Tatsusuke Sato

Novel L-amino acid oxidase with antibacterial activity against methicillin-resistant Staphylococcus aureus isolated from epidermal mucus of the flounder Platichthys stellatus

Fish produce mucus substances as a defensive outer barrier against environmental xenobiotics and predators. Recently, we found a bioactive protein in the mucus layer of the flounder Platichthysu2003stellatus, which showed antibacterial activity against Staphylococcusu2003epidermidis, Staphylococcusu2003aureus and methicillin‐resistant S.u2003aureus. In this study, we isolated and identified the antibacterial protein from the mucus components of P.u2003stellatus using a series of column chromatography steps. We then performed gel electrophoresis and cDNA cloning to characterize the protein. The antibacterial protein in the mucus had a molecular mass of approximately 52u2003kDa with an isoelectric point of 5.3, and cDNA sequencing showed that it corresponded completely with the peptide sequence of antibacterial protein from the gill. A BLAST search suggested that the cDNA encoded an antibacterial protein sharing identity with a number of l‐amino acid oxidases (LAAOs) and possessing several conserved motifs found in flavoproteins. RT‐PCR using a specific primer, and immunohistochemical analysis with anti‐LAAO IgG, demonstrated tissue‐specific expression and localization in the gill. Moreover, the anti‐LAAO IgG was able to neutralize the antibacterial activity of the protein against methicillin‐resistant S.u2003aureus. Thus, we demonstrated that this antibacterial protein, identified from P.u2003stellatus‐derived epidermal mucus, is a novel LAAO‐like protein with antibacterial activity, similar to snake LAAOs.

Invadopodia formation by bladder tumor cells.

A major cause of death in patients with bladder tumors is recurrence with metastasis. Bladder tumor metastasis is largely dependent upon the invasive capacity of tumor cells. Tumor cell invasion is mainly mediated by actin-rich protrusive membrane structures called invadopodia. The formation of invadopodia was observed in various types of invasive tumors such as breast cancer and melanomas. However, invadopodia formation so far has not been described in bladder tumor cells. We here report that human bladder tumor cells form functionally active invadopodia. By using a confocal laser scanning microscope, we demonstrated that invasive bladder tumor cell lines, YTS-1 and T24, with high Matrigel degradation activity form invadopodia but that noninvasive bladder tumor cell lines, RT4 and KK-47, form no detectable invadopodia. Invadopodia formed by YTS-1 cells had the ability to secrete matrix metalloproteases and degrade extracellular matrix to invade surrounding areas. Moreover, we observed that primary tumor cells obtained from patients with invasive bladder tumors also form invadopodia, validating the results from bladder tumor cell lines. Our results provide evidence that invasive human bladder tumor cells form invadopodia for tumor invasion.

Suppression of matrix metalloproteinase-9 by 4-methylumbelliferone

OHK cells, a human lymphoma cell line, are known to produce large amounts of hyaluronan. We investigated the effect of 4‐methylumbelliferone, an inhibitor of hyaluronan synthesis, on the activity of matrix metalloproteinases in OHK cells. Matrix metalloproteinase‐9 was detected on gelatin zymography as the main metalloproteinase excreted into the medium of cultured OHK cells, and 4‐methylumbelliferone added to the medium decreased the activity of the enzyme in a dose‐dependent manner. Addition of Streptomyces hyaluronidase to the medium during cultivation did not decrease the enzyme activity. Reverse transcription‐polymerase chain reaction revealed that 4‐methylumbelliferone markedly decreased the level of mRNA for matrix metalloproteinase‐9 in cultured OHK cells. A similar decrease of the activity of matrix metalloproteinase‐9 by 4‐methylumbelliferone was also observed in cultured human breast and colon carcinoma cells. These results suggest that 4‐methylumbelliferone suppresses the expression of matrix metalloproteinase‐9 in cultured cancer cells.

4-Methylumbelliferone induces the expression of membrane type 1-matrix metalloproteinase in cultured human skin fibroblasts.

Human skin fibroblasts were cultured in the presence of 4-methylumbelliferone, an inhibitor of hyaluronan synthesis. Gelatinolytic activity excreted in the medium was examined by zymography and gelatinase assay using a fluorogenic substrate. 4-Methylumbelliferone added to the medium activated the latent form of matrix metalloproteinase-2 in a dose- and time-dependent manner. Immunoblot analysis also showed the conversion of the latent form of matrix metalloproteinase-2 to its active form. This activation was observed even when the cells were cultured with both 4-methylumbelliferone and hyaluronan. Addition of Streptomyces hyaluronidase to the medium during cultivation did not activate the latent form of matrix metalloproteinase-2. Reverse transcription-polymerase chain reaction revealed that 4-methylumbelliferone markedly increased the level of mRNA for membrane type 1-matrix metalloproteinase, whereas levels of mRNA for matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 were little affected. These results suggest that 4-methylumbelliferone induces the expression of membrane type 1-matrix metalloproteinase, resulting in activation of matrix metalloproteinase-2, in cultured human skin fibroblasts.

Hepatoid carcinoma of the skin: spontaneous rat skin hepatoid carcinoma with eosinophilic globules and crystals immunoreactive to α-1-antitrypsin

We present a case of hepatoid carcinoma of the abdominal skin in a male Wistar rat. Histopathologically, this carcinoma resembled human hepatocellular carcinoma with respect to trabecular-sinusoidal structures. Carcinoma tissues contain numerous eosinophilic globules and crystals, and in this case, we found the characteristic eosinophilic globules in the hepatoid carcinoma cells and the crystals in the extracellular portions. Vivid carcinoma cells full of eosinophilic globules were present near the necrotic areas in tumor tissue, wherein quadrate crystals unstained with eosin were observed. PAS staining after diastase digestion revealed that the globules were PAS positive and diastase resistant. In addition, we found that the hepatoid carcinoma cells were immunoreactive for α-1-antitrypsin (anti-A1AT) antibody with the globules and crystals staining peripherally, and a central unstained region. Ultrastructural study of intracytoplasmic globules and extracellular crystals revealed that the fringe of each globule and crystal had no limiting membrane and showed the same level of electron density. These findings suggest that the characteristic crystals in this tumor may have originated from the globules that were emitted from the carcinoma cells after their death as a result of saturation with intracytoplasmic globules.