Periodontitis and Porphyromonas gingivalis in Preclinical Stage of Arthritis Patients

Purpose

To determine whether the presence of periodontitis (PD) and Porphyromonas gingivalis (Pg) in the subgingival biofilm associates with the development of rheumatoid arthritis (RA) in treatment naïve preclinical stage of arthritis patients.

Methods

We conducted a prospective cohort study of 72 consecutive patients with arthralgia who had never been treated with any anti-rheumatic drugs or glucocorticoids. Periodontal status at baseline was assessed by dentists. PD was defined stringently by the maximal probing depth≧4 mm, or by the classification by the 5th European Workshop in Periodontology (EWP) in 2005 using attachment loss. Up to eight plaque samples were obtained from each patient and the presence of Pg was determined by Taqman PCR. The patients were followed up for 2 years and introduction rate of methotrexate (MTX) treatment on the diagnosis of RA was compared in patients with or without PD or Pg.

Results

Patients with PD (probing depth≧4mm) had higher arthritis activity (p = 0.02) and higher risk for future introduction of MTX treatment on the diagnosis of RA during the follow up than patients without PD (Hazard ratio 2.68, p = 0.03). Arthritis activity and risk for MTX introduction increased with the severity of PD assessed by EWP, although not statistically significant. On the other hand, presence of Pg was not associated with arthritis activity (p = 0.72) or the risk for MTX introduction (p = 0.45).

Conclusion

In treatment naïve arthralgia patients, PD, but not the presence of Pg, associates with arthritis activity and future requirement of MTX treatment on the diagnosis of RA.     

Noninvasive cross-sectional observation of three-dimensional cell sheet-tissue-fabrication by optical coherence tomography

Cell sheet engineering allows investigators/clinicians to prepare cell-dense three-dimensional (3-D) tissues, and various clinical trials with these fabricated tissues have already been performed for regenerating damaged tissues. Cell sheets are easily manipulated and 3-D tissues can be rapidly fabricated by layering the cell sheets. This study used optical coherence tomography (OCT) to noninvasively analyze the following processes: (1) adhesions between layered cell sheets, and (2) the beating and functional interaction of cardiac cell sheet-tissues for fabricating functional thicker 3-D tissues. The tight adhesions and functional couplings between layered cell sheets could be observed cross-sectionally and in real time. Importantly, the noninvasive and cross-sectional analyses of OCT make possible to fabricate 3-D tissues by confirming the adherence and functional couplings between layered cell sheets. OCT technology would contribute to cell sheet engineering and regenerative medicine.     

Gentisate 1,2-dioxygenase from Xanthobacter polyaromaticivorans 127W

A putative gentisate 1,2-dioxygenase was encoded in the dibenzothiophene degradation gene cluster (dbd) from Xanthobacter polyaromaticivorans 127W. The deduced amino acid sequence showed high sequence similarity with gentisate dioxygenases from Pseudomonas alcaligenes (AAD49427, 65% identical), Bradyrhizobium japonicum (NP_766750, 64%), and P. aeruginosa (ZP_00135722, 54%), and moderate similarity with 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp. KP7 (BAA31235, 33%) and salicylate dioxygenase from Pseudaminobacter salicylatoxidans (AAQ91293, 33%). The enzyme, GDOxp, was heterologously produced in Escherichia coli and purified to homogeneity. GDOxp formed a tetramer and exhibited high dioxygenase activity against 1,4-dihydroxy 2-naphthoate as well as gentisate, suggesting unusually broad substrate specificity. GDOxp easily released ferrous ion under unfavorable temperature and pH conditions to become an inactive monomer protein. An inactive monomer protein can reconstitute a tetramer structure and restore enzyme activity in a cooperative manner upon the addition of ferrous ion. Chymotryptic digestion and protein truncation experiments suggested that the N-terminal region is important for the tetramerization of GDOxp.     

Evidence of local antibody response against Alloiococcus otitidis in the middle ear cavity of children with otitis media

Alloiococcus otitidis is a recently discovered bacterium frequently associated with otitis media. However, no study is available as to whether A. otitidis has a pathogenic role and induces local immune response in the middle ear as a true pathogen. Whole bacterial sonicate of A. otitidis was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane. Then, Western blot analysis was performed with supernatant of the middle ear effusions from children with A. otitidis-positive otitis media. SDS-PAGE of the bacterial sonicate showed several protein bands, designated A1-A11. Western blot analysis revealed the presence of IgG, secretory IgA, IgG2, and IgM against A. otitidis in the middle ear effusions. Absorption of the specimens with sonicates of other major middle ear pathogens did not alter the reactivity of antibodies against the alloiococcal antigens. The results suggest that specific local immune response against A. otitidis is induced during middle ear infection of the organism as a true pathogen. A5, A6 or A11 is expected to be a main antigenic determinant. This is the first report to show evidence of local antibody response against A. otitidis and to disclose antigenic components of A. otitidis.     

Cloning and characterization of an autophagy-related gene, ATG12, from the three-host tick Haemaphysalis longicornis

Ticks are obligate hematophagous ectoparasites with a life cycle characterized by a period of starvation; many ticks spend more than 95% of their life off the host. Autophagy, which is the process of bulk cytoplasmic degradation in eukaryotic cells, is induced by starvation and is essential for extension of the lifespan. Therefore, we hypothesized that autophagy also occurs in ticks; however, there has been no report on autophagy-related (ATG) genes in ticks. Here, we show the homologue of an ATG gene, ATG12, and its expression pattern from the nymphal to adult stages in the three-host tick Haemaphysalis longicornis. The sequence analysis showed that H. longicornis ATG12 (HlATG12) cDNA is 649bp, has a 411bp ORF coding for a 136-amino acid polypeptide with the carboxy-terminal glycine residue, and has a predicted molecular mass of 15.2kDa. Moreover, RT-PCR revealed that HlATG12 was downregulated at the beginning of feeding, upregulated after engorgement, and downregulated again after molting. The expression level of HlATG12 was highest at 3 months after engorgement. By immuno-electron microscopy, it was demonstrated that HlAtg12 was localized to the region around granule-like structures within midgut cells of unfed adults. In conclusion, HlATG12 might function during unfed and molting stages.     

A ROCK inhibitor permits survival of dissociated human embryonic stem cells

Poor survival of human embryonic stem (hES) cells after cell dissociation is an obstacle to research, hindering manipulations such as subcloning. Here we show that application of a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, to hES cells markedly diminishes dissociation-induced apoptosis, increases cloning efficiency (from approximately 1% to approximately 27%) and facilitates subcloning after gene transfer. Furthermore, dissociated hES cells treated with Y-27632 are protected from apoptosis even in serum-free suspension (SFEB) culture and form floating aggregates. We demonstrate that the protective ability of Y-27632 enables SFEB-cultured hES cells to survive and differentiate into Bf1(+) cortical and basal telencephalic progenitors, as do SFEB-cultured mouse ES cells.     

Role of growth factor receptor bound protein 7 in hepatocellular carcinoma

The human growth factor receptor-bound protein 7 (Grb7) is an adaptor molecule and is related to cell invasion. In this present study, we investigated the clinical and biological significance of Grb7 expression in human hepatocellular carcinoma (HCC). We reviewed 64 consecutive patients who had undergone liver resection for HCC, and we investigated the correlation between Grb7 expression and clinical outcome. To analyze the biological behavior of Grb7 in vitro and in vivo, we established Grb7 stable knockdown HCC cells using RNA interference technology. The positive staining of Grb7 protein was correlated with portal venous invasion (P < 0.01), hepatic venous invasion (P < 0.01), and intrahepatic metastasis (P < 0.05). Positive expression of Grb7 was significantly correlated with focal adhesion kinase (FAK) protein levels in HCC (P < 0.01). The Grb7- and FAK-positive group showed a significantly poorer prognosis as compared with the Grb7- and FAK-negative group (P < 0.05). Grb7 knockdown HCC cells exhibited significantly lower levels of invasion potential (P < 0.05) and motility (P < 0.05) than the control cells in vitro; moreover, Grb7 knockdown HCC cells showed delayed onset of the tumors compared with the control cells in vivo. Grb7 expression can modulate the invasive phenotype of HCC. Grb7 plays an important role in HCC progression and is strongly associated with expression of FAK. Grb7 could be a therapeutic target in HCC.     

Local conformational transition of Hydrogenobacter thermophilus cytochrome c552 relevant to its redox potential

In order to elucidate the molecular mechanisms responsible for the apparent nonlinear behavior of the temperature dependence of the redox potential of Hydrogenobacter thermophilus cytochrome c552 [Takahashi, Y., Sasaki, H., Takayama, S. J., Mikami, S., Kawano, S., Mita, H., Sambongi, Y., and Yamamoto, Y. (2006) Biochemistry 45, 11005-11011], its heme active site structure has been characterized using variable-temperature and -pressure NMR techniques. The study revealed a temperature-dependent conformational transition between protein structures, which slightly differ in the conformation of the loop bearing the Fe-bound axial Met residue. The heme environment in the protein structure which arises at lower temperature was found to be more polar, as a result of the altered orientation of the loop with respect to the heme due to its conformational change, than that arising at higher temperature. The present study demonstrated the importance of the structural and dynamic properties of the polypeptide chain in close proximity to the heme for redox regulation of the protein.     

2-Aminoethyl diphenylborinate analogues: selective inhibition for store-operated Ca2+ entry

Capacitative calcium entry (CCE), the mechanism that replenishes the internal Ca2+ stores with Ca2+ from the extracellular milieu in response to depletion of the store, is mediated by Ca2+ channels in the plasma membrane generally referred to as store-operated channels (SOCs). However, the roles of SOCs in the more physiological context have been fully elucidated. 2-Aminoethyl diphenylborinate (2-APB) strongly inhibits SOCs, as well as inositol-1,4,5 trisphosphate (IP3) receptors. In the present study, we screened a library of 166 2-APB analogues for effects on CCE and IP3-induced Ca2+ release in order to discover specific SOC inhibitors, and found that some blocked both store-operated and receptor-operated Ca2+ influx more strongly and selectively than 2-APB. Indeed, these new compounds ceased the prolonged intracellular Ca2+ oscillations induced by a low concentration of ATP in CHO-K1 cells. These novel SOC inhibitors will be valuable pharmacological and biochemical tools for elucidating the physiological roles.