Reactive oxygen species (ROS) have been implicated in the pathogenesis of temporomandibular disorders. In the present study, we provide the first evidence of ROS generation in the synovial fluid from human temporomandibular disorder patients, as shown by electron spin resonance (ESR) and spin trapping. Three distinct ESR spectra of DMPO spin adducts were observed in the synovial fluid. They corresponded to three free radical species: hydroxyl radical (HO(*)), hydrogen radical (H(*)), and carbon-center radical (R(*)). Among them, the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OH spectrum was the most prominent, suggesting that HO(*) was dominantly generated in the synovial fluid from temporomandibular disorder patients. Desferrioxamine (DFO), an iron chelator, strongly depressed the DMPO-OH signal intensity in the synovial fluid from patients with temporomandibular disorders. We successfully demonstrated ROS-induced oxidative stress in the synovial fluid from temporomandibular disorder patients. ROS generation in the temporomandibular joint could lead to exacerbation of inflammation and activation of cartilage matrix degrading enzymes that proceed to degenerative change of the temporomandibular joint. Thus, iron-dependent generation of HO( *) might have a crucial role in the pathogenesis of temporomandibular disorders.

Poster Sessions C285 to discover CARM1 specific inhibitors and their binding mode. Our last results concerning the development of news selective inhibitors of PRMTs will be also presented. Four-way DNA junctions (Holliday junctions) are key intermediates formed during homologous recombination, which elegantly explain how the exchange of genetic material between two DNA molecules can occur. Their resolution back to two separate duplexes is catalysed by various classes of DNA junction-resolvases, which specifically cleave Holliday junctions, but critically have no nucleolytic activity against double stranded DNA. Junction resolution proceeds via the sequential nicking of the DNA backbone at two positions close to the centre of the junction and productive resolution is ensured by a significant enhancement of the rate of nicking of the second DNA strand relative to the first. Previously, we have solved the crystal structure at 3.2 Å of a complex between a slow cleaving mutant of Endonuclease I (the resolvase from bacteriophage T7) and a synthetic Holliday junction to ascertain the structural basis for junction specificity [1]. The structure reveals the DNA junction to be bound in a highly distorted conformation relative to the free junction, the protein interacts solely with the DNA backbone suggesting that junction recognition is structure rather than sequence specific, and the active site structure suggests that an activated water molecule is positioned for in-line attack with the scissile phosphate. We have recently solved the structure of wild-type EndoI and are using this crystal system to understand the molecular basis of the increased rate of DNA nicking for the second strand by attempting to follow junction cleavage in the crystals and cryotrapping the complex at different stages of the reaction. We have also crystalllised Endonuclease I with a junction pre-nicked in one of the strands mimicking a partially cleaved intermediate. Keywords

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This study investigated whether the presence of Mycoplasma fermentans (M. fermentans) in the temporomandibular joint (TMJ) could be associated with the pathology of temporomandibular joint disorders (TMD). One hundred fifteen synovial fluid (SF) samples from patients with TMD were evaluated for the presence of DNA of M. fermentans by polymerase chain reaction (PCR) assay. Specific antibody against M. fermentans was also detected in the SF as well as sera by Western blot analysis. M. fermentans DNA was identified in 37.4％ of the SF samples from the TMD patients. There was no difference between PCR‐positive and ‐negative rate regarding sex and disease categories, e.g., internal derangement (ID) and osteoarthritis (OA). However, the prevalence of M. fermentans DNA in ID patients was higher in elderly patients (73.3％) than in younger patients(31.8％). Anti‐M. fermentans immunoreactivities (IgG) specific for lipoproteins with various molecular sizes, 56 kilo‐Dalton (kDa), 48 kDa, 38 kDa, and 29 kDa, were also identified in the SF. The immunoreactivity was also detected in the patients’ sera. The reactivity patterns of the anti‐M. fermentans antibodies were, however, different between the SF and the sera ; reactivities to 48 kDa and 29 kDa lipoproteins were prominent in the former, while the reactivities to those of 56 kDa, 48 kDa, and 29 kDa were evidently increased in the latter. The presence of specific DNA and antibody for M. fermentans in the TMJ implies that M. fermentans could possibly induce joint specific immunoreaction, thus perpetuating the inflammatory reaction in the diseased TMJ.