Insights into the structures of DNA damaged by hydroxyl radical: crystal structures of DNA duplexes containing 5-formyluracil.

Hydroxyl radicals are potent mutagens that attack DNA to form various base and ribose derivatives. One of the major damaged thymine derivatives is 5-formyluracil (fU), which induces pyrimidine transition during replication. In order to establish the structural basis for such mutagenesis, the crystal structures of two kinds of DNA d(CGCGRATfUCGCG) with R = A/G have been determined by X-ray crystallography. The fU residues form a Watson-Crick-type pair with A and two types of pairs (wobble and reversed wobble) with G, the latter being a new type of base pair between ionized thymine base and guanine base. In silico structural modeling suggests that the DNA polymerase can accept the reversed wobble pair with G, as well as the Watson-Crick pair with A.

Expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic cartilage.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)1, 4, 5, 8, 9 and 15, members of the ADAMTS gene family, have the ability to degrade a major cartilage proteoglycan, aggrecan, at the specific sites, and thus are called 'aggrecanases'. The expression of these ADAMTS species was examined in human osteoarthritic articular cartilage on reverse transcription-polymerase chain reaction. The results demonstrated the predominant expression of ADAMTS4 in osteoarthritic cartilage, while ADAMTS5 was constitutively expressed in osteoarthritic and normal cartilage. ADAMTS9 was expressed mainly in normal cartilage, whereas no or negligible expression of ADAMTS1, 8 and 15 was observed in either osteoarthritic or normal cartilage. In situ hybridization for ADAMTS4 indicated that chondrocytes in osteoarthritic cartilage expressed the mRNA. Two monoclonal antibodies to ADAMTS4 were developed, and immunolocalized ADAMTS4 to chondrocytes in the proteoglycan-depleted zones of osteoarthritic cartilage, showing a direct correlation with the Mankin scores. Immunoblotting indicated a major protein band of 58 kDa in the chondrocyte culture media and osteoarthritic cartilage tissue homogenates. These data demonstrate that among the six ADAMTS species, ADAMTS4 is mainly expressed in an active form in osteoarthritic cartilage, and suggest that ADAMTS4 may play an important role in the degradation of aggrecan in human osteoarthritic cartilage.

Serum levels of matrix metalloproteinase 3 (stromelysin 1) for monitoring synovitis in rheumatoid arthritis.

CONTEXT: Matrix metalloproteinase 3 (MMP-3) is expressed in synovial tissues and involved in cartilage destruction in rheumatoid arthritis and osteoarthritis. OBJECTIVE: To study whether measurement of MMP-3 serum concentrations is useful to monitor the activity of rheumatoid synovitis. DESIGN: Levels of MMP-3 in serum and synovial tissue samples obtained from 29 rheumatoid arthritis patients and 20 osteoarthritis patients were measured by the 1-step sandwich enzyme immunoassay system. RESULTS: Levels of MMP-3 in the serum and synovial samples were significantly higher in rheumatoid arthritis than in osteoarthritis (P < .001), and the levels correlated directly with each other (r = 0.712, P < .001; N = 49). Immunohistochemistry demonstrated almost exclusive localization of MMP-3 to the lining cells in rheumatoid synovium. The immunoreactivity correlated directly with the scores of synovial inflammatory cell infiltration (r = 0.606, P < .001; n = 29) and the MMP-3 levels in the synovial tissues (r = 0.564, P = .001; n = 29) and those in the serum samples (r = 0.529, P = .003; n = 29) in rheumatoid arthritis. Levels of MMP-3 in rheumatoid serum samples dropped to low values at 1 and 2 weeks after total knee arthroplasty, while the levels of C-reactive protein increased at 1 week and the erythrocyte sedimentation rate and counts of white blood cells and platelets were unchanged at 1 and 2 weeks postoperative. CONCLUSIONS: Our results demonstrate that MMP-3 levels in the serum of rheumatoid arthritis patients correlate with the levels produced by the synovial lining cells and suggest that the activity of rheumatoid synovitis can be monitored by measuring serum levels of MMP-3.

Structural significances of pseudouridination and methylation of uridine residues in tRNA: X-ray analyses of DNA dodecamers containing uridine and pseudouridine.

Pseudouridine (psi) and 5-methyluridine (T) residues found in tRNA are prepared through modification of the uridine residues by specific enzymes just after transcription. On the other hands, thymidine residues in DNA are incorporated using dTTP which is derived from UTP before replication. In order to investigate the necessity of such modification and the structural properties of these residues, we have determined two X-ray structures of DNA dodecamers containing dU or d psi, and compared with that containing dT. There is found remarkable difference in arrangement of water molecules hydrogen-bonded to these residues, which form a Watson-Crick type base pair. The psi residue stabilizes the phosphate-backbone conformation of the phosphate group via water mediated hydrogen bond networks, while dT residues seem to prevent from attacking of water molecules.