MALT1 Inhibition of Oral Carcinoma Cell Invasion and ERK/MAPK Activation.

The expression of mucosa-associated lymphoid tissue 1 (MALT1) that activates nuclear factor (NF)-κB in lymphocyte lineages is rapidly inactivated in oral carcinoma cells at the invasive front and the patients with worst prognosis. However, its mechanism to accelerate carcinoma progression remains unknown, and this study was carried out to examine the role in invasion. HSC2 oral carcinoma cells stably expressing wild-type MALT1 (wtMALT1) reduced the invasion of basement membrane matrices and collagen gels, and the dominant-negative form (∆MALT1)-expressing cells aggressively invaded into collagen gels. MALT1 decelerated proliferation and migration of cells and downregulated expression of matrix metalloproteinase 2 and 9, which were confirmed by short interfering RNA transfections. Reporter assays and immunoblot analysis showed that MALT1 does not affect the NF-κB pathway but inhibits ERK/MAPK activation. This was confirmed by endogenous MALT1 expression in oral carcinoma cell lines. Orthotopic implantation of ∆MALT1-expressing HSC2 cells in mice grew rapid expansive and invasive tongue tumors in contrast to an absence of tumor formation by wtMALT1-expressing cells. These results demonstrate that MALT1 suppresses oral carcinoma invasion by inhibiting proliferation, migration, and extracellular matrix degradation and that the ERK/MAPK pathway is a target of MALT1 and further suggests a role as a suppressor of carcinoma progression.

BDM (2,3-butanedione monoxime), an inhibitor of myosin-actin interaction, suppresses myofibrillogenesis in skeletal muscle cells in culture.

During the initial phase of myofibrillogenesis in developing muscle cells, the majority of thin filaments lie parallel to, and exhibit correct polarity and spatial position with thick filaments, as in mature myofibrils. Since myosin is known to function as an accelerator of actin polymerization in vitro, it has been postulated that myosin-actin interaction is important in the initial phase of myofibrillogenesis. To clarify further the role of actin-myosin interaction in myofibril formation during development, BDM (2, 3-butanedione 2-monoxime), an inhibitor of myosin ATPase, was applied to primary cultures of skeletal muscle to inhibit myosin activity during myofibrillogenesis, and myofibril formation was examined. When 10 mM BDM was added to the myotubes just after fusion and the cultures were maintained for a further 4 days, cross-striated myofibrils were scarcely observed by fluorescence microscopy when examined by staining with antibodies to actin, myosin, troponin and alpha-actinin, whereas in the control myotubes not exposed to BDM, typical sarcomeric structures were detected. Electron microscopy revealed a disorganized arrangement of myofilaments and incomplete sarcomeric structures in the BDM-treated myotubes. Thus, formation of cross-striated myofibrils was remarkably suppressed in the BDM-treated myotubes. When the myotubes cultured in BDM-containing media were transferred to control media, sarcomeric structures were formed in 2-3 days, suggesting that the inhibitory effect of BDM on myotubes is reversible. These results suggest that actin-myosin interaction plays a critical role in the early process of myofibrillogenesis.

Sexual dimorphism of porcine amelogenins: male-specific amelogenins have strong adsorption properties onto apatite crystals.

The present studies were undertaken to investigate the sexual dimorphism of porcine amelogenins and to gain information as to whether excesses of male amelogenins, if any, possess functional significance in protein-crystal interactions. Enamel proteins, including the intact full-length amelogenins and their degraded polypeptides, were isolated from the secretory enamel of male and female pigs. To identify the amelogenins among the separated pools of male- and female-matrix proteins, rabbit anti-C13 and C25 peptide sera were used, which reacted specifically with the conserved C-terminal domain. Immunoblotting showed that a few extra members of the amelogenins, sharing common epitopes at the C-terminus, were recognized in male products. The apparent yield of the male amelogenins was only marginal, on the basis of their stained intensities on the gel, but the secreted male amelogenins demonstrated selective (probably the strongest among the amelogenins) adsorption properties onto apatite crystals. Reflecting the general symmetric electrophoretic profiles of the male- and female-enamel proteins in toto, there were no sex-linked differences in the protein-crystal interaction and the resulting regulatory function of crystal precipitation.

Generation of functional beta-actinin (CapZ) in an E. coli expression system.

beta-actinin (CapZ) is a heterodimeric actin-binding protein which caps the barbed end of action filaments and nucleates actin-polymerization in a Ca2+ -independent manner. In myofibrils it is localized in the Z-lines. As judged by these properties of b-actinin, it is conceivable that beta-actinin is involved in the regulation of actin assembly, especially in the formation of I-Z-I complex during myofribrillogenesis. In this study, we devised a system to produce functional beta-actinin in E. Coli. The cDNAs of beta I' and beta II subunits of beta-actinin were obtained by RT-PCR methods using the published sequence as references, and subcloned in a pET vector. When the proteins were produced with the cDNA of either beta I' and beta II in E. coli, the proteins were insoluble and non-functional. However, when the cDNAs encoding the two subunits were cloned into a single vector and both proteins were expressed simultaneously, the proteins became soluble and purified as a functional heterodimer The activity of the purified proteins was not distinguishable from that of beta-actinin purified from skeletal muscle.

Organization of connectin/titin filaments in sarcomeres of differentiating chicken skeletal muscle cells.

Very long, elastic connectin/titin molecules position the myosin filaments at the center of a sarcomere by linking them to the Z line. The behavior of the connectin filaments during sarcomere formation in differentiating chicken skeletal muscle cells was observed under a fluorescent microscope using the antibodies to the N terminal (located in the Z line), C terminal (M line), and C zone (myosin filament) regions ofconnectin and was compared to the incorporation of alpha-actinin and myosin into forming sarcomeres. In early stages of differentiating muscle cells, the N terminal region of connectin was incorporated into a stress fiber-like structure (SFLS) together with alpha-actinin to form dots, whereas the C terminal region was diffusely distributed in the cytoplasm. When both the C and N terminal regions formed striations in young myofibrils, the epitope to the C zone of A-band region, that is the center between the A-I junction and the M-line, initially was diffuse in appearance and later formed definite striations. It appears that it took some time for the N and C terminal regions of connectin to form a regular organization in a sarcomere. Thus the two ends of the connectin filaments were first fixed followed by the specific binding of the middle portion onto the myosin filament during sarcomere formation.