Genomic variation in Streptococcus mutans: deletions affecting the multiple pathways of β‐glucoside metabolism

The genome of Streptococcus mutans UA159 contains two phospho‐β‐glucosidase genes, bglA and celA, which occur in operon‐like arrangements along with genes for components of phosphotransferase transport systems and a third phospho‐β‐glucosidase encoded by the arb gene, which does not have its own associated transport system but relies on uptake by the bgl or cel systems. Targeted inactivation of each of the phospho‐β‐glucosidase genes revealed that bglA is involved in aesculin hydrolysis, celA is essential for utilisation of cellobiose, amygdalin, gentobiose and salicin, and arb is required for utilisation of arbutin. Inactivation of genes for the phosphotransferase systems revealed an overlap of specificity for transport of β‐glucosides and also indicated that further, unidentified transport systems exist. The cel and arb genes are subject to catabolite repression by glucose, but the regM gene is not essential for catabolite repression. Screening a collection of isolates of S. mutans revealed strains with deletions affecting the msm, bgl and/or cel operons. The phenotypes of these strains could largely be explained on the basis of the results obtained from the knockout mutants of S. mutans UA159 but also indicated the existence of other pathways apparently absent from UA159. The extensive genetic and phenotypic variation found in β‐glucoside metabolism indicates that there may be extensive heterogeneity in the species.     

Effects of interleukin‐1β and tumor necrosis factor‐α on macrophage inflammatory protein‐3α production in synovial fibroblast‐like cells from human temporomandibular joints

Background

Interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) are key mediators of the intracapsular pathological conditions of the temporomandibular joint (TMJ). Therefore, the gene expression profiles in synovial fibroblast‐like cells (SFCs) from patients with internal derangement of the TMJ were examined after they were stimulated with IL‐1β or TNF‐α to determine which genes were altered.

Methods

Ribonucleic acid was isolated from SFCs after IL‐1β or TNF‐α treatment. Gene expression profiling was performed using oligonucleotide microarray analysis. On the basis of the results of this assay, we investigated the kinetics of macrophage inflammatory protein‐3α (MIP‐3α) gene expression using PCR, and protein production in TMJ SFCs stimulated by IL‐1β or TNF‐α using an ELISA. Inhibition experiments were performed with MAPK and NFκB inhibitors. SFCs were stimulated with IL‐1β or TNF‐α after treatment with inhibitors. The MIP‐3α levels were measured using an ELISA.

Results

Macrophage inflammatory protein‐3α was the gene most upregulated by IL‐1β‐ or TNF‐α stimulation. The mRNA and protein levels of MIP‐3α increased in response to IL‐1β in a time‐dependent manner. In contrast, during TNF‐α stimulation, the MIP‐3α mRNA levels peaked at 4 h, and the protein levels peaked at 8 h. In addition, the IL‐1β‐ and TNF‐α‐stimulated MIP‐3α production was potently reduced by the MAPK and NFκB signaling pathway inhibitors.

Conclusion

Interleukin‐1β and TNF‐α increased the MIP‐3α production in SFCs via the MAPK and NFκB pathways. These results suggest that the production of MIP‐3α from stimulation with IL‐1β or TNF‐α is one factor associated with the inflammatory progression of the internal derangement of the TMJ.     

Utilization and acid production of β‐galactosyllactose by oral streptococci and human dental plaque

β‐Galactosyllactose is a trisaccharide containing the β‐galactosidic linkage at the nonreducing end. The purpose of this study was to determine whether certain oral streptococci could utilize four kinds of β‐galactosyllactoses. Three of four β‐galactosyllactoses were unable to support growth of the oral streptococci and to be a substrate for producing acid from the cell suspensions and dental plaque. 4′‐β‐Galactosyllactose supported growth of Streptococcus sanguis ATCC 35105, ATCC 49298, Streptococcus mitis ATCC 15914, Streptococcus oralis ATCC 35037, ATCC 10557 and Streptococcus milleri 10707 and produced acid from dental plaque. Although β‐galactosidase activities were observed in all the strains, 4′‐β‐galactosyllactose could not be used as a carbon source for the growth of mutans streptococci. Enzymes metabolizing 4′‐β‐galactosyllactose were induced when S. oralis ATCC 10557 was cultured in medium containing galactose. These results suggested that 4′‐β‐galactosyllactose could be as cariogenic as lactose if it is consumed frequently and retained for a long period in the mouth.     

人牙髓干细胞条件培养基通过AMPK/PGC-1α途径改善氯化钴诱导的颏舌肌成肌细胞低氧损伤

目的： 研究氯化钴 (CoCl₂) 模拟的低氧对颏舌肌成肌细胞活性和氧化应激的影响,探讨人牙髓干细胞 （human dental pulp stem cells,hDPSCs）条件培养基 (conditioned medium,CM)保护作用的机制与AMPK/PGC-1α通路的关系。方法： 分离、培养及鉴定hDPSCs,通过超滤浓缩法提取条件培养基;分离、培养小鼠颏舌肌成肌细胞,分为对照组、CM组、CoCl₂组、CoCl₂+CM组。采用CCK-8法检测颏舌肌成肌细胞的活性,分别使用DCFH-DA和MitoSOX Red评估细胞内和线粒体活性氧 (reactive oxygen species,ROS) 水平,实时定量PCR分析NRF-1、NRF-2线粒体相关基因的表达,Western 印迹法检测PGC-1α、p-AMPK、总AMPK蛋白表达。采用SPSS 22.0软件包对数据进行统计学分析。结果： 颏舌肌成肌细胞经过200 μmol/L CoCl₂处理24 h后增殖显著下降（P<0.05）,细胞内和线粒体内ROS含量显著上升（P<0.05）;与CoCl₂组相比,加入hDPSCs-CM后,细胞活性显著增加（P<0.05）,胞内和线粒体内ROS含量显著降低 (P<0.05);hDPSCs-CM显著上调颏舌肌成肌细胞中pAMPK和PGC-1α蛋白的表达水平以及PGC-1α下游的线粒体效应物NRF-1、NRF-2的mRNA表达水平 (P<0.05)。结论： 人牙髓干细胞条件培养基能减轻CoCl₂诱导的颏舌肌成肌细胞低氧损伤,其机制可能与AMPK/PGC-1α通路的调节有关。