Porphyromonas gingivalis is a black-pigmented asaccharolytic anaerobe and a major causative agent of periodontitis. Here, we report the complete genome sequence of P. gingivalis strain TDC60, which was recently isolated from a severe periodontal lesion in a Japanese patient.Porphyromonas gingivalis is a Gram-negative asaccharolytic bacterium which causes destructive periodontal diseases that result in a weakening of the tooth-supporting tissue and loss of teeth (7). Complete genomic sequences of two P. gingivalis laboratory strains, W83 and ATCC 33277, have been available for several years (11,12). However, there is a need to analyze recently isolated strains without extensive subculture, because reports indicate a loss of virulence by a number of pathogenic bacteria after several passages in laboratory culture (1). P. gingivalis TDC60 was isolated from a severe periodontal lesion at Tokyo Dental College in Japan. Strain TDC60 exhibited higher pathogenicity in causing abscesses in mice than strains W83 and ATCC 33277 and other strains tested in the college. Thus, analyzing the genomic information of TDC60 is expected to provide new insights into the mechanisms of the onset and progression of periodontitis.The complete genome sequence of TDC60 was determined by a combination of pyrosequencing (22,277,778-bp sequences; 9-fold coverage) and the Sanger method (16,934,400-bp sequences; 7-fold coverage). The pyrosequencing reads and the Sanger reads were assembled using the Newbler and Phred/ Phrap/Consed, respectively. Gaps between adjacent contigs were closed by sequencing PCR amplicons from genomic DNA. Protein-coding sequences (CDSs) over length of 90 bp were predicted using a combination of MetaGeneAnnotator (13), GLIMMER (4), and the IMCGE software (In Silico Biology Co., Ltd., Japan). Functional annotation of CDSs was based on the results of the BLASTP searches against the NCBI nonredundant protein database. Insertion sequences (ISs), miniature inverted-repeat transposable elements (MITEs), conjugative transposons (CTns), and clustered regularly interspaced short palindromic repeats (CRISPRs) were identified using ISfinder (15), a combination of MUST (2) and MITEhunter (8), a combination of Mauve (3) and GenomeMatcher (14), and CRISPRFinder (6), respectively. Nontranslated genes were predicted using the tRNAscan-SE (10), RNAmmer (9), and Rfam (5).The genome of P. gingivalis TDC60 contained a single circular chromosome (2,339,898 bp; 48.34% GC content). The chromosome contained 2,220 CDSs, four rRNA operons, 53 tRNA sequences, and nine noncoding RNAs.All-to-all BLASTP analysis of W83 and ATCC 33277 protein sequences showed that TDC60 possessed 382 strain-specific CDSs, and approximately two-thirds were annotated as hypothetical proteins. Of the CDSs encoding hypothetical proteins, 87 CDSs were on mobile elements, while some showed high homology to proteins encoded by genes of the CytophagaFlavobacteria-Bacteroides (CFB) group of bacteria. Strain TDC60 may have acquired these CDSs via horizontal gene transfer fro...

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These results indicate that the PG1385 protein is involved in P. gingivalis virulence and that the method used here is useful when investigating the P. gingivalis proteins responsible for virulence.

Porphyromonas gingivalis is a Gram-negative anaerobic pathogen associated with chronic periodontitis. Although anaerobic, P. gingivalis exhibits a high degree of aerotolerance, which enables it to survive within periodontal pockets. The aim of the present study was to examine the effect of oxidative stress on protein expression in P. gingivalis to obtain a better understanding of the mechanism underlying its aerotolerance. To accomplish this, P. gingivalis cells were grown under conditions of hemin limitation (0.01 microg/mL) to avoid the oxygen protective effect of hemin on oxidative stress. The proteins were then extracted from cultures either left untreated or subjected to oxidative stress and separated by 2-DE. The resultant protein expression profiles were examined by image scanning, and those found to differ depending on the presence or absence of aeration were subjected to MALDI-MS and then analyzed using the ORF database of P. gingivalis W83 from The Institute of Genomic Research. Oxidative stress was found to affect the expression of numerous proteins in P. gingivalis cells. In particular, the levels of HtpG, GroEL, DnaK, AhpC, TPR domain protein, and trigger factor were substantially increased.

Neuroblastoma is the most common solid tumor in children. Despite aggressive chemotherapy, the prognosis of patients with advanced neuroblastoma is still very poor. Our recent study showed that xanthoangelol, a major chalcone constituent of the stem exudates of Angelica keiskei, induced caspase-3-dependent apoptosis in neuroblastoma cells. However, details of the mechanism underlying its apoptotic action are still unclear. Here we show that xanthoangelol triggers oxidative stress by generation of reactive oxygen species and induces apoptosis through release of cytochrome c and activation of caspase-9 in IMR-32 cells. Pretreatment with an antioxidant, vitamin E, prevented the increase of reactive oxygen species and apoptosis induced by xanthoangelol. Proteomic analysis using 2-dimensional electrophoresis and MALDI-TOF-MS revealed that DJ-1 protein was involved in xanthoangelol-induced apoptosis. DJ-1 responded to its oxidative stress status by being oxidized itself. Furthermore, DJ-1 was down-regulated by xanthoangelol, leading to loss of antioxidant function and acceleration of apoptosis. We also show that xanthoangelol has a cytotoxic effect on drug-resistant LA-N-1 and NB-39 cells as well as drug-sensitive IMR-32 and SK-N-SH cells. These findings suggest that xanthoangelol induces apoptosis by increasing reactive oxygen species and targeting DJ-1, and such mechanism may be an effective therapeutic approach for advanced neuroblastoma.