Abnormal HLA-G expression occurs in various diseases such as melanoma, renal cell carcinoma, asthma, and classic Hodgkin's lymphoma. The purpose of this study was to determine whether HLA-G gene is linked with oral squamous cell carcinoma (OSCC). To investigate the possible link with susceptibility to OSCC, 54 OSCC patients and 120 healthy controls were enrolled in this study. HLA-G 14bp insertion/deletion polymorphism is in 3′-untranslated region of HLA-G gene. HLA-G 14bp insertion/deletion polymorphism was analyzed using the polymerase chain reaction (PCR) method. For the analysis of genetic data, SPSS18.0 program was used. Logistic regression models were performed for odds ratio (OR), 95 percent confidence interval (CI), and P value. There was a significant difference in distribution allele between OSCC patients and control subjects (OR=0.018, 95% CI=0.002-0.131, p<0.001). Our results suggest that HLA-G 14bp insertion/deletion polymorphism may be linked with susceptibility to OSCC in the Korean population.
Alleles ; Asthma ; Carcinoma, Renal Cell ; Carcinoma, Squamous Cell* ; Epithelial Cells* ; Exons* ; HLA-G Antigens* ; Hodgkin Disease ; Humans ; Logistic Models ; Melanoma ; Methods ; Odds Ratio ; Polymerase Chain Reaction

BMP-2 is a well-known TGF-beta related growth factor, having a significant role in bone and cartilage formation. It has been employed to promote bone formation in some clinical trials, and to differentiate mesenchymal stem cells into osteoblasts. However, it is difficult to obtain this protein in its soluble and active form. hBMP-2 is expressed as an inclusion body in the bacterial system. To continuously supply hBMP-2 for research, we optimized the refolding of recombinant hBMP-2 expressed in E. coli, and established an efficient method by using detergent and alkali. Using a heparin column, the recombinant hBMP-2 was purified with the correct refolding. Although combinatorial refolding remarkably enhanced the solubility of the inclusion body, a higher yield of active dimer form of hBMP-2 was obtained from one-step refolding with detergent. The refolded recombinant hBMP-2 induced alkaline phosphatase activity in mouse myoblasts, at ED₅₀ of 300-480ng/ml. Furthermore, the expressions of osteogenic markers were upregulated in hPDLSCs and hDPSCs. Therefore, using the process described in this study, the refolded hBMP-2 might be cost-effectively useful for various differentiation experiments in a laboratory.
Alkalies ; Alkaline Phosphatase ; Animals ; Cartilage ; Detergents ; Heparin ; Humans* ; Inclusion Bodies ; Mesenchymal Stromal Cells ; Methods ; Mice ; Myoblasts ; Osteoblasts ; Osteogenesis ; Solubility ; Stem Cells* ; Transforming Growth Factor beta

Selecting an appropriate antigen with optimal immunogenicity and physicochemical properties is a pivotal factor to develop a protein based subunit vaccine. Despite rapid progress in modern molecular cloning and recombinant protein technology, there remains a huge challenge for purifying and using protein antigens rich in hydrophobic domains, such as membrane associated proteins. To overcome current limitations using hydrophobic proteins as vaccine antigens, we adopted in silico analyses which included bioinformatic prediction and sequence-based protein 3D structure modeling, to develop a novel periodontitis subunit vaccine against the outer membrane protein FomA of Fusobacterium nucleatum. To generate an optimal antigen candidate, we predicted hydrophilicity and B cell epitope parameter by querying to web-based databases, and designed a truncated FomA (tFomA) candidate with better solubility and preserved B cell epitopes. The truncated recombinant protein was engineered to expose epitopes on the surface through simulating amino acid sequence-based 3D folding in aqueous environment. The recombinant tFomA was further expressed and purified, and its immunological properties were evaluated. In the mice intranasal vaccination study, tFomA significantly induced antigen-specific IgG and sIgA responses in both systemic and oral-mucosal compartments, respectively. Our results testify that intelligent in silico designing of antigens provide amenable vaccine epitopes from hard-to-manufacture hydrophobic domain rich microbial antigens.
Animals ; Cloning, Molecular ; Computational Biology ; Computer Simulation* ; Epitopes ; Epitopes, B-Lymphocyte ; Fusobacterium nucleatum* ; Fusobacterium* ; Hydrophobic and Hydrophilic Interactions ; Immunoglobulin A, Secretory ; Immunoglobulin G ; Membrane Proteins ; Mice ; Periodontitis ; Solubility ; Vaccination

Recent studies indicate that mitochondria are an important source of reactive oxygen species (ROS) in the spinal dorsal horn. In our previous study, application of malate, a mitochondrial electron transport complex I substrate, induced a membrane depolarization, which was inhibited by pretreatment with ROS scavengers. In the present study, we used patch clamp recording in the substantia geletinosa (SG) neurons of spinal slices, to investigate the cellular mechanism of mitochondrial ROS on neuronal excitability. DNQX (an AMPA receptor antagonist) and AP5 (an NMDA receptor antagonist) decreased the malate-induced depolarization. In an external calcium free solution and addition of tetrodotoxin (TTX) for blockade of synaptic transmission, the malateinduced depolarization remained unchanged. In the presence of DNQX, AP5 and AP3 (a group I metabotropic glutamate receptor (mGluR) antagonist), glutamate depolarized the membrane potential, which was suppressed by PBN. However, oligomycin (a mitochondrial ATP synthase inhibitor) or PPADS (a P2 receptor inhibitor) did not affect the substrates-induced depolarization. These results suggest that mitochondrial substrate-induced ROS in SG neuron directly acts on the postsynaptic neuron, therefore increasing the ion influx via glutamate receptors.
Animals ; Calcium ; Electron Transport Complex I ; Glutamic Acid ; Membrane Potentials ; Membranes ; Mitochondria ; Mitochondrial Proton-Translocating ATPases ; N-Methylaspartate ; Neurons* ; Oligomycins ; Rats* ; Reactive Oxygen Species ; Receptors, AMPA ; Receptors, Glutamate ; Receptors, Metabotropic Glutamate ; Spinal Cord Dorsal Horn ; Substantia Gelatinosa* ; Synaptic Transmission ; Tetrodotoxin

Anthriscus sylvestris (L.) Hoffm. is a perennial herb found widely distributed in various regions of Korea, Europe, and New Zealand. The root of A. sylvestris have been extensively used in the treatment for antitussive, antipyretic, cough remedy in Oriental medicine, but the physiologically active function of the leaf of A. sylvestris is as yet unknown. In this study, we investigated the anti-cancer activity and the mechanism of cell death of water extracts of leaf of Anthriscus sylvestris (WELAS), on human FaDu hypopharyngeal squamous carcinoma cells. Our data showed that WELAS treatment inhibited cell viability in a concentration- and time-dependent manner. In addition, the treatment of WELAS markedly induced apoptosis in FaDu cells, as determined by the viability assay, DAPI stain and FACS analysis. WELAS also increased the proteolytic cleavage of procaspase-3, -9 and PARP (poly(ADP-ribose) polymerase). In addition, exposure to WELAS decreased the expression of Bcl-2 (an anti-apoptotic factor), but increased the expression of Bax (a pro-apoptotic factor), suggesting that mitochondria-dependent apoptotic pathways are mediated in WELAS-induced apoptosis. Taken together, these results indicate that water extracts of leaf of A. sylvestris inhibits cell growth and induces apoptosis via the mitochondrial-dependent apoptotic pathway in FaDu human hypopharyngeal squamous carcinoma cells. Therefore, we propose that the water extracts of leaf of A. sylvestris is a novel chemotherapeutic drug, having growth inhibitory properties and induction of apoptosis in human oral cancer cells.
Apoptosis* ; Carcinoma, Squamous Cell* ; Caspase 3 ; Cell Death ; Cell Survival ; Cough ; Europe ; Humans* ; Hypopharynx* ; Korea ; Medicine, East Asian Traditional ; Mouth Neoplasms ; New Zealand ; Water*

Metformin (1,1-dimethylbiguanide hydrochloride), derived from French lilac (Galega officinalis), is a first-line anti-diabetic drug prescribed for patients with type 2 diabetes. However, the role of metformin in odontoblastic cell differentiation is still unclear. This study therefore undertook to examine the effect of metformin on regulating odontoblast differentiation in MDPC-23 mouse odontoblastic cells derived from mouse dental papilla cells. As compared to controls, metformin significantly accelerated the mineralization, significantly increased and accelerated the expressions of ALP and Col I mRNAs, and significantly increased the accelerated expressions of DSPP and DMP-1 mRNAs, during differentiation of MDPC-23 cells. There was no alteration in cell proliferation of MDPC-23 cells, on exposure to metformin. These results suggest that the effect of metformin on MDPC-23 mouse odontoblastic cells derived from mouse dental papilla cells, facilitates the odontoblast differentiation and mineralization, without altering the cell proliferation.
Animals ; Cell Differentiation ; Cell Proliferation ; Dental Papilla ; Humans ; Metformin* ; Mice ; Miners ; Odontoblasts* ; RNA, Messenger

The presence of distinct bacterial species is found to be dependent on age, diet, and disease. We compared the detection rate of several oral bacterial strains in a cohort of 36 subjects including healthy volunteers, periodontal patients, and oral cancer patients. Gargling samples were obtained from these subjects from which DNA was then extracted. Specific primers for 29 bacterial species were used for PCR detection. In the oral cancer patients, Capnocytophaga ochracea, Gemella morbillorum, and Streptococcus salivarius were detected more frequently compared with the healthy volunteers and periodontitis patients. Fusobacterium nucleatum/polymorphym and Prevotella nigrescens were significantly less prevalent in oral cancer patients than the other groups. In periodontitis patients, Porphyromonas gingivalis and Treponema denticola were more frequently found compared with the healthy volunteers. In the healthy volunteer group, Peptostreptococcus anaerobius was more frequently found than the other groups. The detection rate of several oral bacterial species was thus found to differ between healthy volunteers, periodontitis patients and oral cancer patients.
Capnocytophaga ; Cohort Studies ; Diet ; DNA ; Fusobacterium ; Gemella ; Healthy Volunteers* ; Humans ; Microbiota* ; Mouth Neoplasms* ; Peptostreptococcus ; Periodontitis* ; Polymerase Chain Reaction ; Porphyromonas gingivalis ; Prevotella nigrescens ; Streptococcus ; Treponema denticola

Periodontal disease is a major oral disorder and comprises a group of infections that lead to inflammation of the gingiva and the destruction of periodontal tissues. PPARgamma plays an important role in the regulation of several metabolic pathways and has recently been implicated in inflammatory response pathways. However, its effects on periodontal inflammation have yet to be clarified. In our current study, we evaluated the anti-inflammatory effects of PPARgamma on periodontal disease. Human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS) showed high levels of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), matrix metalloproteinase-2 (MMP-2), and -9 (MMP-9). Moreover, these cells also showed upregulated activities for extracellular signal regulated kinase (ERK1/2), inducible nitric oxide synthase (iNOS) and cyclooxygnase-2. However, cells treated with Ad/PPARgamma and rosiglitazone in same culture system showed reduced ICAM-1, VCAM-1, MMP-2, -9 and COX-2. Finally, the anti-inflammatory effects of PPARgamma appear to be mediated via the suppression of the ERK1/2 pathway and consequent inhibition of NF-kB translocation. Our present findings thus suggest that PPARgamma indeed has a pivotal role in gingival inflammation and may be a putative molecular target for future therapeutic strategies to control chronic periodontal disease.
Fibroblasts ; Gingiva ; Humans ; Inflammation ; Intercellular Adhesion Molecule-1 ; Matrix Metalloproteinase 2 ; Metabolic Networks and Pathways ; NF-kappa B ; Nitric Oxide Synthase Type II ; Periodontal Diseases ; Phosphotransferases ; PPAR gamma ; Thiazolidinediones ; Vascular Cell Adhesion Molecule-1

This study was undertaken to develop species-specific forward and universal reverse PCR primers for the detection of Streptococcus sobrinus. These primers target the variable regions of the 16S ribosomal RNA coding gene (rDNA) and their specificity was tested against 10 strains of S. sobrinus strains and 20 different species of oral bacteria using serial dilutions of the purified genomic DNA of S.sobrinus ATCC 33478T . Our data show that species-specific amplicons were obtained from all the S. sobrinus strains tested but not from other species. Both direct and nested PCR could detect as little as 400 pg and 4 fg of genomic DNA from S. sobrinus ATCC 33478T , respectively. This result suggests that these PCR primers are highly specific and sensitive and applicable to the detection of S. sobrinus.
Bacteria ; Clinical Coding ; Collodion ; DNA ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; Sensitivity and Specificity ; Streptococcus ; Streptococcus sobrinus

The DNA probes Pn17 and Pn34 were evaluated for their ability to specifically detect clinical strains of P. intermedia and P. nigrescens from a Korean population by dot blot hybridization. These probes were sequenced by extension termination and their specificity was determined by Southern blot analysis. The results revealed that the Pn17 sequence (2,517 bp) partially encodes an RNA polymerase beta subunit (rpoB) and that Pn34 (1,918 bp) partially encodes both rpoB (1-169 nts) and the RNA polymerase beta subunit (rpoB'; 695-1918 nts). These probes hybridized with both HindIII- and PstI-digested genomic DNAs from the strains of P. intermedia and P. nigrescens used in this study. Interestingly, each of the hybrid bands generated from the HindIII-digested genomic DNAs of the two bacterial species could be used to distinguish between them via restriction fragment length polymorphism. These results thus indicate that Pn17 and Pn34 can simultaneously detect P. intermedia and P. nigrescens.
Blotting, Southern ; Chimera ; DNA ; DNA Probes ; DNA-Directed RNA Polymerases ; Polymorphism, Restriction Fragment Length ; Prevotella ; Prevotella intermedia ; Prevotella nigrescens ; Sensitivity and Specificity