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Tetsuya Mizutani

Publications and source records attributed to Tetsuya Mizutani.

At least 19 recordsLinked to original sources
PubMed2025

Multivariate Effects of SNPs on Environmental Streptococcal Mastitis Evaluated With an NGS-Based Association Study Using Targeted Resequencing in the Bovine MHC Region.

Mastitis is an inflammatory reaction caused by bacterial infection of the teat, and a relationship between its onset and cattle major histocompatibility complex (BoLA) region has been reported. However, no comprehensive genetic analysis of mastitis caused by environmental streptococci has been reported. Here, we resequenced the BoLA region using a hybridisation capture target next-generation sequencing (NGS) method to identify disease susceptibility markers mapped to the BoLA region in environmental streptococcal mastitis. This study examined 75 cows with mastitis caused by environmental streptococci selected from 1641 cows with mastitis and 222 healthy cows without mastitis in Japan. Targeted sequences obtained from MiSeq NGS were aligned to the bovine reference genome (ARS-UCD1.2/bosTau9), and 2,920,355 variants were detected within the BoLA region of the 297 Holstein cattle. In an association study using 2264 variants after quality control, the top 20 variants with the lowest P values were selected and assigned to the 18 surrounding candidate genes, and a gene network analysis of these genes resulted in the narrowing down of five candidate genes POU5F1, IER3, GNL1, ABCF1, and PRR3. Multivariate effect analysis of all 6 SNPs associated with these 5 genes revealed that they were significantly correlated with mastitis, indicating that they were useful for classification of mastitis-resistant and mastitis-susceptible cattle. This is the first report to identify SNPs associated with environmental streptococcal mastitis with an NGS-based association study using targeted resequencing in the BoLA region, and understanding host factors may provide important clues for mastitis control.

Animals↗
PubMed2006

Co-activator p120 is increased by gonadotropins in the rat ovary and enhances progesterone receptor activity.

BACKGROUND: Ovarian follicular development is primarily dependent on pituitary gonadotropins. Identification of gonadotropin-inducible genes in the ovary is one of the effective approaches for the study of follicular development. In this study we identify rat homologue of p120, a nuclear transcription co-activator, as one of the FSH inducible genes in the rat granulosa cells. METHODS: A full-length cDNA encoding rat p120 was cloned, and expression of the gene in the ovary was examined by Northern blotting. Tissue localization of p120 was examined by in situ hybridization. Cellular functions of p120 were studied by co-transfection of rat p120 gene together with estrogen receptor (ER)-alpha, ER-beta, androgen receptor (AR), or progesterone receptor (PR) genes. RESULTS: A full-length cDNA encoding rat p120 was characterized as a protein with 957 amino acid residues. Rat p120 was expressed ubiquitously, but strongly in the ovary and the testis. Expression of p120 mRNA was also induced in vivo by PMSG or PMSG/hCG treatment. Strong expression of p120 mRNA was observed in the granulosa cells of pre-ovulatory large antral follicles. Progesterone receptor was co-localized with p120 in the large antral follicles. Co-transfection experiments revealed that rat p120 activated AR, ER-alpha, ER-beta, and PR in the presence of their respective ligands. CONCLUSION: These observations suggest that rat p120 is strongly induced in the ovarian granulosa cells, and may work together with PR in the granulosa cells of ovulatory follicles to promote the ovulation process.

Animals↗
PubMed2006

Granulosa cells promote differentiation of cortical stromal cells into theca cells in the bovine ovary.

Formation of a theca cell (TC) layer is an important physiologic event that occurs during early follicular development. Nevertheless, little is known concerning the nature and regulation of the formation of the TC layer during follicular growth. Using an established coculture system in this study, we examined the hypothesis that stromal cells differentiate into TCs during early follicular development and that this process involves interaction with granulosa cells (GCs). Ovarian stromal cells from the bovine ovarian cortex (S(C)) and medulla (S(M)) were cultured with or without GCs from small antral follicles. The presence of GCs increased the number of lipid droplets and mitochondria, and it stimulated androstenedione production in S(C) and S(M). However, luteinizing hormone/choriogonadotropin receptor (LHCGR) mRNA abundance and hCG-induced cAMP and androstenedione production were increased in S(C) but not in S(M) by the presence of GCs. The present results indicate that GCs are involved in the functional differentiation and the acquisition of LH responsiveness in stromal cells of the ovarian cortex. We suggest that GC-S(C) interaction is important in the formation of the TC layer during early follicular development, although the nature of this interaction remains to be determined.

Androstenedione↗
PubMed2006

Mechanisms of establishment of persistent SARS-CoV-infected cells.

Previously, we reported the establishment of cells with persistent SARS-CoV infection after apoptotic events and showed that both JNK and PI3K/Akt signaling pathways are important for persistence by treatment with inhibitors at the early stages of SARS-CoV infection. However, the mechanisms of establishment of persistent infection are still unclear. In this study, we investigated which signaling pathways play important roles in escape from apoptosis in cells infected with SARS-CoV. In persistently infected cells at 50h.p.i., PI3K/Akt, JNK, p38 MAPK and Bcl-2 were phosphorylated and the protein levels of Bcl-2 and Bcl-xL were increased. When surviving cells were treated with the JNK-specific inhibitor, SP600125, at 50h.p.i., all cells died, suggesting that the JNK signaling pathway is necessary for maintenance of persistently infected cells. Among the signaling pathways in persistently infected cells, Akt and JNK were phosphorylated in SARS-CoV-nucleocapsid (N) protein-expressing Vero E6 cells using vaccinia viral vector (DIs), strongly suggesting that N protein-induced phosphorylation of Akt and JNK are necessary to establish persistence. These results indicated that at least four proteins, Akt, JNK, Bcl-2 and Bcl-xL, are necessary for survival of persistently SARS-CoV-infected cells.

Animals↗
PubMed2006

Differentiation of adult stem cells derived from bone marrow stroma into Leydig or adrenocortical cells.

Adult stem cells from bone marrow, referred to as mesenchymal stem cells or marrow stromal cells (MSCs), are defined as pluripotent cells and have the ability to differentiate into multiple mesodermal cells. In this study, we investigated whether MSCs from rat, mouse, and human are able to differentiate into steroidogenic cells. When transplanted into immature rat testes, adherent marrow-derived cells (including MSCs) were found to be engrafted and differentiate into steroidogenic cells that were indistinguishable from Leydig cells. Isolated murine MSCs transfected with green fluorescence protein driven by the promoter of P450 side-chain cleaving enzyme gene (CYP11A), a steroidogenic cell-specific gene, were used to detect steroidogenic cell production in vitro. During in vitro differentiation, green fluorescence protein-positive cells, which had characteristics similar to those of Leydig cells, were found. Stable transfection of murine MSCs with a transcription factor, steroidogenic factor-1, followed by treatment with cAMP almost recapitulated the properties of Leydig cells, including the production of testosterone. Transfection of human MSCs with steroidogenic factor-1 also led to their conversion to steroidogenic cells, but they appeared to be glucocorticoid- rather than testosterone-producing cells. These results indicate that MSCs represent a useful source of stem cells for producing steroidogenic cells that may provide basis for their use in cell and gene therapy.

3-Hydroxysteroid Dehydrogenases↗
PubMed2006

Induction of protective immunity against severe acute respiratory syndrome coronavirus (SARS-CoV) infection using highly attenuated recombinant vaccinia virus DIs.

SARS-coronavirus (SARS-CoV) has recently been identified as the causative agent of SARS. We constructed a series of recombinant DIs (rDIs), a highly attenuated vaccinia strain, expressing a gene encoding four structural proteins (E, M, N and S) of SARS-CoV individually or simultaneously. These rDIs elicited SARS-CoV-specific serum IgG antibody and T-cell responses in vaccinated mice following intranasal or subcutaneous administration. Mice that were subcutaneously vaccinated with rDIs expressing S protein with or without other structural proteins induced a high level of serum neutralizing IgG antibodies and demonstrated marked protective immunity against SARS-CoV challenge in the absence of a mucosal IgA response. These results indicate that the potent immune response elicited by subcutaneous injection of rDIs containing S is able to control mucosal infection by SARS-CoV. Thus, replication-deficient DIs constructs hold promise for the development of a safe and potent SARS vaccine.

Animals↗
PubMed2006

Development of an enzyme-linked immunosorbent assay for serological diagnosis of tick-borne encephalitis using subviral particles.

The similarity of symptoms produced by tick-borne encephalitis (TBE) and Japanese encephalitis (JE) and the high degree of cross-reactivity between TBE and JE viruses by serological tests make the development of a differential diagnostic test a priority. In this study, recombinant prM/E proteins of TBE virus strain Oshima 5-10 expressed in mammalian cells resulted in the release of subviral particles (SPs) into the culture medium. Using the SPs as antigens, enzyme-linked immunosorbent assay (ELISA) systems were developed to detect TBE virus-specific IgM and IgG antibodies, designated SP-IgG and SP-IgM ELISAs, respectively. Of 83 serum samples from encephalitis patients in Khabarovsk, Russia, which were positive with the neutralization test (NT), 82 were positive by the SP-IgG ELISA, for a sensitivity of 98.8%, which was higher than that of a commercial ELISA kit. All 12 NT-negative samples were also negative by the SP-IgG ELISA (specificity, 100%). Of 17 patient samples that were NT-positive, 16 (94.1%) were positive by the SP-IgM ELISA. Of 15 paired serum samples that yielded equivocal results by NT, 11 had positive results with the SP-IgM ELISA, indicating a diagnosis of TBE infection. The SP-IgG and SP-IgM ELISAs showed no cross-reactivity with antibodies to the JE virus. The results indicate that these ELISAs will be useful for the detection of TBE-specific antibodies.

Animals↗
PubMed2006

Regulation of p90RSK phosphorylation by SARS-CoV infection in Vero E6 cells.

The 90 kDa ribosomal S6 kinases (p90RSKs) are a family of broadly expressed serine/threonine kinases with two kinase domains activated by extracellular signal-regulated protein kinase in response to many growth factors. Our recent study demonstrated that severe acute respiratory syndrome (SARS)-coronavirus (CoV) infection of monkey kidney Vero E6 cells induces phosphorylation and dephosphorylation of signaling pathways, resulting in apoptosis. In the present study, we investigated the phosphorylation status of p90RSK, which is a well-known substrate of these signaling pathways, in SARS-CoV-infected cells. Vero E6 mainly expressed p90RSK1 and showed weak expression of p90RSK2. In the absence of viral infection, Ser221 in the N-terminal kinase domain was phosphorylated constitutively, whereas both Thr573 in the C-terminal kinase domain and Ser380 between the two kinase domains were not phosphorylated in confluent cells. Ser380, which has been reported to be involved in autophosphorylation by activation of the C-terminal kinase domain, was phosphorylated in confluent SARS-CoV-infected cells, and this phosphorylation was inhibited by , which is an inhibitor of p38 mitogen-activated protein kinases (MAPK). Phosphorylation of Thr573 was not upregulated in SARS-CoV-infected cells. Thus, in virus-infected cells, phosphorylation of Thr573 was not necessary to induce phosphorylation of Ser380. On the other hand, Both Thr573 and Ser380 were phosphorylated by treatment with epidermal growth factor (EGF) in the absence of p38 MAPK activation. Ser220 was constitutively phosphorylated despite infection. These results indicated that phosphorylation status of p90RSK by SARS-CoV infection is different from that by stimulation of EGF. This is the first detailed report regarding regulation of p90RSK phosphorylation by virus infection.

Animals↗
PubMed2006

Evaluation of a novel vesicular stomatitis virus pseudotype-based assay for detection of neutralizing antibody responses to SARS-CoV.

Severe acute respiratory syndrome (SARS)-coronavirus (SARS-CoV) is the causative agent of SARS. The S protein of SARS-CoV is a major target for neutralizing antibodies (Nabs) in infected patients. We developed a neutralization assay using a recombinant vesicular stomatitis virus (VSV) bearing SARS-CoV-S protein (VSV-SARS-St19). A total of 56 serum samples collected from 22 healthcare workers in the Hanoi French Hospital during the SARS epidemic in 2003 were evaluated and compared to the conventional neutralizing assay using infectious SARS-CoV. The results of the neutralization assay using VSV-SARS-St19 pseudotype showed good correlations with those using infectious SARS-CoV. The newly developed neutralization assay was more sensitive to low antibody titers in serum samples. Thus, the VSV-SARS-St19 is a useful tool for detecting Nabs against SARS-CoV.

Animals↗
PubMed2006

Effects of ovarian theca cells on granulosa cell differentiation during gonadotropin-independent follicular growth in cattle.

We investigated the effects of theca cells or FSH on granulosa cell differentiation and steroid production during bovine early follicular growth, using a co-culture system in which granulosa and theca cells were cultured on opposite sides of a collagen membrane. Follicular cells were isolated from early antral follicles (2-4 mm) that were assumed to be in gonadotropin-independent phase and just before recruitment into a follicular wave. Granulosa cells were cultured under serum-free conditions with and without theca cells or recombinant human FSH to test their effects on granulosa cell differentiation. Messenger RNA levels for P450 aromatase (aromatase), P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), LH receptor (LHr), and steroidogenic acute regulatory protein (StAR) in granulosa cells were measured by real-time quantitative RT-PCR analysis. FSH enhanced aromatase mRNA expression in granulosa cells, but did not alter estradiol production. FSH also enhanced mRNA expression for P450scc, LHr, and StAR in granulosa cells, resulting in an increase in progesterone production. In contrast, theca cells enhanced aromatase mRNA expression in granulosa cells resulting in an increase in estradiol production. Theca cells did not alter progesterone production and mRNA expression in granulosa cells for P450scc, 3beta-HSD, LHr, and StAR. The results of the present study indicate that theca cells are involved in both rate-limiting steps in estrogen production, i.e., androgen substrate production and aromatase regulation, and that theca cell-derived factors regulate estradiol and progesterone production in a way that reflects steroidogenesis during the follicular phase of the estrous cycle.

3-Hydroxysteroid Dehydrogenases↗
PubMed2006

Inhibition of cell proliferation by SARS-CoV infection in Vero E6 cells.

Severe acute respiratory syndrome (SARS) is caused by SARS-coronavirus (SARS-CoV). Infection of Vero E6 cells with SARS-CoV inhibits cell proliferation. Our previous study indicated that Akt, which is poorly phosphorylated in confluent cultures of Vero E6 cells, is phosphorylated and then dephosphorylated upon infection by SARS-CoV. In the present study, we showed that a serine residue of Akt was phosphorylated in Vero E6 cells in subconfluent culture and that Akt was dephosphorylated rapidly after SARS-CoV infection without up-regulation of its phosphorylation. Phosphorylation of glycogen synthase kinase-3beta, which is one of the downstream targets of Akt, was prevented in SARS-CoV-infected cells. However, treatment with glycogen synthase kinase-3beta small interfering RNA indicated that the glycogen synthase kinase-3beta signaling pathway was not related to inhibition of cell proliferation. Treatment of Vero E6 cells with the phosphatidylinositol 3'-kinase/Akt inhibitor, LY294002, which induces dephosphorylation of Akt, inhibited cell proliferation. As shown in our previous studies, apoptosis occurred in virus-infected cells within 18 h postinfection. Cellular mRNA transcription, which was reported to be up-regulated in SARS-CoV-infected Caco-2 cells, was not up-regulated in virus-infected Vero E6 cells, partially as a result of apoptosis. These results suggested that inhibition of cell proliferation is regulated by both the phosphatidylinositol 3'-kinase/Akt signaling pathway and by apoptosis in SARS-CoV-infected Vero E6 cells. This is the first study to analyze SARS-CoV-induced cell growth inhibition.

Animals↗
PubMed2006

LC16m8, a highly attenuated vaccinia virus vaccine lacking expression of the membrane protein B5R, protects monkeys from monkeypox.

The potential threat of smallpox as a bioweapon has led to the production and stockpiling of smallpox vaccine in some countries. Human monkeypox, a rare but important viral zoonosis endemic to central and western Africa, has recently emerged in the United States. Thus, even though smallpox has been eradicated, a vaccinia virus vaccine that can induce protective immunity against smallpox and monkeypox is still invaluable. The ability of the highly attenuated vaccinia virus vaccine strain LC16m8, with a mutation in the important immunogenic membrane protein B5R, to induce protective immunity against monkeypox in nonhuman primates was evaluated in comparison with the parental Lister strain. Monkeys were immunized with LC16m8 or Lister and then infected intranasally or subcutaneously with monkeypox virus strain Liberia or Zr-599, respectively. Immunized monkeys showed no symptoms of monkeypox in the intranasal-inoculation model, while nonimmunized controls showed typical symptoms. In the subcutaneous-inoculation model, monkeys immunized with LC16m8 showed no symptoms of monkeypox except for a mild ulcer at the site of monkeypox virus inoculation, and those immunized with Lister showed no symptoms of monkeypox, while nonimmunized controls showed lethal and typical symptoms. These results indicate that LC16m8 prevents lethal monkeypox in monkeys, and they suggest that LC16m8 may induce protective immunity against smallpox.

Animals↗
PubMed2006

Marburgvirus nucleoprotein-capture enzyme-linked immunosorbent assay using monoclonal antibodies to recombinant nucleoprotein: detection of authentic Marburgvirus.

There have recently been large outbreaks of Marburg hemorrhagic fever (MHF) caused by Marburgvirus (MARV) in the Democratic Republic of Congo and Angola. The development of reliable diagnostic systems for MHF is urgently needed. An antigen-capture enzyme-linked immunosorbent assay (Ag-capture ELISA) using either of the two monoclonal antibodies (2A7 and 2H6) produced by immunizing mice with recombinant nucleoprotein of MARV was described (Journal of Medical Virology, 76, 111-118, 2005). In the present study, it was revealed that the Ag-capture ELISA specifically detected authentic MARV antigen and that the sensitivity of the Ag-capture ELISA was at a level similar to that of reverse-transcription polymerase chain reaction. These results suggest that the Ag-capture ELISA using the monoclonal antibodies, 2A7 and 2H6, is applicable to the diagnosis of MHF.

Animals↗
PubMed2005

Activation of the lutropin/choriogonadotropin receptor in MA-10 cells leads to the tyrosine phosphorylation of the focal adhesion kinase by a pathway that involves Src family kinases.

We show that activation of the endogenous or recombinant lutropin/choriogonadotropin receptor (LHR) in mouse Leydig tumor cells (MA-10 cells) leads to the tyrosine phosphorylation of the focal adhesion kinase (FAK) and one of its substrates (paxillin). Using specific antibodies to the five tyrosine residues of FAK that become phosphorylated, we show that activation of the LHR increases the phosphorylation of Tyr576 and Tyr577, but it does not affect the phosphorylation of Tyr397, Tyr861, or Tyr925. Because FAK is a prominent substrate for the Src family of tyrosine kinases (SFKs) we tested for their involvement in the LHR-mediated phosphorylation of FAK-Tyr576. Src is not detectable in MA-10 cells, but two other prominent members of this family (Fyn and Yes) are present. The LHR-mediated phosphorylation of FAK-Tyr576 is readily inhibited by PP2 (a pharmacological inhibitor of SFKs) and by dominant-negative mutants of SKFs. Moreover, activation of the LHR in MA-10 cells results in the stimulation of the activity of Fyn and Yes, and overexpression of either of these two tyrosine kinases enhances the LHR-mediated phosphorylation of FAK-Tyr576. Studies involving activation of other G protein-coupled receptors, overexpression of the different Galpha-subunits, and the use of second messenger analogs suggest that the LHR-induced phosphorylation of FAK-Tyr576 in MA-10 cells is mediated by SFKs, and that this family of kinases is, in turn, independently or cooperatively activated by the LHR-induced stimulation of Gs and Gq/11-mediated pathways.

Animals↗