三浦 惠二

Anti-endothelial cell antibodies (AECA) are frequently detected in patients with systemic lupus erythematosus (SLE), but their pathological role remains unclear. We recently developed a solubilized cell surface protein capture enzyme-linked immunosorbent assay (CSPELISA) to detect antibodies against membrane proteins involved in autoimmune reactions. In this study, sera from 51 patients with biopsy-proven lupus nephritis (LN), 25 with SLE without renal involvement (non-LN SLE), 42 disease control (DC) subjects, and 80 healthy control (HC) subjects were tested for IgG-and IgA-AECA for human umbilical vein endothelial cells (HUVEC) and human glomerular EC (HGEC) by using CSP-ELISA. IgG-and IgA-AECA titers were significantly higher in LN and non-LN SLE patients than in the DC or HC (P < 0.001) groups. IgG-and IgA-AECA titers for HUVEC corresponded well with those for HGEC. The IgA-AECA level correlated with the SLE disease activity index and with histological evidence of active lesions (cellular proliferations, hyaline thrombi and wire loops, leukocytic infiltration, and fibrinoid necrosis) in LN patients (P < 0.001). The sensitivity of IgA-AECA as a diagnostic test for histological evidence of active lesions in LN patients was 0.92, with a specificity of 0.70. The significant correlation of IgA-AECA with glomerular hypercellularity indicates that IgA-AECA are associated with endothelial damage in LN.

This article describes a novel method for detecting anti-endothelial cell antibodies (AECAs). Sera from patients with systemic vasculitis or inflammatory conditions have been reported to contain antibodies (Abs) that bind to endothelial cells (EC), i.e., AECAs. AECAs are known to play immunogenic effects by triggering EC activation and vascular damage, but the immunopathological role of AECAs is not clear. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting have previously been used for detecting target antigens of AECAs. However, we assumed that these methods are not appropriate for searching genuine target antigens (Ags) on cell surface, and developed a novel solubilized cell surface protein-capture ELISA (CSP-ELISA). Ags were obtained as cell surface proteins from the plasma membrane of human umbilical vein endothelial cells (HUVECs); these cell surface proteins were biotinylated, solubilized with detergent, and captured on ELISA wells coated with NeutrAvidin (TM) biotin binding protein (NeuAvi). AECA titers in serum from 126 autoimmune disease patients and 122 healthy donors were tested. AECAs were detected in 28 of 36 (78%) of systemic lupus erythematosus (SLE) patients; in 13 of 16(81%) of mixed connective tissue disease (MCTD) patients; and in 5 of 9 (56%) of systemic sclerosis (SSc) patients. Relatively weak denaturation of antigens on ELISA wells caused loss of binding of these autoantibodies (autoAbs). Thus, this newly developed CSP-ELISA method enables the detection of Abs to the labile epitopes of autoantigens (autoAgs) such as membrane proteins, and this method is generally applicable to various kinds of membrane proteins and the Abs against them. We propose CSP-ELISA for measuring AECAs in serum samples for routine laboratory testing. (C) 2012 Elsevier B.V. All rights reserved.

We isolated BAC clones that cover the entire hox gene loci in the medaka fish Oryzias latipes. The BAC clones were characterized by the Southern hybridization with many hox gene probes isolated in our previous study and by PCR using primers designed for selective amplification of respective hox genes. Then, the BAC clones have been subjected to shotgun sequencing. The results revealed the organization of the entire hox gene loci. Forty-six hox genes in total are encoded in seven clusters as follows: 10 hox genes in Aa cluster; 5 in Ab; 9 in Ba; 4 in Bb; 10 in Ca; 6 in Da; and 2 in Db. Together with the information on the hox gene loci registered in the Fugu genome database and in the Danio genome database, the physical maps of three fish genomes were constructed and compared one another. Not only numbers of hox genes but also the distances between the neighboring hox genes are highly similar between medaka and fugu. As for six clusters, Aa, Ab, Ba. Bb, Ca and Da that are commonly present in the three fishes, only few or no differences were found in each cluster. Thus, the hox gene sets should have been well conserved once they had been established in respective species. (c) 2005 Elsevier B.V All rights reserved.

Nucleobindin (Nuc) is a DNA- and calcium-binding protein that was originally identified as an anti-DNA antibody-enhancing factor in MRL/MpJ-lpr/lpr (MRL/lpr) mice. In MRL/lpr mice, both expression of Nuc mRNA in enlarged lymph nodes and serum concentration of Nuc protein are shown to increase as disease progresses. Administration of recombinant (r) Nuc to young MRL/MpJ-+/+ and normal BALB/c mice leads to augmentation or induction of IgG anti-double-stranded (ds) DNA autoantibodies. In this study, spleen cells prepared From MRL/lpr mice were found to show a proliferative response to rNuc, indicating existence of T cells that are specific to this autoantigen in peripheral lymphoid tissues. Furthermore, CD4(+) T cell lines were generated from a BALB/c mouse that had been producing anti-dsDNA antibodies as a result of repeated injections of rNuc. These T cell lines were confirmed to be autoreactive, because they proliferated in culture with syngeneic but not with allogeneic spleen cells without addition of any exogenous antigens. Their proliferation was enhanced by rNuc, and inhibited by an anti-MHC class II monoclonal antibody. Upon in vivo inoculation, these T cells provided help for rNuc-injected BALB/c mice to produce anti-DNA antibodies. These results suggest that Nuc is able to activate autoreactive peripheral T cells through an MHC-class II pathway leading to acceleration or induction of anti-DNA antibody production when it is excessively produced in lupus-prone mice or experimentally administered into normal mice. (C) 2001 Elsevier Science B.V. All rights reserved.

Saccharomyces cerevisiae cho1/pss mutants, which are severely impaired in phosphatidylserine (PS) synthesis, do not have detectable amounts of PS in their lipid fractions. Their derivatives with mutations that cause defects in tryptophan synthesis grew poorly in a medium containing 5 mu g/ml of L-tryptophan, a concentration that met the requirements of tryptophan-auxotrophic CHO1/PSS strains. The rates of tryptophan uptake of trp1 cho1/pss mutants were low at low tryptophan concentrations. This defect in the use of tryptophan was restored either by expression of CHO1/PSS or by introduction of a gene encoding tryptophan transporter, TAT1 or TAT2. These results indicate that PS synthesis is required for the maximal tryptophan-transporting activity of S. cerevisiae at low tryptophan concentrations.

Nucleobindin (Nuc) was originally found to be an enhancement factor of anti-DNA antibody production secreted by a lymphoid cell line derived from a lymphoproliferative MRL/lpr mouse. It has been shown that Nuc has a unique structure containing a DNA- and two calcium-binding domains, and a leucine zipper motif, but its biological roles have not yet been fully elucidated. Expression of Nuc was first studied in human lymphocytes. Expression of Nuc mRNA in normal peripheral blood mononuclear cells was significantly increased upon mitogen stimulation. Anti-human Nuc monoclonal antibody H-1D8 immunoprecipitated Nuc protein in the nuclear extract of Molt-4 cells, Furthermore, in the immunohistochemical staining of tumor specimens from 108 patients with non-Hodgkin's lymphoma (NHL) with H-1D8, H-1D8-positive cells were observed in nearly all cases in varying frequency. According to the Working Formulation, the percentage of cases in which more than 90% of the tumor cells were stained with H-1D8 was 65% in the high grade of the histological malignancy, 54% in the intermediate grade, and 22% in the low grade; however, normal cells surrounding the tumor cells were virtually negative for H-1D8. These results showed that the level of Nuc expression in human lymphocytes reflects the status of activation or proliferation of the cells, thus providing a clue for the further investigation into biological roles of Nuc. In addition, it might be applicable to the clinicopathological estimation of NHL as a novel indicator.

We expressed the extracellular domain (20-408 aa, (T)) of human TSH receptor (TSHR) in E. coli to detect TSHR autoantibodies (TRAb) and, moreover, we expressed the two portions (20-218 aa (5') and 217-408 aa (3')) of the extracellular domain thought to distinguish thyroid stimulating antibodies (TSAb) from blocking antibodies (TSBAb), using pGEX.3X as the expression vector. Using Western blotting analysis of the sera from patients with autoimmune thyroid disease, sera from Graves' patients and patients with idiopathic myxedema who had TSBAb reacted with the fusion protein (T), but none of the control sera reacted with it. We further evaluated whether or not the positive sera for T recognized fusion proteins (5') or (3'). The sera from Graves' patients reacted with both fusion proteins (5') and (3'). The sera from patients with idiopathic myxedema did not react with either of fusion proteins (5') or (3'). These findings suggest that these recombinant TSHR proteins could be used as antigens to detect TRAb, and differentiate TSBAb from patients with idiopathic myxedema. (C) 1997 Elsevier Science B.V.

Nucleobindin (Nuc) was originally identified as a 55 kDa protein that enhanced anti-DNA antibody production in cultures of autoimmune MRL/lpr mouse spleen cells. cDNA cloning and the production of recombinant protein (rNuc) have revealed that rNuc binds to DNA and Ca2+ by means of leucine zipper and EF-hand motif structures. In vitro and in vivo effects of rNuc to induce autoantibodies including anti-dsDNA antibody in normal mice have been demonstrated; however, whether Nuc is involved in the state of autoimmunity occurred in MRL/lpr, C3H/gld and male BXSB mice has not been elucidated. Here we report that the expression of Nuc mRNA is upregulated with age in the lymphatic organs and is positively correlated to the serum levels of Nuc in these lupus-prone strains of mice. Upon immunization with KLH in Freund's complete adjuvant, normal BALB/c mice also showed an elevated level of Nuc in their serum and elevated Nuc mRNA in their lymphatic organs. In addition, in vitro stimulation of BALB/c splenocytes with Con A or LPS remarkably enhanced Nuc mRNA expression. These results suggest that upregulation of Nuc mRNA in lymphatic organs and serum Nuc of lupus-prone mice is related to spontaneous activation of immunocompetent cells; the present data are also consistent with our previous hypothesis on the role of Nuc in the induction or enhancement of autoimmunity in lupus models of mice.

Nucleobindin (Nuc) was first identified as a secreted protein of 55 kDa that promotes production of DNA-specific antibodies in lupus-prone MRL/lpr mice. Analysis of cDNA that encoded Nuc revealed that the protein is composed of a signal peptide, a DNA-binding site, two calcium-binding motifs (EF-hand motifs), and a leucine zipper. In the present study, we analyzed the organization of the human gene for Nuc (NUC). It consists of 13 exons that are distributed in a region of 32 kb. The functional motifs listed above are encoded in corresponding exons. NUC was expressed in all organs examined. Comparison of nucleotide sequences in the promoter regions between human and mouse NUC genes revealed several conserved sequences. Among them, two Sp1- binding sites and a CCAAT box are of particular interest. The promoter is of the TATA-less type, and transcription starts at multiple sites in both the human and the mouse genes. These features suggest that NUC might normally play a role as a housekeeping gene. NUC was located at human chromosome 19q13.2-q13.4.

Long-term administration of low doses (5 mu g) of recombinant nucleobindin (rNuc), which is an MRL/lpr/lpr (MRL/l) mouse-derived DNA-binding protein, induces autoimmunity in both young lupus-prone MRL/+/+ (MRL/n) and normal BALB/c mice. In relation to this autoimmunity, using agarose gel electrophoresis we found an approximately 160 bp mono-nucleosomal DNA (nsDNA) in the sera of 6-week-old normal mice 15 h after i.p. injection of 50 mu g rNuc. Co-injection of rNuc (50 mu g) and anti-CD3 monoclonal antibody (MAb, 50 mu g) further accelerated the appearance of nsDNA in the serum together with DNA fragmentation (apoptosis) in the thymus, which had not been so clearly induced by either double amounts of rNuc or anti-CD3 mAb alone. Acceleration of the appearance of nsDNA in the serum by co-injection was also found in age-matched MRL/n and MRL/l mice, indicating the close association of apoptosis in the thymus with the appearance of nsDNA in the serum. Furthermore, we have detected naturally occurring tri-, di- and mono-nsDNAs from immune complexes (IC) of the sera of 20 similar to 22-week-old MRL/l mice, which indicates that apoptosis in the lymphoid tissues, including the thymus, is the source of serum nsDNA that may trigger or continue production of anti-nuclear antibodies. Evidence that clearance of nsDNA from the circulation is retarded in the presence of rNuc in BALB/c mice may give rationale to the induction of autoimmunity in normal mice by long-term administration of even low doses (5 mu g) of rNuc after all. Taken together, the unusual appearance of natural Nuc in the circulation may help to trigger and/or maintain the autoimmune status in view of providing nuclear constituents into the immune system through the apoptotic process.

Our previous works have shown that nucleobindin (Nuc) or recombinant (r) Nuc not only augments anti-DNA antibody production in vitro but also accelerates autoimmune response in vivo in MRL/ + / + (MRL/n) mice which are the substrain of autoimmune MRL/lpr/lpr (MRL/l) mice. To investigate whether rNuc can induce autoimmune response similarly in naive mice, we carried out intraperitoneal (i.p.) injection of rNuc (5 mu g) without adjuvant into 8-week-old female BALB/c mice and continued injection twice a week for 12 weeks. About 5 weeks after the first injection, all the mice began to show IgG hypergammaglobulinemia (HG) followed by elevation of a number of autoantibodies of the IgG class such as anti-double-stranded (ds) DNA, anti-U1 ribonuclear protein (RNP), anti-ssB(La) and anti-Fc antibodies (RF), but not by anti-Sm antibodies. However, the IgG anti-dsDNA antibody response and histopathological changes in the kidney of these BALB/c mice were not so noticeable as those in MRL/n mice induced by rNuc in our previous experiment. In contrast, the IgG anti-rNuc antibody response of normal BALB/c mice induced by rNuc was stronger than that of MRL/n mice induced by rNuc. Since the titers of each autoantibody of BALB/c mice induced by rNuc were not always associated with the level of IgG HG, and either IgG HG or IgG autoantibodies could not be induced by control administration of extracts (5 mu g) of Escherichia coli with or without harboring plasmid alone, polyclonal B cell activation (PBA) appeared not to be the mechanism of this autoimmunity. With the strong autoantigenicity of rNuc in both normal and MRL/n mice as a momentum, we found naturally occurring autoantibodies against rNuc in the sera of autoimmune MRL/l mice. Since Nuc is present in the sera of autoimmune MRL/l mice ranging from 100 to 600 ng/ml, but under the detectable level in MRL/n mice, Nuc and/or the IgG response against Nuc may play a crucial role in the development of systemic autoimmunity. Taken together, co-factor(s) other than Nuc and/or more amounts of Nuc seemed to be necessary for the development of full-blown systemic autoimmunity in naive mice.

A total of 100 gastric adenocarcinomas, comprising 50 cases with lymph node metastasis and 50 cases without lymph node metastasis, were examined for immunohistochemical reactivity with the monoclonal antibody to urokinase-type plasminogen activator (u-PA), Le(x) related 4C9 antigen, Jun, or to nucleobindin (Nuc), In tumors with lymph node metastasis, 41 (82%) were positive for u-PA and 28 (56%) were positive for Nuc, In tumors without lymph node metastasis, 26 (52%) were positive for u-PA and five (10%) were positive for Nuc, The percentage of cases positive for u-PA or Nuc was significantly higher in tumors with lymph node metastasis than that in tumors without lymph node metastasis (P < 0.01), The expression of u-PA was found to be significantly correlated with that of Nuc (P < 0.001), mode of infiltrative growth (P < 0.05), depth of invasion (P < 0.01), and grade of lymphatic invasion (P < 0.01). However, the expression of Nuc was found to be significantly correlated with the expression of Jun (P < 0.05), depth of invasion (P < 0.01), and grade of lymphatic invasion (P < 0.001), These results suggest that immunohistochemical examination for the expression of u-PA and Nuc in tumor cells may help evaluate the potential of adenocarcinomas of the stomach for lymph node metastasis.

The porifera represent the most primitive phylum of the metazoa. We identified three homeobox-containing genes in the freshwater sponge (Ephydatia fluviatilis). Genomic DNA of the sponge was subjected to amplification by PCR with two primers that corresponded to the helix-1 and helix-3 regions of the homeodomain. Using the amplified products as probes, we isolated two homeobox genes, designated prox1 and prox2. The amino acid sequences of the homeodomains of prox1 and prox2 were 72% and 62% identical to those of the NK-3 and Om(1D) genes of Drosophila, respectively. Screening of a sponge genomic library with degenerate oligonucleotides that corresponded to helix 3 further revealed the presence of one more homeobox gene, prox3. The amino acid sequence of the homeodomain of the prox3 product was 77% identical to that of the msh gene product of human. These results indicate that, when the metazoa appeared during the course of evolution, the multiple and distinct classes of homeobox-containing genes that have been identified in higher organisms already existed.

In a previous paper (Biochem. Biophys. Res. Commun. 187, 375-380, 1992), we reported cloning of a gene for nucleobindin (Nuc) from mouse and human. Nuc was identified as a secreted protein with DNA-binding properties. We noticed that Nuc includes two units of sequence similar to the EF hand motif, a calcium-binding motif, between a region rich in basic amino acids, which is presumably a DNA-binding site, and a leucine zipper which is a dimerization motif. In the present study, we examined the calcium-binding activity of Nuc. Mouse and human recombinant Nuc (rNuc), as well as two mutant rNuc proteins, were synthesized in Escherichia coli. Mouse and human rNuc and one mutant rNuc, in which the region from the second EF hand motif to the C-terminus had been deleted, all had calcium-binding activity. By contrast, another mutant rNuc in which both EF hand motifs had been deleted lacked this activity. Analysis by circular dichroism spectrometry indicated that addition of Ca2+ ions induces a structural change in Nuc, presumably an increase in the amount of alpha helix. However, addition of Ca2+ ions did not seem to cause any change in DNA-binding activity. (C) 1994 Academic Press, Inc.

We previously purified a 55 kDa protein that preferentially expands anti-DNA antibody production both in vitro and in vivo across the H-2 barrier from culture supernatants of KML(1)-7 cells, cloned from a lupus-prone MRL/lpr mouse. By using the purified protein, termed nucleobindin (Nuc), we cloned cDNA and produced recombinant(r) Nuc in Escherichia coli. To elucidate the function of rNuc in vivo, we initially injected intraperitoneally 5 mu g of rNuc without adjuvant into female MRL/n mice at 8 weeks of age and continued injection twice a week. As early as 5 weeks after administration, all mice treated showed an increase in IgG anti-double stranded (ds) DNA antibodies accompanied by IgG hypergammaglobulinemia (HG). Of particular interest was that these mice also produced anti-U1RNP antibodies and rheumatoid factor (RF) of IgG class, but not anti-Sm antibodies. Histopathologically, hypercellularity with occasional crescents in the glomeruli was observed, but evidence for lupus nephritis was lacking, indicating that some factors other than Nuc are necessary for the development of a lupus syndrome observed in MRL/lpr mice. Similar administration of lipopolysaccharide into MRL/n mice failed to induce autoantibodies except for a slight increase in serum IgG, suggesting that these autoimmune responses are not due simply to polyclonal B-cell activation. The presence of rNuc will give us a clue for further understanding of autoimmunity.

A Saccharomyces cerevisiae mutant that lacked phosphatidylserine synthase EC 2.7.8.8] (CDP-1,2-diacyl-sn-glycerol: L-serine O-phosphatidyltransferase) completely was constructed by disrupting its structural gene, CHO1. Over two-thirds of its coding region, from the starting to the 200th codon, was replaced with a LEU2 DNA fragment. This new cho1 mutant showed no detectable synthesis of phosphatidylserine but grew slowly in a medium that contained either ethanolamine or choline. These results indicate that phosphatidylserine synthase and most probably phosphatidylserine are dispensable in S. cerevisiae but necessary for its optimal growth. Additional supplementation with myo-inositol raised the cellular content of phosphatidylinositol and improved the growth of the mutant, suggesting the importance of the negative charges of the membrane surface. The CHO1-disrupted mutant, when grown on choline, accumulated phosphatidylethanolamine to a significant level even after extensive dilution of the initial culture. It segregated prototrophic revertants that could synthesize phosphatidylethanolamine without recovery of phosphatidyl serine synthesis. These results imply the presence of a route(s) for the formation of ethanolamine or its phosphorylated derivative in S. cerevisiae. © 1988 COPYRIGHT 1988 BY THE JOURNAL OF BIOCHEMISTRY.