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Naokazu Inoue

Publications and source records attributed to Naokazu Inoue.

10 recordsLinked to original sources
PubMed2005

Wild-type measles virus infection in human CD46/CD150-transgenic mice: CD11c-positive dendritic cells establish systemic viral infection.

We generated transgenic (TG) mice that constitutively express human CD46 (huCD46) and/or TLR-inducible CD150 (huCD150), which serve as receptors for measles virus (MV). These mice were used to study the spreading and pathogenicity of GFP-expressing or intact laboratory-adapted Edmonston and wild-type Ichinose (IC) strains of MV. Irrespective of the route of administration, neither type of MV was pathogenic to these TG mice. However, in ex vivo, limited replication of IC was observed in the spleen lymphocytes from huCD46/huCD150 TG and huCD150 TG, but not in huCD46 TG and non-TG mice. In huCD150-positive TG mouse cells, CD11c-positive bone marrow-derived myeloid dendritic cells (mDC) participated in MV-mediated type I IFN induction. The level and induction profile of IFN-beta was higher in mDC than the profile of IFN-alpha. Wild-type IC induced markedly high levels of IFN-beta compared with Edmonston in mDC, as opposed to human dendritic cells. We then generated huCD46/huCD150 TG mice with type I IFN receptor (IFNAR1)-/- mice. MV-bearing mDCs spreading to draining lymph nodes were clearly observed in these triple mutant mice in vivo by i.p. MV injection. Infectious lymph nodes were also detected in the double TG mice into which MV-infected CD11c-positive mDCs were i.v. transferred. This finding suggests that in the double TG mouse model mDCs once infected facilitate systemic MV spreading and infection, which depend on mDC MV permissiveness determined by the level of type I IFN generated via IFNAR1. Although these results may not simply reflect human MV infection, the huCD150/huCD46 TG mice may serve as a useful model for the analysis of MV-dependent modulation of mDC response.

Animals↗
PubMed2005

The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs.

Representing the 60 trillion cells that build a human body, a sperm and an egg meet, recognize each other, and fuse to form a new generation of life. The factors involved in this important membrane fusion event, fertilization, have been sought for a long time. Recently, CD9 on the egg membrane was found to be essential for fusion, but sperm-related fusion factors remain unknown. Here, by using a fusion-inhibiting monoclonal antibody and gene cloning, we identify a mouse sperm fusion-related antigen and show that the antigen is a novel immunoglobulin superfamily protein. We have termed the gene Izumo and produced a gene-disrupted mouse line. Izumo-/- mice were healthy but males were sterile. They produced normal-looking sperm that bound to and penetrated the zona pellucida but were incapable of fusing with eggs. Human sperm also contain Izumo and addition of the antibody against human Izumo left the sperm unable to fuse with zona-free hamster eggs.

Amino Acid Sequence↗
PubMed2004

Lineage-specific cell disruption in living mice by Cre-mediated expression of diphtheria toxin A chain.

We have established a transgenic mouse line in which floxed neomycin resistant cassette was inserted between the CAG promoter and EGFP. When these transgenic mice were mated with Cre-expressing transgenic animals, the offspring obtained were fluorescent green. We then established a transgenic mouse line in which EGFP in the above construct was replaced by diphtheria toxin A chain (DT). When the latter transgenic mice were mated with mice expressing Cre restricted to germ cells, we obtained healthy but sterile offspring due to a disruption of germ line cells by DT expression. We predict that this strategy will be useful for the construction of new animal models for human diseases, featuring a variety of missing cell lineages produced by disruption with DT.

Animals↗
PubMed2004

A short consensus repeat-containing complement regulatory protein of lamprey that participates in cleavage of lamprey complement 3.

The prototype of the short consensus repeat (SCR)-containing C regulatory protein is of interest in view of its evolutionary significance with regard to the origin of the C regulatory system. Lamprey is an agnathan fish that belongs to the lowest class of vertebrates. Because it does not possess lymphocytes, it lacks Ig and consequently the classical C pathway. We identified an SCR-containing C regulatory protein from the lamprey. The primary structure predicted from the cDNA sequence showed that this is a secretary protein consisting of eight SCRs. This framework is similar to the alpha-chain of C4b-binding protein (C4bp). SCR2 and -3 of human C4bp are essential for C4b inactivation, and this region is fairly well conserved in the lamprey protein. However, the other SCRs of this protein are similar to those of other human C regulatory proteins. The lamprey protein binds to the previously reported lamprey C3b/C3bi deposited on yeast and cleaves lamprey C3b-like C3 together with a putative serum protease. The scheme resembles the C regulatory system of mammals, where factor I and its cofactor inactivate C3b. Unlike human cofactors, the lamprey protein requires divalent cations for C3b-like C3 cleavage. Its artificial membrane-anchored form protects host cells from lamprey C attack via the lectin pathway. Thus, the target of this protein appears to be C3b and/or its family. We named this protein Lacrep, the lamprey C regulatory protein. Lacrep is a member of SCR-containing C regulators, the first of its kind identified in the lowest vertebrates.

Amino Acid Sequence↗
PubMed2003

Disruption of mouse CD46 causes an accelerated spontaneous acrosome reaction in sperm.

Human membrane cofactor protein (MCP, CD46) is a ubiquitously expressed protein known to protect cells from complement attack. Interestingly, when we examined the expression of mouse CD46, which we recently cloned, the message was found only in testis and the protein was found on the inner acrosomal membrane of sperm. In order to elucidate the function of CD46, we produced mice carrying a null mutation in the CD46 gene by using homologous recombination. Despite the absence of CD46, the mice were healthy and both sexes were fertile. However, to our surprise, the fertilizing ability of males appeared to be facilitated by disruption of the CD46 gene, as the average number of pups born from CD46(-/-) males was significantly greater than that of wild-type males. It was also revealed that the incidence of the spontaneous acrosome reaction doubled in CD46(-/-) sperm compared to that in wild-type sperm. It was assumed that this increase caused the heightened fertilizing ability found in CD46(-/-) sperm. These data suggest that CD46 may have some role in regulating sperm acrosome reaction.

Acrosome↗
PubMed2002

Characterization of mineral deposits formed in cultures of a hamster tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell line (CCP).

We have established tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell lines (CCP-2, CCP-7, CCP-8) from hamster bone marrow. Accumulation of mineral deposits was observed on the dishes when the clones were cultured in McCoy's 5A medium supplemented with 20% fetal calf serum. The materials were dissolved in 0.05 N HCl, and proteins found in the acid extracts were identified by N-terminal amino acid sequencing. The major components were bovine fetuin and prothrombin precursor. In addition, several cell-derived proteins, such as high mobility group 1 protein (HMG1), secretory leukocyte protease inhibitor (SLPI) and EPV20, a 2.0-kDa milk glycoprotein, were identified. HMG1 was detected, by immunostaining, on the cell surface of all the CCP clones. Metabolically labeled cellular sphingomyelin, sialyllactosylceramide, and proteoglycans were also found in the mineral deposits. Reverse transcription/polymerase chain reaction of CCP-2 mRNA revealed that the cells synthesized alkaline phosphatase, bone sialo protein, and osteonectin, but not matrix Gla protein, osteopontin, and type I collagen. CCP-2 cells formed tumors when injected subcutaneously into nude mice. In the tumor tissue, Alizarin-red-positive nodules surrounded by TRAP- and ALP-positive cells were observed, indicating CCP-2 cells can also induce calcification in vivo.

3T3 Cells↗
PubMed2002

Mechanism of host cell protection from complement in murine cytomegalovirus (CMV) infection: identification of a CMV-responsive element in the CD46 promoter region.

Mouse cells ubiquitously express CRRY, which is a functional orthologue of human decay-accelerating factor (DAF; CD55) and membrane cofactor protein (MCP; CD46), and thus protects cells from homologous complement. NIH3T3 cells expressed minute levels of mouse CD46 (mCD46) mRNA but barely produced mCD46 protein. mCD46 message and protein levels were markedly increased during mouse cytomegalovirus (mCMV) infection. Consistently, mCD46-expressing cells became resistant to mouse complement; primary-cultured fibroblasts from mCD46 gene-disrupted mice showed no increase in protection, resulting in complement-dependent cytolysis. Thus, the marked up-regulation of mCD46 in mouse fibroblast cells/cell lines by mCMV infection participates in host cell protection from complement. By mCD46 promoter deletion assay, the region necessary for induction of the promoter activity by mCMV infection was shown to be restricted to a sequence of 19 bp, which was homologous to the corresponding portion in human CD46, and the promoter regions of early-inducible human CMV UL36 and human herpesvirus 6 UL29. The results were confirmed by mutation analysis of this 19-bp region. We designated this sequence as the CMV-responsive element (CMVRE). Electrophoretic mobility shift assay demonstrated the existence of a CMVRE-binding factor, expression of which was significantly increased after mCMV infection. Thus, mCMV up-regulates the gene expression of mCD46 via CMVRE and CMVRE-binding factor, resulting in mCD46 protein expression on mCMV-infected cells. Since both the membrane and soluble mCD46retained complement regulatory activity, mCD46 induced by mCMV infection may act as a regulator of systemic complement activation. This represents a unique strategy of mCMV survival in host cells with sufficient replication by circumventing host complement attack.

3T3 Cells↗
PubMed2002

Cloning of hamster type XVII collagen cDNA, and pathogenesis of anti-type XVII collagen antibody and complement in hamster bullous pemphigoid.

Bullous pemphigoid is an inflammatory subepidermal blistering skin disease associated with an IgG autoimmune response to the type XVII collagen. The immunopathologic features of bullous pemphigoid can be reproduced in mice by the passive transfer of anti-type XVII collagen antibodies. In this model, it is thought that blister formation depends upon complement activation, neutrophil recruitment, and some proteolytic enzymes. In this study, we cloned hamster type XVII collagen cDNA, which contains a 4296 bp coding region and which is predicted to be a transmembrane protein with an extracellular collagenous domain, residing in type II orientation. Antipeptide antibodies (anti-1191 IgG) were obtained against a segment of hamster type XVII collagen homologous with the human type XVII collagen autoantibody-reactive site. The antipeptide antibodies were passively transferred to neonatal Syrian hamsters. The injected hamsters developed a microscopic subepidermal blister as seen previously in the mice. In order to test whether antigen-antibody complexes and complement initiate the subepidermal blister formation, we carried out experiments in vitro on condition that inflammatory cells were completely eliminated. Complement activation in sera was inhibited either by heating (at 56 degrees C for 30 min) or by preincubating with cobra venom factor. When the hamster skin was incubated with fresh anti-1191 antisera, separation of dermal-epidermal junction was observed. The anti-1191 IgG failed to induce C3 deposition and dermal-epidermal junction separation, however, if the anti-1191 IgG was added alone or complement activation in sera was inhibited. Under these conditions, IgG but not C3 was deposited on the basement membrane. These results strongly suggest that antigen-antibody complexes and complement initiate dermal-epidermal junction separation.

Amino Acid Sequence↗
PubMedDate not supplied

Inhibition of murine osteosarcoma cell proliferation by glucocorticoid.

The effects of glucocorticoid (GC) on the proliferation of Dunn Osteosarcoma (OS) cells were examined under in vitro culture conditions. Dexamethasone (Dex) inhibited the proliferation of Dunn OS cells in a dose-dependent manner, while the addition of anti-GC, RU486, to the culture medium in part recovered Dex-induced growth inhibition. The number of maximum binding sites (Bmax) and the dissociation constant (Kd) value of glucocorticoid receptor (GR) in Dunn OS cells were 19,560 sites/cell and 5.2 +/- 0.8 nM, respectively. RU486 competed with labeled Dex against GR at a concentration of 10(-6) M. Western blot analysis of [3H]Dex-mesylate-labeled cell homogenate and immunohistochemical staining against GR further confirmed the presence of GR. Dex treatment of Dunn OS cells resulted in apoptosis with the characteristic internucleosomal DNA cleavage shown by the DNA ladder pattern in agarose gel electrophoresis. These data demonstrate that GC inhibits the proliferation of Dunn OS cells via GR, for which one possible mechanism in vitro is induction of apoptosis.

Animals↗