Cloning and expression of SmDXS2 gene in Swertia mussotii / 中国中药杂志

1-deoxy-D-xylulose-5-phosphate synthase2(DXS2) is the first key enzyme of the MEP pathway,which plays an important role in terpene biosynthesis of plants. According to the data of Swertia mussotii transcriptome, DXS2 gene(Gen Bank number MH535905) was cloned and named as Sm DXS2. The bioinformatics results showed that Sm DXS2 has no intron,with a 2 145 bp open reading frame encoding a polypeptide of 714 amino acids. They are belonging to 20 kinds of amino acids,and the most abundant amino acids include Ala,Gly and Trp. The predicted protein molecular weight was 76. 91 k Da and its theoretical isoelectric point(p I) was6. 5,which belonging to a hydrophilic protein. α-Helix and loop were the major motifs of predicted secondary structure of DXS2. The three function domains are TPP＿superfamily,Transket＿pyr＿ superfamily and Transketolase＿C superfamily,respectively. The Sm DXS2 protein shared high identity with other DXS2 proteins of plants. Phylogenetic analysis showed that Sm DXS2 protein is grouped with the gentian DXS2 protein. The recombinant protein of Sm DXS2 gene in Escherichia coli was approximately 92. 00 k Da(containing sumo-His tag protein 13 k Da),which was consistent with the anticipated size.This work will provide a foundation for further functional research of Sm DXS2 protein and increasing the product of iridoid compound by genetic engineering in S. mussotii.

Cloning and bioinformatics analysis of SmMK gene in Swertia mussotii / 中草药

Objective: In order to identify the function of the mevalonate kinase (MK) which is a key enzyme of the mevalonate pathway (MVA) in Swertia mussotii, and to improve the study of MVA in S. mussotii. Methods: According to the SmMK gene sequence of transcriptome of S. mussotii, the specific primers were designed, the cDNA complete sequences was obtained by RT-PCR and the sequence was analyzed using bioinformatics. Prokaryotic expression vector MBP-SmMK was constructed and transformed into Escherichia coli Rosetta (DE3) for expression. Results: The results showed that SmMK cDNA complete sequences had a length of 1 164 bp encoding 387 amino acid residues. The SmMK protein shared high identity with other MK proteins of plants. And the protein signal peptide, transmembrane region, location, secondary, and tertiary structures were analyzed and forecasted. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size, which was 40 970. Conclusion: This work will provide a foundation for research the SmMK protein functional and study MCA in S. mussotii. At the same time, it will supply the basis to improve the production of the isoprenoids.

Analysis of the correlation between 11β-HSDs expression in the ovarian granulosa cells and cortisol metabolism in the ovary of PCOS / 上海交通大学学报（医学版）

Objective: To explore the relationship of 11β-hydroxysteroid dehydrogenases (11β-HSDs) expression in the ovarian granulosa cells and cortisol regeneration in the ovary of polycystic ovary syndrome (PCOS). Methods: In accordance with the revised Rotterdam inclusion/exclusion criteria (2003), 24 patients in the PCOS group were included, meanwhile 21 patients in the control group were included. Follicular fluid and luteinized granulosa cells were collected from patients underwent in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) in the Center for Reproductive Medicine of Renji Hospital. Enzyme linked immunosorbent assay (ELISA) was used to detect the cortisol concentration and the cortisone levels of follicular fluid. Real-time quantitative PCR (RT-qPCR) was used to measure 11β-HSDs abundance in the luteinized granulosa cells. The expression of 11β-HSD1 and 11β-HSD2 in the luteinized granulosa cells were verified by immunofluorescence. The correlation of local cortisol level in the ovary with 11β-HSDs expression in the luteinized granulosa cells was further analyzed. Results: Compared with the control group, the cortisol level in the follicular fluid was elevated in the PCOS group [(477.3±35.3) nmol/L vs (292.0±36.4) nmol/L, P=0.014], while no change was observed in the cortisone levels in two groups. Both 11β-HSD1 and 11β-HSD2 were detected in the luteinized granulosa cells. Compared with the control group, the 11β-HSD1 mRNA abundance (P=0.033) and reductase activity (P=0.006) were increased significantly in the PCOS group, and there was no significant difference in the 11β-HSD2 expression and oxidase activity between two groups. Consistently, Pearson correlation analysis showed that the 11β-HSD1 but not 11β-HSD2 mRNA levels in the luteinized granulosa cells was correlated with the cortisol level in the follicular fluid (r2=0.347 6, P=0.001). Conclusion: Elevation of local cortisol in the ovary of PCOS patients may be related to the increased expression of 11β-HSD1 and reductase activity in the ovarian granulosa cells.

Piperine regulates glucose metabolism disorder in HepG2 cells of insulin resistance models via targeting upstream target of AMPK signaling pathway / 中国中药杂志

To investigate the effect of piperine on the disorder of glucose metabolism in the cell model with insulin resistance (IR) and explore the molecules mechanism on intervening the upstream target of AMPK signaling pathway. The insulin resistance models in HepG2 cells were established by fat emulsion stimulation. Then glucose consumption in culture supernatant was detected by GOD-POD method. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of leptin(LEP) and adiponectin(APN) in culture supernatant; Real-time quantitative PCR was used to assess the mRNA expression of APN and LEP; and the protein expression levels of LepR, AdipoR1, AdipoR2 and the activation of AMPK signaling pathway were detected by Western blot analysis. The results showed that piperine, rosiglitazone and AMPK agonist AICAR could significantly elevate the glucose consumption in insulin resistance cell models, enhance the level of APN, promote APN mRNA transcripts and increase the protein expression of Adipo receptor. Meanwhile,AMPKα mRNA and р-AMPKα protein expressions were also increased in piperine treated cells, but both LEP mRNA expression and LepR protein expressions were decreased in piperine treated group. The results indicated that piperine could significantly ameliorate the glucose metabolism disorder in insulin resistance cell models through regulating upstream molecules (APN and LEP) of AMPK signaling pathway, and thus activate the AMPK signaling pathway.

Genotoxicity of organic bentonite particles in vitro / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the genotoxicity induced by organic bentonite particles in vitro.</p><p><b>METHODS</b>Human B lymphoblast cells (HMy2.CIR) were exposed to organic bentonite particles at the doses of 0, 1.88, 3.75, 7.50 and 15.00 µg/ml for 24, 48 and 72 h, calcium sulfate (30 µg/ml) and SiO2 (30 and 240 µg/ml) served as negative and positive controls, respectively. The genotoxicity of organic bentonite particles and soluble fraction was detected using comet assay and Cytokinesis-block micronucleus (CBMN) assay.</p><p><b>RESULTS</b>The results of comet assay indicated that % tail DNA increased with the exposure doses and time in organic bentonite group, % tail DNA at the dose of 15.00 µg/ml for 24 h, 48 h and 72 h in organic bentonite group were 3.20 ± 0.19, 4.63 ± 0.88 and 9.49 ± 1.31 respectively which were significantly higher than those in calcium sulfate group (1.40 ± 0.11, 1.37 ± 0.22 and 0.90 ± 0.16) and those in 30 µg/ml SiO2 group (1.83 ± 0.21, 1.41 ± 0.27 and 2.48 ± 0.25) (P < 0.01). The results of CBMN assay showed that micronucleus frequencies (MNF) in organic bentonite group (except for 1.88 µg/ml for 24 h) were significantly higher than those in 30 µg/ml calcium sulfate group (MNF for 24, 48 and 72 h were 1.33‰ ± 0.58‰, 1.33‰ ± 1.15‰ and 1.33‰ ± 0.58‰) and those in 30 µg/ml SiO2 group (2.00‰ ± 0.00‰, 1.68‰ ± 0.58‰ and 2.33‰ ± 0.58‰) (P < 0.01). The results of two assays demonstrated that the soluble fraction of organic bentonite did not induce the genotoxicity.</p><p><b>CONCLUSION</b>The organic bentonite dusts can induce the genotoxicity in vitro, which may be from the particle fraction.</p>

Comparative study of cytotoxicity induced by two kinds of bentonite particles in vitro / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study comparatively the cytotoxicity induced by acid bentonite and organic bentonite.</p><p><b>METHODS</b>The cytotoxicity of two kinds of bentonite was detected using CCK8 assay, neutral red uptake (NRU) assay, lactate dehydrogenase (LDH) leakage assay, apoptosis assay and hemolysis assay. In hemolysis assay human erythrocytes served as target cells and were exposed to the two kinds of bentonite at the doses of 0, 0.3125, 0.6250, 1.2500 and 2.5000 mg/ml for ten min. In other four assays, human B lymphoblast cells (HMy2.CIR) served as target cells and were exposed to the two kinds of bentonite at the doses of 0, 10, 20, 30, 60, 120 and 180 microg/ml for four h.</p><p><b>RESULTS</b>In hemolysis assay, the hemolysis rates induced by two kinds of bentonite at all doses were significantly higher than that of control (P<0.05); in CCK-8 assay, the cellular activities in acid bentonite group at the doses > or =30 microg/ml and in organic bentonite group at the doses > or =20 microg/ml were significantly lower than that of control (P<0.01); the similar results appeared in NRU assay and LDH assay, and the dose-effect relationship was observed in above 4 assays. In apoptosis assay, the early apoptosis cell rates in acid bentonite group at the dose of 180 microg/ml and in organic bentonite group at the doses of 120,180 microg/ml were significantly higher than that of control (P<0.05). Moreover, the results of five in vitro assays indicated the cytotoxicity induced by organic bentonite was higher than that induced by acid bentonite.</p><p><b>CONCLUSION</b>Two kinds of bentonite could induce cytotoxicity, such as apoptosis and damage of cell membrane. The cytotoxicity of organic bentonite is higher than that of acid bentonite due to the different industrial treatment and characteristics of two kinds of bentonite particles.</p>

Cyto-genotoxicity induced by cigarette smoke condensates in human peripheral blood lymphocytes in vitro / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the cyto-genotoxicity of cigarette smoke condensates (CSCs) in human peripheral blood lymphocytes with different assays in vitro.</p><p><b>METHODS</b>Human lymphocytes were exposed to particle matter of cigarette smoke combined with or without S9 mixtures at doses of 25, 50, 75, 100 and 125 microg/ml for 3 h. The cytotoxicity induced by CSCs was detected by CCK-8 assay. The DNA damage, DNA repair (repair time: 30, 60, 90, 120 and 240 min, respectively) and the somatic cell mutations induced by 75 microg/ml CSCs were measured by comet assay, hprt gene and TCR gene mutation tests, respectively.</p><p><b>RESULTS</b>CCK-8 assay indicated that the cell viability decreased with CSCs doses. At the doses of 100, 125 microg/ml, the cell viability of CSCs +S9 group was significantly higher than that of CSCs -S9 group (P < 0.05, P < 0.01). In comet assay, DNA damage significantly increased in a dose-dependent manner, as compared with controls (P < 0.01). Moreover, there was significant difference between -S9 group and +S9 group (P < 0.05, P < 0.01). The Mf-TCR at each dose group was significantly higher than that of controls (P < 0.05, P < 0.01). The Mf-hprt at high-dose groups were significantly higher than that of controls (P < 0.01), and significant difference of Mf-TCR and Mf-hprt at high doses of CSCs between -S9 group and +S9 group (P < 0.05, P < 0.01). The DNA damage induced by CSCs +S9 or CSCs -S9 could be repaired, but DNA repair speed was different between -S9 group and +S9 group (P < 0.05, P < 0.01).</p><p><b>CONCLUSION</b>CSCs may induce cyto-genotoxicity in human peripheral blood lymphocytes in vitro, but S9 mix could reduce the toxicity of CSCs and impact DNA repair speed.</p>