Hiroyuki Horiuchi

Tributyltin disturbs bovine adrenal steroidogenesis by two modes of action

Tributyltin, an environmental pollutant, affected adrenal steroid hormone biosynthesis by two modes of action. Treatment of bovine adrenal cultured cells with 10-100 nM tributyltin for 48 h suppressed cortisol and androstenedione secretion, but induced the accumulation of 17alpha-hydroxyprogesterone and deoxycortisol, indicating that the P450(C21) and P450(11beta) activities were specifically suppressed. Direct inhibition of the enzymatic activities due to tributyltin was not observed in isolated organelles of untreated cells at concentrations less than 10 microM. Western blotting experiments using specific antibodies against steroidogenic enzymes showed that treatment with 1-100 nM tributyltin caused a decrease in cellular P450(C21) and P450(11beta) protein levels, and real-time PCR experiments showed that the decrease in protein content was attributable to decreases in mRNA of the enzymes. Tributyltin at concentrations higher than 100 nM suppressed all steroid biosynthesis in the adrenal cells. This suppression was closely correlated to the decrease in steroidogenic acute regulatory protein. Since nanomolar concentrations of tributyltin disturbed steroidogenesis in mammalian cells, there is the possibility that steroid hormone synthesis in polluted wild animals is affected by this compound.

Role of bursin in the development of B lymphocytes in chicken embryonic Bursa of Fabricius

Localization and role of bursin during Bursa of Fabricius (BF) ontogeny were examined by immunohistochemical staining and by in ovo injection with anti-bursin antibody. Mouse monoclonal anti-bursin antibody HU2 was generated by immunization with synthetic bursin. It recognized reticular cells (REC), follicular associated epithelium (FAE), FAE-supporting cells, and the basal layer of interfollicular epithelium (IFE) in the mature BF. Bu-1(+) cells were first detectable in the mesenchyme area at 13 days of embryogenesis (E13) before bud formation, then lined up along the bud, and homed into the bud at around E15. IgM(+) cells were detected in the bud after E13. Bursin was first observed at the under edge of the bud. Injection of HU2 into embryonal vein at E13 suppressed the appearance of IgM(+) cells in the Bursa at E17. These results indicate that bursin exists beneath the bud and may act on the appearance of IgM(+) cells during BF ontogeny.

Construction of recombinant monoclonal antibodies from a chicken hybridoma line secreting specific antibody

The chicken is a useful animal for the development of the specificantibodies against the mammalian conserved proteins. We generated twotypes of recombinant chicken monoclonal antibodies (mAbs), using a phagedisplay technique from a chicken hybridoma HUC2-13 which secreted themAb to the N-terminal of the mammalian prion protein (PrP). Althoughthe mAb HUC2-13 is a useful antibody for the prion research, thehybridoma produces a low level of antibody production. In order to producea large amount of the mAb, we have constructed a single chain fragmentvariable region (scFV) mAb by using the variable heavy(VH) and light (VL)genes which were amplified by using the two primer pairs and theflexible linker. The two phage display mAbs (HUC2p3 and HUC2p5)expressed on a M13 filamentous phage and their soluble type mAbs(HUC2s3 and HUC2s5) were reacted with the PrP peptide antigen in theELISA. In the Western blot analysis, the mAbs HUC2p3 and HUC2s3 wereas reactive to PrPc from mouse brains as the mAb HUC2-13 was. The nucleotide sequences of VH and VL genes from HUC2-13 and the two cloneswere identical except for only one residue. These results indicate that themethods presented here provide an effective tool for the improvement ofthe low levels of antibody production in the chicken hybridoma system.

Immunobiology of chicken germinal center: I. Changes in surface Ig class expression in the chicken splenic germinal center after antigenic stimulation

The germinal center (GC) develops after antigenic stimulation and is thought to occur at the site of various immune responses. We separated a single GC from chicken spleen after antigenic stimulation. Flow cytometric analysis of the cells derived from a single GC and RT-PCR analysis of Ig mRNA expression in GC was performed. Direct evidence indicates that: (1) there was a considerable difference in the cell population of each GC, (2) the ratio of CD3(+) cells in a GC remains constant at 10-20%, (3) the highest proportion of sIgY(+) cells in a GC occurs 1 week after the time of highest proportion of sIgM(+) cells, and (4) RT-PCR analysis was used to detect IgY mRNA expression in a GC. The continuous existence of CD3(+) cells, the alterations in sIgM(+) and sIgY(+) cell ratios, and the expression of IgY mRNA strongly suggest that Ig class switching occurs in the GC during an immune response.

(免疫学)ニワトリ胚におけるバイエル氏板,盲腸扁桃(腸管付属リンパ組織)の発生

It is well known that chicken B cells develop in the bursa of Fabricius (BF), which is categorized as gut-associated lymphoid tissue (GALT). Chicken GALT also includes Peyers patch (PP) and cecal tonsil (CT). The relationship between these tissues in GALT during B cell development is currently unknown. In this study, we conducted comparative examination of PP, CT and BF development during embryogenesis using immunohistochemical staining. On day 13 of embryogenesis (E13), accumulation of MHC class II(+) cells was observed in the intestine. Thereafter, Bu-1(+) cells and IgM(+) cells appeared, and their number continuously increased at the same sites where MHC class II(+) cells were present. Similar results were obtained in the CT. The locations of embryonic PP were limited to two sites; near the Meckels diverticulum and the ileocecal junction. Anlage of bursal follicles first appeared at E13 and developed thereafter. Immigration of Bu-1(+) cells to bursal follicles began at E13, and the number of Bu-1(+) cell subsequently increased. When the follicle of BF was eliminated from the embryo by treatment with testosterone, development of PP and CT were observed. We concluded therefore that the development of PP and CT start during late embryogenesis at the same time as the follicle of BF, and that appearance of surface IgM(+) cells in PP and CT is independent form the development of the follicle of BF.

Generation of Antibodies Against Prion Protein by Scrapie-Infected Cell Immunization of PrP0/0 Mice

Four monoclonal antibodies (MAbs) specific for prion protein (PrP) were generated by using PrP-knockout mice immunized with a scrapie-infected mouse neuroblastoma cell line (N2a/22L). The MAbs reacted with both the cellular form (PrP(C)) and the protease K-treated form (PrP(Sc)) on Western blotting. Of the four MAbs, three recognized mouse and hamster PrP, while the remaining MAb recognized mouse, sheep, and bovine PrPs. In addition, these MAbs were shown to react only with the unglycosylated and monoglycoslated forms of PrP(Sc) in N2a/22L, but reacted with all glycosylated forms of PrP(C) and PrP(Sc) from mouse brain. This study was the first to report the development of anti-PrP MAbs using scrapie-infected cells as an immunogen and provides one approach for the generation of PrP-specific MAbs.

Manipulating the Avian Epiblast and Epiblast-Derived Stem Cells

Compared to eutherian mammals, birds retain a primitive form of epiblast development. Molecular studies of the avian epiblast can provide valuable insight for mammalian epiblast research. Here, we introduce several basic techniques in handling epiblast-stage embryos of the chick, the major model organism for avian developmental biology studies. We describe how to collect embryos for RNA extraction and gene expression analysis, to set up ex ovo New culture for overexpression, bead graft and small molecule-based inhibitor studies, and to carry out whole-mount RNA in situ hybridization analysis. We introduce a novel and simple method for molecular perturbation of the epiblast differentiation in ovo. We also describe how to perform primary chicken epiblast cell culture, to establish stable epiblast stem cell (Epi-SC) lines, and to assay for pluripotency in primary epiblast cells and Epi-SCs.

Cloning and characterization of a chicken platelet-derived growth factor B-chain cDNA.

Avian thrombocytes are nucleated blood cells homologous in function to mammalian platelets. In the present study, we obtained a cDNA from chicken thrombocyte polyadenylated RNA [Poly(A)+RNA], which coded for the chicken PDGF-B chain. The sequence was 1083-bp long and had an open reading frame (ORF) of 753-bp. At the amino acid level, the predicted mature protein showed 69% homology with the processed coding region of human PDGF-B. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that PDGF-B mRNA was expressed at high levels in thrombocytes and in the lung. The expression of PDGF-B chain mRNA in thrombocytes reached its maximum level 12h following type 1 collagen treatment. These results suggest that chicken PDGF-B chain may play an important role in the vascular system and in healing wounded tissue.

Generation and characterization of chicken monoclonal antibodies against human LOX-1

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is the major receptor for oxidized LDL (oxLDL), and plays a key role in the pathogenesis of atherosclerosis and cardiovascular diseases. Monoclonal antibodies (mAbs) specific for human LOX-1 (hLOX-1) were generated by a phage display technique using chickens immunized with recombinant hLOX-1 (rhLOX-1). A total of 53 independent scFv clones reactive for rhLOX-1 were obtained. Of the 53 clones, 49 recognized the C-type lectin-like domain (CTL domain), which contributes to the binding of oxLDL. Of these, 45 clones inhibited oxLDL-binding with LOX-1. Furthermore, some of these clones cross-reacted with rabbit, pig and/or mouse LOX-1. For possible application as therapeutic agents in the future, two cross-reactive mAbs were re-constructed as chicken-human chimeric antibodies. The chimeric antibodies showed similar characteristics compared to the original antibodies, and inhibited oxLDL binding to LOX-1 expressed on CHO cells. The results obtained in this study indicate that anti-LOX-1 mAbs might be useful tools for functional analyses and development of therapeutic agents for cardiovascular indications such as atherosclerosis.

Maintenance of chicken embryonic stem cells in vitro.

In this chapter, we describe the methods we have used to show that chicken leukemia inhibitory factor (LIF) maintains chicken embryonic stem (ES) cells in an undifferentiated state in culture. Recombinant chicken LIF (rchLIF) was expressed as a fusion protein linked to glutathione S-transferase (GST) and purified to greater than 90% purity in two chromatography stages, the first an affinity step using the GST tail, which was cleaved before further purification by gel chromatography. Chicken ES cells were obtained by culturing chicken blastodermal cells isolated from stage X embryos of freshly laid chicken eggs. These cells can be maintained in media containing rchLIF for at least 9 d without any other cytokines or feeder cells. Chicken ES cells were characterized by the expression of alkaline phosphatase activity, stage-specific embryonic antigen (SSEA)-1 and embryonal carcinoma cell monoclonal antibody-1. In addition, the phosphorylation of signal transducers and activators of transcription-3 by LIF, which is sufficient to maintain the undifferentiated state of ES cells, was detected by Western blotting analysis.