Human parvovirus B19 (B19) DNA was detected in the synovial tissues in 30 of 39 patients with rheumatoid arthritis (RA), and infrequently in those with osteoarthritis and traumatic joints. On the other hand, the expression of the B19 antigen VP-1 was specific (

Human parvovirus B19 frequently causes acute and chronic arthritis in adults. The molecular mechanism of B19 arthritis, however, remains poorly understood. We previously showed that the transmission of B19 from rheumatoid synoviocytes to monocytic cells is associated with enhanced secretion of tumor necrosis factor alpha (TNF-␣), which triggers inflammation, and interleukin-6. To determine the role of B19 in the production of TNF-␣, we focused on the function of its nonstructural protein, NS1, and established monocytic U937 lines transduced with the NS1 gene under the control of an inducible promoter. Production of TNF-␣ mRNA and protein was elevated in a manner associated with NS1 expression. Reporter assays revealed that AP-1 and AP-2 motifs on the TNF-␣ promoter were responsible for NS1-mediated up-regulation. Electrophoretic mobility shift assay showed specific binding of nuclear proteins from NS1 gene-transduced cells with the AP-1 or AP-2 probe. Antibodies against transcription factors AP-1 and AP-2 and anti-NS1 antibody inhibited the binding of nuclear proteins to the corresponding probes. These data indicate that NS1 up-regulates TNF-␣ transcription via activation of AP-1 and AP-2 in monocytic cells. The molecular mechanisms of NS1-mediated TNF-␣ expression would explain the pathogenesis of B19-associated inflammation.Human parvovirus B19 belongs to the family Parvoviridae and the genus Erythrovirus. The B19 genome includes three major open reading frames coding for the nonstructural protein NS1 in the left half and structural proteins VP1 and VP2 in the right half (44). B19 is the only parvovirus that has been clearly linked with disease in humans. Common manifestations caused by B19 infection include transient aplastic crisis in patients with histories of chronic hemolytic anemia (37), erythema infectiosum (7), nonimmune hydrops fetalis (9), chronic pure red cell aplasia in patients with immunosuppression (24), and arthralgia or arthropathy (59). Acute onset of polyarthritis is common in adults (mostly women) (60). Joint symptoms last for 1 to 3 weeks, although they may persist for months or years. B19 arthritis often meets clinical diagnostic criteria for rheumatoid arthritis and can be erosive (12,19,35,53,59). Because it has been observed that joint symptoms caused by B19 infection coincide with the appearance of specific immunoglobulin G (IgG) and the disappearance of viremia (2) and joint symptoms occur in chronically infected subjects after treatment with immunoglobulin (17), immune complex is thought to cause acute polyarthropathy. However, molecular mechanisms for the involvement of immune complexes in joint inflammation are unclear.Tumor necrosis factor alpha (TNF-␣), a representative proinflammatory cytokine, is an important mediator in the inflammatory process associated with many types of infections and autoimmune diseases through induction of a variety of cytokines, chemokines, and proteases in autocrine and paracrine pathways (16,55). Although various cell types are capable of producing T...

MCM2-7 proteins are essential for eukaryotic DNA replication and are the most likely candidates for the replicative DNA helicase responsible for unwinding DNA at the replication forks [1][2][3]. Consistent with their primary amino acid sequences, a subcomplex of MCM4 ⁄ 6 ⁄ 7 functions as DNA helicase in vitro [4]. It has been suggested that MCM2, -3 and -5 play a regulatory role in the function of MCM4 ⁄ 6 ⁄ 7 DNA helicase, because addition of MCM2 or MCM3 ⁄ 5 to MCM4 ⁄ 6 ⁄ 7 complex resulted in inhibition of the MCM4 ⁄ 6 ⁄ 7 DNA helicase [5,6]. Thus MCM2-7 complex, a major MCM complex on chromatin during the G 1 phase, has to be activated to show DNA helicase activity. It is possible that several proteins, including CDC7 kinase and CDC45 are involved in this activation. Evidence suggests that MCM2-7 proteins may have additional functions during the cell cycle [3]. Cyclin-dependent kinases (CDK), which play a critical MCM4, a subunit of a putative replicative helicase, is phosphorylated during the cell cycle, at least in part by cyclin-dependent kinases (CDK), which play a central role in the regulation of DNA replication. However, detailed characterization of the phosphorylation of MCM4 remains to be performed. We examined the phosphorylation of human MCM4 at Ser3, Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110 using anti-phosphoMCM4 sera. Western blot analysis of HeLa cells indicated that phosphorylation of MCM4 at these seven sites can be classified into two groups: (a) phosphorylation that is greatly enhanced in the G 2 and M phases (Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110), and (b) phosphorylation that is firmly detected during interphase (Ser3). We present data indicating that phosphorylation at Thr7, Thr19, Ser32, Ser88 and Thr110 in the M phase requires CDK1, using a temperature-sensitive mutant of mouse CDK1, and phosphorylation at sites 3 and 32 during interphase requires CDK2, using a dominant-negative mutant of human CDK2. Based on these results and those from in vitro phosphorylation of MCM4 with CDK2 ⁄ cyclin A, we discuss the kinases responsible for MCM4 phosphorylation. Phosphorylated MCM4 detected using anti-phospho sera exhibited different affinities for chromatin. Studies on the nuclear localization of chromatin-bound MCM4 phosphorylated at sites 3 and 32 suggested that they are not generally colocalized with replicating DNA. Unexpectedly, MCM4 phosphorylated at site 32 was enriched in the nucleolus through the cell cycle. These results suggest that phosphorylation of MCM4 has several distinct and site-specific roles in the function of MCM during the mammalian cell cycle.Abbreviations CDK, cyclin-dependent kinases.

Once-daily GL injection added on to OHAs was equally safe and effective compared with twice-daily injection of aspart 70/30 premix replacing SU in type 2 patients insufficiently controlled with OHAs.

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