2,6-and 3,5-dimethylaniline-induced mutagenesis in Chinese hamster ovary cells expressing human cytochrome P450 1A2 and sulfotransferase

Aim: The aim of this study was to test the hypothesis that human cytochrome P450 1A2 (CYP1A2) and sulfotransferase (SULT) contribute to the phase I and II bioactivation of 2,6-dimethylaniline (2,6-DMA) and 3,5-dimethylaniline (3,5-DMA) in affecting the incidence of genotoxicity.Methodology: 5P3H1 cells carrying cytochrome P450 1A2 (CYP1A2) and SULT cells were treated with various concentrations of 2,6-and 3,5-DMA for 48 hr or their N-hydroxyl and aminophenol metabolites for 1 hr in the absence or presence of 2,6-Dichloro-4-nitrophenol (DCNP). Cell lethality was assayed by trypan blue exclusion and induced mutagenesis of adenine phosphoribosyl transferase (aprt) gene was also evaluated. Results: A significant dose-dependent increase in cytotoxicity and mutant fraction was observed after treatment with 2,6- and 3,5-DMA, and their metabolites; N-hydroxy and aminophenol metabolites are more potent than the parent compounds. Addition of sulfotransferase inhibitor DCNP decreased the cytotoxic and mutagenic effects of 2,6- and 3,5-DMA, and their metabolites in a dose-dependent manner. Interpretation: This research indicate that 2,6 and 3,5-DMA are mutagenic, and their toxicity in model systems depend on metabolic activation. This activation is mediated by CYP1A2 and SULT enzymes

Construction and analysis of competitive endogenous RNA regulatory network related to gastric cancer / 中华肿瘤杂志

Objective@#To construct the competitive endogenous RNA (ceRNA) network related to gastric cancer and explore the molecular mechanism.@*Methods@#The expression profiles of lncRNA, miRNA and mRNA in gastric cancer and paracancer tissues were analyzed by biochip technology, edgeR package in R software was used to filtrate differential expression genes (multiple change of >1.5 times, P<0.05) and volcano map was drawn. Based on the online miRNA-lncRNA prediction tool lncBase database and the miRNA Target gene prediction database (miRTarBase, target-scan, miRDB, starBase), the relationship between miRNA, lncRNA and mRNA was predicted. Cytoscape software was used to construct lncRNA-miRNA-mRNA ceRNA network and key genes (hub genes) were identified based on cytohubba calculation of degree score of each node. Then Hub genes related to the prognosis of gastric cancer were verified in the TCGA database. The GO and KEGG enrichment analysis of differentially expressed mRNA was performed using the online biological information annotation database DAVID, P<0.05 and false discovery rate (FDR)<0.05 were used as cut-off criteria. R software was used to download the RNA sequencing data and mirna-seq data of gastric cancer and adjacent tissues in TCGA database, edgeR package was used to screen out differentially expressed mRNA, miRNA and lncRNA, and some differentially expressed genes in our data were verified. In OncoLnc database, STAD project of TCGA data was selected and hub gene was input. Patients were divided into two groups based on the median value for hub genes and Kaplan-meier analysis was performed.@*Results@#The differentially expressed 766 mRNA, 110 lncRNA and 10 miRNA were screened out, among them 90 mRNA, 4 lncRNA and 6 miRNA were used to construct the ceRNA network, and 2 of the 20 hub genes were related to the prognosis of patients. MLK7-AS1, SPP1, SULF1, hsa-miR-1307-3p were upregulated in gastric cancer tissues from our biochip, while MT2A, MT1X were downregulated, which were consistent with the results of TCGA gastric cancer database. The differentially expressed mRNAs were significantly enriched in the biological process (BP) and the mineral absorption pathway. CHST1 was negatively correlated while miR-183-5p was positively corelated with the survival of patients.@*Conclusion@#The establishment of ceRNA network for gastric cancer is conducive to further understanding of the molecular biological mechanism. CHST1 and miR-183-5p can be used as prognostic factors of gastric cancer.

Cloning and in vitro expression of sulfotransferase genes from Ilex asprella / 中草药

Objective: The medicinal plant Ilex asprella contains various biologically active sulfonated triterpenoids and triterpenoid saponins. The objective of this study was to clone sulfotransferases (ST) from I. asprella and facilitate the elucidation of the sulfonation mechanism therein. Methods: The physicochemical properties, secondary structure and tertiary structure of two ST candidates of IaST1 and IaST2 were forecasted and analyzed using related software. IaST1 and IaST2 were cloned by RT-PCR and expressed in Escherichia coli. Results: The open reading frame (ORF) of IaST1 was 1 002 bp long and encoded a protein of 333 amino acids with the calculated molecular weight of 55 500, while the ORF of IaST2 was 993 bp long and encoded a protein of 330 amino acids with the calculated molecular weight of 54 700. Both contain the five highly conserved domains of ST. Phylogenetic analysis revealed that IaST1 and IaST2 were genetically closely related and clustered together with flavonol C-3 ST of Flaveria bidentis. Conclusion: This is the first report on the cloning of STs from I. asprella, providing an important basis for further investigations into their functions and roles in the biosynthesis of sulfonated triterpenoids in I. asprella.

Arenobufagin is a novel isoform-specific probe for sensing human sulfotransferase 2A1

Human cytosolic sulfotransferase 2A1 (SULT2A1) is an important phase II metabolic enzyme. The detection of SULT2A1 is helpful for the functional characterization of SULT2A1 and diagnosis of its related diseases. However, due to the overlapping substrate specificity among members of the sulfotransferase family, it is difficult to develop a probe substrate for selective detection of SULT2A1. In the present study, through characterization of the sulfation of series of bufadienolides, arenobufagin (AB) was proved as a potential probe substrate for SULT2A1 with high sensitivity and specificity. Subsequently, the sulfation of AB was characterized by experimental and molecular docking studies. The sulfate-conjugated metabolite was identified as AB-3-sulfate. The sulfation of AB displayed a high selectivity for SULT2A1 which was confirmed by reaction phenotyping assays. The sulfation of AB by human liver cytosols and recombinant SULT2A1 both obeyed Michaelis-Menten kinetics, with similar kinetic parameters. Molecular docking was performed to understand the interaction between AB and SULT2A1, in which the lack of interaction with Met-137 and Tyr-238 of SULT2A1 made it possible to eliminate substrate inhibition of AB sulfation. Finally, the probe was successfully used to determine the activity of SULT2A1 and its isoenzymes in tissue preparations of human and laboratory animals.

Optimized expression of heparin sulfotransferases and their application in sulfation of animal derived heparin / 生物工程学报

Heparin is a very important anticoagulant drug. Currently, heparin is mainly extracted from porcine mucosa. However, animal-derived heparin shows low anticoagulant activity due to the low proportion of the anticoagulant active unit, the GlcNS6S-GlcA-GlcNS6S3S-Ido2S-GlcNS6S pentasaccharide. In this study we proposed an enzymatic strategy to sulfate the animal-sourced heparin to increase the proportion of anticoagulant pentasaccharide and the anticoagulant activity. First, three sulfotransferases HS2ST, HS6ST, and HS3ST were expressed tentatively in Escherichia coli and Pichia pastoris. After measuring the sulfotransferase activity, we confirmed P. pastoris GS115 is the better host for sulfotransferases production. Then, the maltose binding protein (MBP) and thioredoxin (TrxA) were fused separately to the N-terminal of sulfotransferases to increase enzyme solubility. As a result, the yields of HS2ST and HS6ST were increased to (839±14) U/L and (792±23) U/L, respectively. Subsequent sulfation of the animal-sourced heparin with the recombinant HS2ST, HS6ST and HS3ST increased the anticoagulant activity from (76±2) IU/mg to (189±17) IU/mg.

Molecular characterization of CHST11 and its potential role in nacre formation in pearl oyster Pinctada fucata martensii

Background: C4ST-1 catalyzes the transfer of sulfate groups in the sulfonation of chondroitin during chondroitin sulfate synthesis. Chondroitin sulfate consists of numerous copies of negatively charged sulfonic acid groups that participate in the nucleation process of biomineralization. In the present study, we obtained two CHST11 genes (PmCHST11a and PmCHST11b) which encoded the C4ST-1 and explored the functions of these genes in the synthesis of chondroitin sulfate and in the formation of the nacreous layer of shells. Results: Both PmCHST11a and PmCHST11b had a sulfotransferase-2 domain, a signal peptide and a transmembrane domain. These properties indicated that these genes localize in the Golgi apparatus. Real-time PCR revealed that both PmCHST11a and PmCHST11b were highly expressed in the central zone of the mantle tissue. Inhibiting PmCHST11a and PmCHST11b via RNA interference significantly decreased the expression levels of these genes in the central zone of the mantle tissue and the concentration of chondroitin sulfate in extrapallial fluid. Moreover, shell nacre crystallized irregularly with a rough surface after RNA interference. Conclusions: This study indicated that PmCHST11a and PmCHST11b are involved in the nacre formation of Pinctada fucata martensii through participating in the synthesis of chondroitin sulfate.

Effect of miRNA-146a on life cycle of hepatitis B virus in vitro / 第三军医大学学报

Objective To determine the effect of microRNA-146a (miR-146a) on the life cycle of hepatitis B virus (HBV) and investigate the underlying mechanisms.Methods The miRNA expression profiles were compared by miRNA array between HepG2 and HepG2.2.15 cells.Then miR-146a was chosen as objective,and its expression level was further confirmed by RT-PCR.After miR-146a mimic and inhibitor were transfected into HepG2.2.15 cells respectively,the quantification of HBV replication was determined by RT-PCR,and the levels of HBsAg and HBeAg in the supernatant were measured by ELISA,and the expression of HS3ST3B1 at mRNA and protein levels were tested by RT-PCR and Western blotting.Dualluciferase reporter assay was used to detect the interaction between miR-146a and potential target HS3ST3B1.Results The expression levels of totally 72 miRNAs were changed in HepG2.2.15 cells,with 27 upregulated and 45 down-regulated.RT-PCR showed the expression level of miR-146a was significantly higher in HepG2.2.15 cells than HepG2 cells (1.55-± 0.13 vs 1.00 ± 0.01,P ＜ 0.05).Transfection of miR-146a mimic into HepG2.2.15 cells resulted in significantly increased HBV replication and levels of HBsAg and HBeAg (P ＜ 0.05),while the transfection of its inhibited caused opposite results (P ＜ 0.05).Bioinformatic analysis showed that HS3ST3B1 was a potential target of miR-146a.The reporter luciferase reporter system indicated that the reported fluorescence intensity of HS3ST3B1 wild type vector was significantly lower than that of the control group (P ＜ 0.05),but showed no significant difference between HS3ST3B1 mutant vector and control group (P ＞0.05).The mRNA level of HS3ST3B1 was not significantly changed in HepG2.2.15 cells transfected with miR-146a mimic (P ＞ 0.05),but its protein level was significantly decreased (P ＜ 0.05).Conclusions miR-146a affects the life cycle of HBV,which may be through suppressing the translation of HBV inhibitory factor HS3ST3B1 3'UTR.

A Case of Korean Patient with Macular Corneal Dystrophy Associated with Novel Mutation in the CHST6 Gene

To report a novel mutation within the CHST6 gene, as well as describe light and electron microscopic features of a case of macular corneal dystrophy. A 59-year old woman with macular corneal dystrophy in both eyes who had decreased visual acuity underwent penetrating keratoplasty. Further studies including light and electron microscopy, as well as DNA analysis were performed. Light microscopy of the cornea revealed glycosaminoglycan deposits in the keratocytes and endothelial cells, as well as extracellularly within the stroma. All samples stained positively with alcian blue, colloidal iron, and periodic acid-Schiff. Electron microscopy showed keratocytes distended by membrane-bound intracytoplasmic vacuoles containing electron-dense fibrillogranular material. These vacuoles were present in the endothelial cells and between stromal lamellae. Some of the vacuoles contained dense osmophilic whorls. A novel homozygous mutation (c.613 C>T [p.Arg205Trp]) was identified within the whole coding region of CHST6. A novel CHST6 mutation was detected in a Korean macular corneal dystrophy patient.

Expressions of peripheral lymph node addressin and GlcNAc-6-sulfotransferase in endometrium and their impacts on implantation / 北京大学学报(医学版)

Objective:To investigate the expressions of peripheral lymph node addressin(PNAd)andGlcNAc-6-sulfotransferase(GlcNAc6ST)in endometrium and their impacts on implantation.Methods:PNAd expression in endometrium was examined by immunohistochemistry and Western Blot from 75women(12 from healthy women,in proliferative phase;63 from sterile women,of whom,27 were inearly-secretory and 36 in mid-secretory phase).GlcNAc6ST mRNA was examined by real-time PCR in41 sterile women.The 63 sterile women had underwent ⅣF-ET and were consequently divided into clini-cal pregnant(29 cases)and nonpregnant(34 cases)groups.Results:(1)PNAd localized to the mem-brane and cytoplasm of luminal and glandular epithelia.Staining was patchy and much less intense duringthe proliferative phase than during the secretory phase.In Western Blot of PNAd,four bands appeared,which were Sgp200,CD34,MAdCAM-1,GlyCAM-1 respectively,and each was positively correlatedwith the others significantly.The former three molecular levels were significantly higher during the secre-tory phase as compared with the proliferative phase.Message RNA of GlcNAe6ST was positive in all ca-ses and showed no correlation with any component of PNAd.(2)The expressions of CD34 and GlyCAM-1,but not Sgp200 and MAdCAM-1,were significantly higher in pregnant women than in nonpregnantones.However,the GlcNAc6ST mRNA level did not differ between groups.(3)No significant differ-ence was found in female age,methods of fertilization,thickness of endometrium on day hCG,cumulativeembryo score(CES)and mean score of transferred embryo(MSTE)between the groups.Conclusion:PNAd expression in the human endometrium fluctuates with the menstrual cycle.Elevated CD34 and Gly-CAM-1 during the secretory phase might be stimulative factors for embryo implantation.Defect in PNAdexpression may account for a portion of unexplained infertility.