[Roles of REV3 in proliferation and genomic stability of colon carcinoma cells].

To investigate the roles of human REV3 gene in proliferation and genomic stability, we experimentally suppressed the REV3 expression in human colon cancer cells (SW480) by the interference RNA technology (RNAi) as monitored by real-time RT-PCR. Compared to untreated or mock-treated cells, ablation of REV3 significantly reduced cell growth rate, micronucleus formation and the frequency of sister chromatid exchange. Whereas the differences between untreated and mock-treated controls were insignificant. Indicators of cell cycle, proliferation and the expression of genetic information in cells of case group, which displayed lower-level expression of REV3, were extremely lower than the control group, and the difference was significant (P<0.05). Differences in the two control groups were not significant. This suggested that the reduced expression of REV3 may affect the growth and proliferation of colon cancer cells (SW480), and, to some extent, contribute to suppression of the genetic instability occurred in micronuclei and sister chromatids. Based on the results from this study, REV3 plays an important role in different cellular growth periods and physiological conditions, and its differential expression directly affects the development of human colon cancer cells.

Clonal mesenchymal stem cells derived from human bone marrow can differentiate into hepatocyte-like cells in injured livers of SCID mice.

There is increasing evidence that human mesenchymal stem cells (hMSCs) can be a valuable, transplantable source of hepatocytes. Most of the hMSCs preparations used in these studies were likely heterogeneous cell populations, isolated by adherence to plastic surfaces or by density gradient centrifugation. Therefore, the participation of other unknown trace cell populations cannot be rigorously discounted. Here we report the isolation and establishment of a cloned human MSC line (chMSC) from human bone marrow primary culture, through which we confirmed the hepatic differentiation capability of authentic hMSCs. chMSCs expressed markers of mesenchymal cells, but not markers of hematopoietic stem cells. In vitro, chMSCs can differentiate into either mesenchymal cells or cells exhibiting hepatocyte-like phenotypes. When transplanted intrasplentically into carbon tetrachloride-injured livers of SCID mice, EGFP-tagged chMSCs engrafted into the host liver parenchyma, exhibited typical hepatocyte morphology, form a three-dimensional architecture, and differentiate into hepatocyte-like cells expressing human albumin and alpha-1-anti-trypsin. By confocal microscopy, ultrafine intercellular nanotubular structures were visible between adjacent transplanted and host hepatocytes. We postulate that these structures may assist in the phenotype conversion of chMSCs, possibly by exchange of cytoplasmic components between native hepatocytes and transplanted cells. Thus, a clonal pure population of hMSCs, which can be expanded in culture, may have potential as a cellular source for substitution damaged cells in hepatic injury.

[Concentration Characteristics and Source Analysis of PM2.5 During Wintertime in Zhengzhou-Xinxiang].

To study the pollution characteristics, sources, and transportation process of PM2.5 and its chemical compositions in the Zhengzhou-Xinxiang region, PM2.5 samples were collected using a middle volume sampler, in Zhengzhou and Xinxiang urban areas for 30 consecutive days during the winter of 2016. The mass concentration of PM2.5 was measured gravimetrically. 17 trace metals were determined by inductively coupled plasma-mass spectrometry (ICP-MS), and 7 water-soluble ions were determined by ion chromatography. The enrichment factor (EF) method and principal component analysis were employed to determine the source apportionment. The results showed that the daily mean PM2.5 mass concentration during the winter sampling period of 2016 in Xinxiang and Zhengzhou was 223.87 µg·m-3 and 226.67 µg·m-3, respectively, which indicated that pollution levels were relatively high in both cities. The concentration of three macro elements (Al, Ca, and Fe) accounted for 50% of the total metal elements in both cities, while the heavy metals concentration was higher in Xinxiang than in Zhengzhou. The EFs of Cd, Ag, and Pb in Xinxiang were far higher than 1000, while only Cd was higher than 1000 in Zhengzhou. NO3-, SO42-, and NH4+ were the main ions in the two cities. They exceeded 94% of total water-soluble ions and existed in the forms of (NH4)2SO4 and NH4NO3. The principle component analysis showed that the main contributors to PM2.5 were a mixture of biomass combustion and secondary aerosol in Xinxiang, and a mixture of coal combustion and traffic emissions in Zhengzhou, accounting for 34.94% and 33.99% of total PM2.5 emissions, respectively.

Conversion of immortal liver progenitor cells into pancreatic endocrine progenitor cells by persistent expression of Pdx-1.

The conversion of expandable liver progenitor cells into pancreatic beta cells would provide a renewable cell source for diabetes cell therapy. Previously, we reported the establishment of liver epithelial progenitor cells (LEPCs). In this work, LEPCs were modified into EGFP/Pdx-1 LEPCs, cells with stable expression of both Pdx-1 and EGFP. Unlike previous work, with persistent expression of Pdx-1, EGFP/Pdx-1 LEPCs acquired the phenotype of pancreatic endocrine progenitor cells rather than giving rise to insulin-producing cells directly. EGFP/Pdx-1 LEPCs proliferated vigorously and expressed the crucial transcription factors involved in beta cell development, including Ngn3, NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6, Isl1, MafA and endogenous Pdx-1, but did not secrete insulin. When cultured in high glucose/low serum medium supplemented with cytokines, EGFP/Pdx-1 LEPCs stopped proliferating and gave rise to functional beta cells without any evidence of exocrine or other islet cell lineage differentiation. When transplanted into diabetic SCID mice, EGFP/Pdx-1 LEPCs ameliorated hyperglycemia by secreting insulin in a glucose regulated manner. Considering the limited availability of beta cells, we propose that our experiments will provide a framework for utilizing the immortal liver progenitor cells as a renewable cell source for the generation of functional pancreatic beta cells.

[Forms Distribution and Ecotoxicity of Three Forms of Sulfonamides in Root-Soil Interface of Maize].

Forms distribution and ecotoxicity of sulfonamides in root-soil interface of maize were studied by chemical analysis and luminescent bacteria toxicity assay. The results indicated that the water soluble residues were the main form in rhizosperic soils, and the organic solvent extractable residues were the main form in far-rhizosperic soils. The three forms of sulfonamides showed relatively lower toxicity to luminescent bacteria with the relative inhibition rates less than 30%. The toxicity of water soluble residues was lower than that of the organic solvent extractable residues, and the bound residues showed the lowest toxicity to luminescent bacteria. The contents and relative inhibition rates of water soluble residues in rhizosperic soils were higher than those in far-rhizosperic soils, while the contents and relative inhibition rates of organic solvent extractable and bound residues in rhizosperic soils were lower than those in far-rhizosperic soils.

[Distribution and Risk Assessment of Sulfonamides Antibiotics in Soil and Vegetables from Feedlot Livestock].

Soil and vegetable samples were collected from 13 different livestock farms of different sizes in Xinxiang of China, and the residues of three sulfonamides including sulfadiazine, sulfamonomethoxine, and sulfamethoxazole were analyzed by HPLC with a fluorimetric detector, The results indicated that the total concentration ranges of the three sulfonamides in soil and vegetable were 7.60-176.26 µg · kg⁻¹ and ND-32, 70 µg · kg⁻¹, respectively. The mean concentrations were 70.73 µg · kg⁻¹ and 7.08 µg · kg⁻¹ for soil and vegetables. The residue levels in soil were all lower than the ecotoxic effect trigger value (100 µg · kg⁻¹) set by the Veterinary Medicine International Coordination Commission, indicating the low risk for organisms in soil. The concentrations of three sulfonamides varied significantly in different kinds of vegetables and were all lower than the acceptable daily intake values [50 µg · (kg ·d)⁻¹] set by Joint FAO/WHO Expert CommIttee on Food Additives. But we cannot neglect the potential ecotoxicity and resistance for human via food chain.

Response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine and its role in mutagenesis.

AIM: To understand the response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and its role in human mutagenesis. METHODS: The response of the human REV3 gene to MNNG was measured in human 293 cells and FL cells by RT-PCR. By using antisense technology, mutation analysis at HPRT locus (on which lesion-targeted mutation usually occurs) was conducted in human transgenic cell line FL-REV3(-) by 8-azaguanine screening, and mutation occurred on undamaged DNA template was detected by using a shuttle plasmid pZ189 as the probe in human transgenic cell lines 293-REV3(-) and FL-REV3(-). The blockage effect of REV3 was measured by combination of reverse transcription-polymerase chain reaction to detect the expression of antisense REV3 RNA and Western blotting to detect the REV3 protein level. RESULTS: The human REV3 gene was significantly activated by MNNG treatment, as indicated by the upregulation of REV3 gene expression at the transcriptional level in MNNG-treated human cells, with significant increase of REV3 expression level by 0.38 fold, 0.33 fold and 0.27 fold respectively at 6 h, 12 h and 24 h in MNNG-treated 293 cells (P<0.05); and to 0.77 fold and 0.65 fold at 12 h and 24 h respectively in MNNG-treated FL cells (P<0.05). In transgenic cell line (in which REV3 was blocked by antisense REV3 RNA), high level of antisense REV3 RNA was detected, with a decreased level of REV3 protein. MNNG treatment significantly increased the mutation frequencies on undamaged DNA template (untargeted mutation), and also at HPRT locus (lesion-targeted mutation). However, when REV3 gene was blocked by antisense REV3 RNA, the MNNG-induced mutation frequency on undamaged DNA templates was significantly decreased by 3.8 fold (P<0.05) and 5.8 fold (P<0.01) respectively both in MNNG-pretreated transgenic 293 cells and FL cells in which REV3 was blocked by antisense RNA, and almost recovered to their spontaneous mutation levels. The spontaneous HPRT mutation was disappeared in REV3-disrupted cells, and induced mutation frequency at HPRT locus significantly decreased from 8.66 x 10(-6) in FL cells to 0.14 x 10(-6) in transgenic cells as well (P<0.01). CONCLUSION: The expression of the human REV3 can be upregulated at the transcriptional level in response to MNNG. The human REV3 gene plays a role not only in lesion-targeted DNA mutagenesis, but also in mutagenesis on undamaged DNA templates that is called untargeted mutation.

Effect of alendronate sodium on the expression of mesenchymal-epithelial transition markers in mice with liver fibrosis.

The aim of this study was to explore whether alendronate sodium regulates tissue remodeling by controlling the transforming growth factor (TGF)-ß1-induced epithelial-mesenchymal transition (EMT) and bone morphogenetic protein (BMP)-7-induced mesenchymal-epithelial transition (MET) in CCl(4)-induced hepatic fibrosis in mice. A mouse model of CCl(4)-induced hepatic fibrosis was evaluated using the hematoxylin and eosin (HE) and Masson's trichrome staining histological methods. The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using an automated biochemical analyzer. The expression of TGF-ß1, α-smooth muscle actin (α-SMA), BMP-7 and E-cadherin in the hepatic tissue was detected using immunohistochemistry. The mRNA and protein levels of TGF-ß1, α-SMA, BMP-7, fibroblast-specific protein 1 (FSP1), E-cadherin and N-cadherin were detected using RT-PCR and western blot analysis. Immunohistochemical and molecular biochemical examination revealed that alendronate sodium significantly arrested the progression of hepatic fibrosis. Alendronate sodium caused significant amelioration of liver injury and reduced the activities of serum ALT and AST (P<0.001). Furthermore, alendronate sodium markedly reduced TGF-ß1 and α-SMA mRNA expression and increased BMP-7 and E-cadherin in the mouse liver tissue (P<0.001). Alendronate sodium significantly arrested the progression of hepatic fibrosis. The underlying mechanism was associated with changes in the redox state, which remains variable in liver fibrosis, and depends on the balance between TGF-ß/smad- and BMP-7-modulated mechanisms which regulate EMT and MET in multifunctional progenitors.

Bone morphogenetic protein-7 regulates Snail signaling in carbon tetrachloride-induced fibrosis in the rat liver.

The aim of this study was to explore the molecular mechanism of the bone morphogenetic protein-7 (BMP-7) downregulation of Snail-mediated E-cadherin repression and mesenchymal-epithelial transition (MET) induction, since little is presently known about this issue. In this study, our aim was to elucidate the underlying mechanism by which cells acquire liver fibrosis characteristics after epithelial-mesenchymal transition (EMT). Cell cultures were exposed to Snail alone or in the presence of BMP-7; control cultures were exposed to medium only. The expression of the mRNA encoding α-smooth muscle actin (α-SMA), Snail and E-cadherin in rat liver epithelial cells was determined by real-time quantitative PCR (RT-PCR) and the main results were confirmed by ELISA. Cell differentiation was determined by analysis of the expression of α-SMA, Snail and E-cadherin by western blotting and co-immunoprecipitation. We demonstrated Snail-induced upregulation of mRNAs encoding α-SMA and downregulation of mRNAs encoding E-cadherin in rat liver epithelial cells when compared with unstimulated cells, and confirmed these results at the protein level. BMP-7 downregulated Snail-induced α-SMA and upregulated E-cadherin release compared with untreated and Snail-treated cells. In summary, we demonstrated that BMP-7 induces MET through decreased downregulation of Snail. In addition, Snail1 directly regulates Nanog promoter activity. Notch signaling is also involved in this process.