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.

No abstract

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...

The temperature‐sensitive mutant cell line tsBN2, was derived from the BHK21 cell line and has a point mutation in the RCC1 gene. In tsBN2 cells, the RCC1 protein disappeared after a shift to the non‐permissive temperature at any time in the cell cycle. From S phase onwards, once RCC1 function was lost at the non‐permissive temperature, p34cdc2 was dephosphorylated and M‐phase specific histone H1 kinase was activated. However, in G1 phase, shifting to the non‐permissive temperature did not activate p34cdc2 histone H1 kinase. The activation of p34cdc2 histone H1 kinase required protein synthesis in addition to the presence of a complex between p34cdc2 and cyclin B. Upon the loss of RCC1 in S phase of tsBN2 cells and the consequent p34cdc2 histone H1 kinase activation, a normal mitotic cycle is induced, including the formation of a mitotic spindle and subsequent reformation of the interphase‐microtubule network. Exit from mitosis was accompanied by the disappearance of cyclin B, and a decrease in p34cdc2 histone H1 kinase activity. The kinetics of p34cdc2 histone H1 kinase activation correlated well with the appearance of premature mitotic cells and was not affected by the presence of a DNA synthesis inhibitor. Thus the normal inhibition of p34cdc2 activation by incompletely replicated DNA is abrogated by the loss of RCC1.

Blockade of the renin-angiotensin system improves the impaired endothelium-dependent relaxations associated with hypertension and aging, partly through amelioration of endothelium-derived hyperpolarizing factor (EDHF)-mediated responses. Although the nature of EDHF is still controversial, recent studies have suggested the involvement of gap junctions in EDHF-mediated responses. Gap junctions consist of connexins (Cx), and we therefore tested whether the expression of Cx in vascular endothelial cells would be altered by hypertension and antihypertensive treatment. Spontaneously hypertensive rats (SHR) were treated with either the angiotensin II type 1 receptor antagonist candesartan or the combination of hydralazine and hydrochlorothiazide for 3 mo from 5 to 8 mo of age. Confocal laser scanning microscopy after immunofluorescent labeling with antibodies against Cx37, Cx40, and Cx43 revealed that the expression of Cx37 and Cx40 in endothelial cells of the mesenteric artery was significantly lower in SHR than in WKY. Treatment with candesartan, but not the combination of hydralazine and hydrochlorothiazide, significantly increased the expression of Cx37 and Cx40, although blood pressure decreased similarly. On the other hand, the expression of Cx43, though scarce and heterogeneous, was increased in SHR compared with WKY, and candesartan treatment lowered the expression of Cx43. These findings suggest that renin-angiotensin system blockade corrects the decreased expression of Cx37 and Cx40 in arterial endothelial cells of hypertensive rats, partly independently of blood pressure, whereas the expression of Cx43 changed in the opposite direction. It remains to be clarified whether these changes in Cx37 and Cx40 are related to endothelial function, particularly that attributable to EDHF. connexin; hypertension; treatment; renin-angiotensin system; mesenteric artery

Ligand-gated chloride channels (LGICs) are important targets for insecticides and parasiticides. Genes encoding subunits of two LGICs, a glutamate-gated chloride channel (MdGluCl-alpha) and a gamma-aminobutyric acid (GABA)-gated chloride channel (MdRdl), were cloned from house-flies (Musca domestica L.). These genes were first expressed independently in Xenopus laevis oocytes by cRNA injection in order to investigate the pharmacology of these ligand-gated channels using two-electrode voltage-clamp electrophysiology. It was found that L-glutamate and GABA activated the MdGluCl-alpha homo-oligomers with an EC(50) value of 30 microM and the MdRdl homo-oligomers with an EC(50) value of 101 microM, respectively. Both channels were chloride ion-permeable, and the MdRdl channel was more sensitive to chloride channel blockers, such as gamma-hexachlorocyclohexane (gamma-HCH), fipronil and picrotoxinin, than the MdGluCl-alpha channel. MdGluCl-alpha required only 1-2 days of incubation after cRNA injection to be expressed in oocytes, whereas 4-7 days of incubation was necessary to achieve MdRdl expression. However, when the cRNA of MdGluCl-alpha was injected at a dose of 1% (w/w) 1 day after the injection of the cRNA of MdRdl, a significant increase in the current amplitude of responses to GABA was observed, and the incubation period necessary for MdRdl expression became shorter. These results suggest that MdGluCl-alpha assists in the expression of MdRdl when the two are coexpressed.