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OBJECTIVE: To determine whether the clinical and laboratory characteristics of anticentromere antibody (ACA) positive, anti-SSA/Ro antibody (SSA) negative primary Sjogren's syndrome (SS) differ from SSA positive, ACA negative primary SS. METHODS: Twelve patients with ACA positive primary SS (ACA SS) and 19 patients with SSA positive primary SS (SSA SS) were examined. We compared the age, laboratory data, proportion with Raynaud's phenomenon (RP), activity of natural killer cells (NK), titer of antibodies against Epstein-Barr virus, and histological findings of minor labial salivary glands. The presence of anti-chromo antibodies (AChA) was evaluated by immunoblotting of patients' sera. RESULTS: The mean age of the ACA SS group was higher than that of the SSA SS group (p < 0.05). Serum IgG level was lower in ACA SS than in SSA SS (p < 0.0001). Serum IgG level of the ACA SS group with one exception was close to the normal range. Leukocytopenia was less frequently observed in ACA SS than in SSA SS (p < 0.05). RP was seen more frequently in the ACA SS group than the SSA SS group (p < 0.05). NK activity of the ACA SS group was higher than that of the SSA SS group (p < 0.05). Most of the ACA SS patients' NK activity was normal, in contrast to the tendency for NK activity in SS to be low. Virus capsid antigen IgA titer of the ACA SS group was lower than that of the SSA SS group (p < 0.05). Histological findings of minor labial salivary glands of both groups showed a similar severity of lymphocytic infiltrates, destruction of normal structures, and pattern of infiltrating lymphocyte subsets. AChA was positive in 11 of the 12 sera of ACA SS patients. CONCLUSION: The results confirm that ACA positive primary SS differs from SSA positive classic SS in several significant respects.

Centromere protein-C (CENP-C), one of the centromere autoantigens and components of the inner plate of the kinetochore, is suggested to make a dimer at the C-terminus. In order to investigate the presence of conformation-specific anti-centromere antibodies (ACA) to the dimer form, the C-terminal 124 amino acids (CF-124) were expressed in Escherichia coli, affinity purified and chemically cross-linked. Immunoblotting was utilized to compare the reactivities between the dimers and the monomers against 58 ACA(+) sera. The reactivities of the dimers were obviously higher in both IgG and IgM responses. The dimer was still more reactive than the glutathione S-transferase-fused monomer in some sera. Two kinds of CF-124 mutant (each contained one amino acid change at the N-terminal region of CF-124) and two cut segments of CF-124 (67 N-terminal amino acids and 58 C-terminal amino acids) were also examined. The former two mutants decreased the dimerization activity. The latter two mutants lost both activities except for the faint dimerization activity of the N-terminal half. Affinity-purified antibodies with CF-124 in a liquid phase containing the co-purified GroE protein of E. coli, GroEL, reacted to the centromere in culture cells. In conclusion, there are heterogeneous autoepitopes including some conformational epitopes at the C-terminal CENP-C.

We have cloned human dymple, a novel dynamin family member. The full-length cDNA sequence encodes a protein composed of 736 amino acids with a molecular mass of 80 kDa. This amino acid sequence most resembles yeast DNM1P and VPS1P. Dymple lacks a proline-rich carboxyl-terminal domain through which dynamin binds to SH3 domains to be activated. Northern blot analysis revealed two transcript sizes of 2.5 and 4.2 kilobases with alternative polyadenylation at the highest levels in brain, skeletal muscle, and testis. It was further established that there are three patterns of alternative splicing producing in-frame deletions in the coding sequence of dymple in a tissue-specific manner. When overexpressed, wild-type dymple exhibited a punctate perinuclear cytoplasmic pattern, whereas an amino-terminal deletion mutant formed large aggregates bounded by a trans-Golgi network marker. Since dynamin participates in clathrin-mediated endocytosis through a well-characterized mechanism, the existence of a dynamin-like molecule in each specific vesicle transport pathway has been predicted. Our findings suggest that dymple may be the first example of such a subfamily in mammalian cells other than dynamin itself, although its precise role and membrane localization remain to be resolved.

A cDNA encoding a novel member of the mitogen-activated protein kinase kinase (MAPKK) family, MAPKK6, was isolated and found to encode a protein of 334 amino acids, with a calculated molecular mass of 37 kDa that is 79% identical to MKK3. MAPKK6 was shown to phosphorylate and specifically activate the p38/MPK2 subgroup of the mitogen-activated protein kinase superfamily and could be demonstrated to be phosphorylated and activated in vitro by TAK1, a recently identified MAPKK kinase. MKK3 was also shown to be a good substrate for TAK1 in vitro. Furthermore, when co-expressed with TAK1 in cells in culture, both MAPKK6 and MKK3 were strongly activated. In addition, co-expression of TAK1 and p38/MPK2 in cells resulted in activation of p38/MPK2. These results indicate the existence of a novel kinase cascade consisting of TAK1, MAPKK6/MKK3, and p38/MPK2.

We report the interaction between a human centromere antigen and an alphoid DNA, a human centromeric satellite DNA, which consists of 170-bp repeating units. A cloned alphoid DNA fragment incubated with a HeLa cell nuclear extract is selectively immunoprecipitated by the anticentromere sera from scleroderma patients. Immunoprecipitation of the DNA made by primer extension defines the 17-bp segment on the alphoid DNA that is required for formation of DNA-antigen complex. On the other hand, when proteins bound to the biotinylated alphoid DNA carrying the 17-bp motif are recovered by streptavidin agarose and immunoblotted, the 80-kD centromere antigen (CENP-B) is detected. DNA binding experiments for proteins immunoprecipitated with anticentromere serum, separated by gel electrophoresis, and transferred to a membrane strongly suggest that the 80-kD antigen specifically binds to the DNA fragment with the 17-bp motif. The 17-bp motif is termed the "CENP-B box." Alphoid monomers with the CENP-B box are found in all the known alphoid subclasses, with varying frequencies, except the one derived from the Y chromosome so far cloned. These results imply that the interaction of the 80-kD centromere antigen with the CENP-B box in the alphoid repeats may play some crucial role in the formation of specified structure and/or function of human centromere.