lowlanders (Chinese Han, Indian Tamils) lived in the Himalayas, and European climbers during Everest expeditions as well as Andean natives. The results found that Hb concentration in Himalayan highlanders was systemically lower than those reported for Andean natives and lowland immigrants. These comparative data demonstrated that a healthy native population may successfully reside at high altitude without a significant elevation in Hb, and the lower Hb levels of Himalayan highlanders than those of migrated lowlanders and Andean natives are an example of favourable adaptation over the generations. In addition, excessive polycythemia has frequently been used as a marker of chronic mountain sickness (CMS). Altitude populations who have a higher Hb concentration also have a higher incidence of CMS. The low Hb in Himalayans suggested as showing adaptation over many generations in Tibetan stock. Recent work in Tibet, suggested that Tibetans there may have adapted to high altitude as a result of evolutionary pressure selecting for genes which give an advantage at altitude. All of the population genomic and statistical analysis indicated that EPAS1 and EGLN1 are mostly likely responsible for high altitude adaptation and closely related to low Hb concentration in Tibetans. These data supported the hypothesis that Himalayan highlanders have evolved a genetically different erythropoietic response to chronic hypoxia by virtue of their much longer exposure to high altitude.
Adaptation, Physiological ; Altitude ; Asian Continental Ancestry Group ; genetics ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; Evolution, Molecular ; Hemoglobins ; genetics ; Humans ; Hypoxia-Inducible Factor-Proline Dioxygenases ; genetics ; Tibet

The aromatic-ring-hydroxylating dioxygenase is a key enzyme that initiates the biodegradation of polycyclic aromatic hydrocarbons in bacteria. In the present study, a novel dioxygenase sequence was cloned from Terrabacter sp. FLO using a genome walking method. The dioxygenase was cloned into pET17 and actively expressed in E.coli BL21 (DE3) in co-expression with electron transfer chain proteins. The recombinant dioxygenase was found to transform phenanthrene, fluorene, pyrene and fluoranthene into the cis-dihydrodiol metabolites.
Actinomycetales ; enzymology ; genetics ; Bacterial Proteins ; genetics ; metabolism ; Biodegradation, Environmental ; Cloning, Molecular ; Dioxygenases ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; metabolism ; Fluorenes ; metabolism ; Hydroxylation ; Molecular Sequence Data ; Phenanthrenes ; metabolism ; Polycyclic Aromatic Hydrocarbons ; metabolism ; Pyrenes ; metabolism ; Recombinant Proteins ; metabolism

OBJECTIVETo study the acireductone dioxygenase (designated as SmARD) gene of Salvia miltiorrhiza through bioinformatics and characterization of its tissue expression and response expression on stress in shoot.

METHODSmARD gene was obtained by sequencing cDNA library constructed by us. BLAST was used for alignment, ORF finder software was applied to find open reading frame, prosite was used to analyze the protein characterization. Semi-quantitative RT-PCR was used to detect the gene expression level.

RESULTThe full -length cDNA of SmRAD was 688 bp long with a 591 bp ORF (open reading frame) that putatively encoded a polypeptide of 196 amino acids; with a predicted molecular mass of 23.27 kDa. The deduced amino acid sequence of SmRAD of gene shared high homology with other known RADs. Semi-quantitative RT-PCR analysis indicated that SmRAD was constitutively expressed in roots, stems, flower and leaves of S. miltiorrhiza, with the high expression in roots. In addition, SmRAD expression level under different stress condition was also analyzed in root, including signaling components for plant defence responses, such as methyl jasmonate, salicylic acid and ABA, as well as drought, cold and salt abiotic stress. The expression of SmRAD was suppressed by water deficit treatment for 3 d, 150 mmol x L(-1) NaCl, 4 degrees C cold and 100 mmol x L(-1) ABA treatment for 1 d, but induced by 100 mmol x L(-1) MJ and 10 mmol x L(-1) ETH.

CONCLUSIONA novel SmARD gene was cloned from S. miltiorrhiza. This study will enable us to further understand the role of SmARD in the defense response under different abiotic stress and in synthesis of active cmpounds in S. miltiorrhiza at molecular level.

Amino Acid Sequence ; Cloning, Molecular ; Dioxygenases ; genetics ; metabolism ; Gene Expression Regulation, Plant ; Molecular Sequence Data ; Phylogeny ; Plant Roots ; genetics ; metabolism ; Salvia miltiorrhiza ; genetics ; metabolism ; Sequence Alignment ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid ; Stress, Physiological