The superoxide-producing NAD(P)H oxidase Nox4 was initially identified as an enzyme that is highly expressed in the kidney and is possibly involved in oxygen sensing and cellular senescence. Although the oxidase is also abundant in vascular endothelial cells, its role remains to be elucidated. Here we show that Nox4 preferentially localizes to the nucleus of human umbilical vein endothelial cells (HUVECs), by immunocytochemistry and immunoelectron microscopy using three kinds of affinity-purified antibodies raised against distinct immunogens from human Nox4. Silencing of Nox4 by RNA interference (RNAi) abrogates nuclear signals given with the antibodies, confirming the nuclear localization of Nox4. The nuclear fraction of HUVECs exhibits an NAD(P)Hdependent superoxide-producing activity in a manner dependent on Nox4, which activity can be enhanced upon cell stimulation with phorbol 12-myristate 13-acetate. This stimulant also facilitates gene expression as estimated in the present transfection assay of HUVECs using a reporter regulated by the Maf-recognition element MARE, a DNA sequence that constitutes a part of oxidative stress response. Both basal and stimulated transcriptional activities are impaired by RNAi-mediated Nox4 silencing. Thus Nox4 appears to produce superoxide in the nucleus of HUVECs, thereby regulating gene expression via a mechanism for oxidative stress response.

The tsBN7 cell line, one of the mutant lines temperature sensitive for growth which have been isolated from the BHK21 cell line, was found to die by apoptosis following a shift to the nonpermissive temperature. The induced apoptosis was inhibited by a protein synthesis inhibitor, cycloheximide, but not by the bcl-2-encoded protein. By DNA-mediated gene transfer, we cloned a cDNA that complements the tsBN7 mutation. It encodes a novel hydrophobic protein, designated DAD1, which is well conserved (100% identical amino acids between humans and hamsters). By comparing the base sequences of the parental BHK21 and tsBN7 DAD1 cDNAs, we found that the DAD1-encoding gene is mutated in tsBN7 cells. The DAD1 protein disappeared in tsBN7 cells following a shift to the nonpermissive temperature, suggesting that loss of the DAD1 protein triggers apoptosis.

Zebrafish have a characteristic horizontal-stripe pigment pattern made by a specific distribution of three types of pigment cells: melanophores, xanthophores, and iridophores. This pattern is a valuable model to investigate how the spatial patterns form during animal development. Although recent findings suggest that the interactions among the pigment cells play a key role, the particular details of these interactions have not yet been clarified. In this report, we performed transmission electron microscopic study to show the distribution, conformation, and how the cells contact with each other in the hypodermis. We found that the pigment cells form complex but ordered, layered structures in both stripe and interstripe regions. The order of the layered structures is kept strictly all through the hypodermal regions. Our study will provide basic information to investigate the mechanism of pigment pattern formation in zebrafish. Developmental Dynamics 227:497-503, 2003.

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The Hakata antigen is a novel, thermolabile ␤ 2 -macroglycoprotein that reacts with sera from patients suffering from systemic lupus erythematosus. In this study we present the structure and the function of the Hakata antigen. We have identified cDNA clones encoding the Hakata antigen and analyzed its function. The cDNA included a possible open reading frame of 897 nucleotides, encoding 299 amino acids. The Hakata antigen consisted of a collagen-like domain in the middle section and a fibrinogen-like domain in the COOH terminus, both of which are homologous to human ficolin-1 and opsonin P35, indicating that these three molecules form a distinct family. The molecular mass of the Hakata antigen expressed in transfected cells was 35 kDa under reduced conditions, and it formed ladder bands under nonreducing conditions compatible with the previous result that the Hakata antigen exists in serum as homopolymers. Purified Hakata antigen sustained lectin activity, showing affinity with GalNAc, GlcNAc, D-fucose as mono/oligosaccharide, and lipopolysaccharides from Salmonella typhimurium and Salmonella minnesota. These results suggest that the Hakata antigen, a new member of the ficolin/opsonin P35 family, plays a role in the serum exerting lectin activity under physiological conditions. Inaba and Okochi (1) reported that sera from patients with systemic lupus erythematosus (SLE) 1 contained an antibody that reacted with normal sera. The antibody was shown to react against a novel thermolabile ␤ 2 -macroglycoprotein, designated the "Hakata antigen" (2). A similar thermolabile substance had been reported by Epstein and Tan (3), but it was not known whether the two proteins are the same. The molecular mass of the Hakata antigen in serum was 650 kDa as determined by gel filtration. The antigen was thermolabile because it lost antigenicity upon heating to 56°C for 1 min. The Hakata antigen was separated as a single band of 35 kDa by SDS-PAGE under reducing conditions. However, under nonreducing conditions it separated as ladder bands from 35 kDa to nearly the top of the gel, suggesting that the Hakata antigen exists in serum as homopolymers consisting of the 35 kDa subunit (2). All sera from 10,050 Japanese healthy blood donors, 99.99% of 751,352 Japanese patients' sera, and 99.98% of 41,430 Swedish patients' sera contained the Hakata antigen (4), thus implying that the Hakata antigen is a normal serum protein. The reference range of the Hakata antigen was 7-23 g/ml (2). The antibody against the Hakata antigen was possessed by 4.3% of 349 SLE patients and 0.3% of 703 patients with other autoimmune diseases (4). Among patients with other autoimmune diseases who possessed the antibody against the Hakata antigen, one patient was found among those with chronic glomerulonephritis and another in the group with primary biliary cirrhosis.In this study, we have cloned and characterized cDNA clones encoding the Hakata antigen revealing that the Hakata antigen is a novel serum protein that has Ca 2ϩ -independent lectin activity. The pri...