Purification of ferredoxins and their reaction with purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Four ferredoxin (Fd) fractions, namely, FdA–D were purified from the green sulfur bacterium Chlorobium tepidum. Their absorption spectra are typical of 2[4Fe–4S] cluster type Fds with peaks at about 385 and 280 nm and a shoulder at about 305 nm. The A385/A280 ratios of the purified Fds were 0.76–0.80. Analysis of the N-terminal amino acid sequences of these Fds (15–25 residues) revealed that those of FdA and FdB completely agree with those deduced from the genes, fdx3 and fdx2, respectively, found in this bacterium (Chung and Bryant, personal communication). The N-terminal amino acid sequences of FdC and FdD (15 residues) were identical, and agree with that deduced from the gene fdx1 (Chung and Bryant, personal communication). The A385 values of these Fds were unchanged when they were stored for a month at −80°C under aerobic conditions and decreased by 10–15% when they were stored for 6 days at 4°C under aerobic conditions, indicating that they are not extremely unstable. In the presence of Fd-NADP+ reductase from spinach, and a purified reaction center (RC) preparation from C. tepidum composed of five kinds of polypeptides, these Fds supported the photoreduction of NADP+ at room temperature with the following Km and Vmax (in μmol NADP+ μmol BChl a−1 h−1): FdA, 2.0 μM and 258; FdB, 0.49 μM and 304; FdC, 1.13 μM and 226; FdD, 0.5 μM and 242; spinach Fd, 0.54 μM and 183. The Vmax value of FdB was more than twice that previously reported for purified RC preparations from green sulfur bacteria

コラーゲンスポンジを用いた三次元培養におけるヒト顎下腺由来腺癌細胞株の遺伝子発現

Expressions of genes and proteins of human salivary gland cells (HSG cells) were investigated on three-dimensional culture comprised in collagen sponge in order to understand the response of simple epithelial cells at the molecular level. HSG cells were seeded in collagen sponges (COL-S)(three-dimensional culture) with an agitated seeding method, and cultured for various times. As a control, HSG cells were cultured on a polystyrene dish (PS) or a collagen coated dish (COL-C)(monolayer culture). The morphological features indicate that HSG cells cultured with COL-S presented a globular shape compared with monolayer culture such as with PS or COL-C. We focused on cytokeratins (CK) that are used as the marker proteins for epithelia to characterize the cultured cells. In HSG cells, CK7, CK8, CK17 and CK18 are expressed, but CK13 which is expressed specifically in mucosal epithelia is not expressed. The expression levels of CK genes were investigated by RT-PCR. The results were that the level of CK8 and CK18 were downregulated. The level of CK13 was upregulated. On the other hand, no alternation of the expression levels of CK7 and CK17 was observed. Western Blotting was carried out to investigate the expression of protein. The bands of CK8 and CK18 were detected. The expression level of CK8 and CK18 protein was reduced as well as the mRNA level. CK13 protein was not detected in this way. The expression level of the Nm23-H1 gene, which is one of tumor suppressor genes and concerned with ERK/MAPK signal transduction, was downregulated. These results suggest that HSG cells were altered by three-dimensional culture for different expression patterns of CKs which were not contained in the simple epithelium

Galectin-1はヒト口腔扁平上皮癌細胞のアノイキスを抑制する

In order to determine the factors that relate to anoikis of the human oral squamous cell carcinoma (hOSCC) cells, we tried to search the proteins using the HSC-3 cell with metastaticity and the HSC-2 cells without metastaticity. The HSC cells were cultured in a dish coated with poly-HEMA to prevent cell adhesion, and then the degree of cell death was examined. The ratio of cell death for HSC-2 cells was significantly higher than that for HSC-3 cells, and the result of TUNEL staining showed that the cell death was apoptosis. The level of anoikis in HSC-2 cells was notably higher than that in HSC-3 cells. The expression level of TrkB, caveolin-1, and galectin-3 genes that are known as factors related to anoikis was investigated by RT-PCR. There was no significant difference in the gene expression between HSC-2 and HSC-3 cells. Galectin-1 was found by the proteome analysis and the protein was expressed more strongly in the HSC-3 cells as compared with the HSC-2 cells. A similar result was obtained in the amount of the mRNA expression. The anoikis of HSC-3 cells was caused strongly by the addition of lactose that inhibits the binding of galectin-1 to the cell membrane. When recombinant-galectin-1 was added to the medium, the level of anoikis in HSC-2 cells was decreased significantly. From these results, it was suggested that galectin-1 is the factor that suppresses anoikis in hOSCC cells

Mycoplasma salivariumにおけるヌクレアーゼの存在とその特性

Mycoplasmas depend on the host cells for various nutrients such as nucleic acid precursors and steroids. It has been mentioned that the acquisition of the nucleotides from the host cells is probably mediated through their own nucleases. However, in Mycoplasma salivarium, the existence of nuclease has not yet been known. In this study, we examined the nuclease activity in M. salivarium, and analyzed the properties of the enzyme. The Triton X-114 solubilized supernatant (Tx) obtained from lysate of M. salivarium was used for the enzymatic analysis. The nuclease activity was detected as a specific band of 25kDa by SDS-PAGE and the following in-gel digestion (SDS-PAGE nuclease assay). The nuclease activity of Tx was strictly dependent on Ca^. The nuclease was heat-stable, and the optimum pH was in the range of 7-9. The nuclease showed a low substrate specificity. From these findings, it was revealed that M. salivarium has a novel type of 25kDa and Ca^ dependent nuclease. Moreover, we examined whether Tx could cleave the chromatin DNA in the nuclei originated from eukaryote cells. Consequently, Tx cleaved the DNA of HS-72 B cell nuclei in vitro. From these results, it has been suggested that M. salivarium in the host cells, obtains nucleotides by the digestion of DNA and RNA from the host cells with their own nuclease, and these cleavage results in injury in host cells and the following various pathogenesis of the oral region

Novel biological activity of ameloblastin in enamel matrix derivative

Objective Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration. However, the effects of EMD on gingival epithelial cells during regeneration of periodontal tissues are unclear. In this in vitro study, we purified ameloblastin from EMD and investigated its biological effects on epithelial cells. Material and Methods Bioactive fractions were purified from EMD by reversed-phase high-performance liquid chromatography using hydrophobic support with a C18 column. The mouse gingival epithelial cell line GE-1 and human oral squamous cell carcinoma line SCC-25 were treated with purified EMD fraction, and cell survival was assessed with a WST-1 assay. To identify the proteins in bioactive fractions of EMD, we used proteome analysis with two-dimensional gel electrophoresis followed by identification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Results Purified fractions from EMD suppressed proliferation of GE-1 and SCC-25. LC-MS/MS revealed that ameloblastin in EMD is the component responsible for inhibiting epithelial cell proliferation. The inhibitory effect of ameloblastin on the proliferation of GE-1 and SCC-25 was confirmed using recombinant protein. Conclusion The inhibitory effects of EMD on epithelial cell proliferation are caused by the biological activities of ameloblastin, which suggests that ameloblastin is involved in regulating epithelial downgrowth in periodontal tissues

EGF Positively Regulates the Proliferation and Migration, and Negatively Regulates the Myofibroblast Differentiation of Periodontal Ligament-Derived Endothelial Progenitor Cells through MEK/ERK- and JNK-Dependent Signals

Background/Aims: Remodeling of fibrous and vascular tissues in the periodontal ligament (PDL) around the tooth root was observed during tooth movement by orthodontic force application. We previously demonstrated that a single cell-derived culture (SCDC) of primarily cultured PDL fibroblasts, called SCDC2, has an endothelial progenitor cell (EPC)-like character and can form endothelial cell (EC) marker-positive blood vessel-like structures. However, the types of molecular mechanisms that control the in vivo kinetic properties and the differentiation of the PDL-derived EPC-like cells into myofibroblasts (MFs), which are known to expand fibrous tissues, require clarification. Methods: Using specific mitogen activated protein kinase (MAPK) inhibitors, we examined how epidermal growth factor (EGF)-mediated MAPK signals affected the proliferation, migration, and MF differentiation of these cells. Results: EGF induced SCDC2 cell proliferation in MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)- and c-Jun N-terminal kinase (JNK)-dependent manners. In addition, EGF suppressed the expression of MF differentiation markers in these cells in a MEK/ERK-dependent manner, and, moreover, stimulated the cell migration in a MEK/ERK-dependent manner. Conclusion: EGF regulates fibrous tissue remodeling in PDLs through MEK/ERK- and JNK-mediated signals by affecting the proliferation, migration, and MF differentiation of the PDL-derived EPC-like cells