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Biomedical subjects

Y Muneta

Publications and source records attributed to Y Muneta.

10 recordsLinked to original sources
PubMed2001

Molecular cloning, characterization, and expression of porcine Fas ligand (CD95 ligand).

We isolated and sequenced cDNA that contained the coding sequence of porcine Fas ligand (FasL). Using mixed oligonucleotide primers based on the 5' and 3' nucleotide sequences conserved among human, murine, and rat FasL, we performed the reverse transcription polymerase chain reaction (RT-PCR) with total RNA prepared from porcine thymocytes stimulated with 5 microg/ml concanavalin A (ConA) to clone the cDNA of porcine FasL. The open reading frame (ORF) of porcine FasL cDNA was 849 base pairs (bp) in length and encoded 282 amino acids. The predicted amino acid sequence was 85.5%, 76.6%, and 75.5% homologous to the predicted human, murine, and rat FasL, respectively. The recombinant porcine FasL expressed by recombinant baculovirus containing the whole coding sequences of porcine FasL showed cytotoxic effect and induced apoptosis in porcine renal tubular cell line PK-15 cells sensitized by cycloheximide (CHX), which was confirmed by MTT assay, DNA fragmentation assay, and TUNEL staining, respectively. Furthermore, the mRNA expression of porcine FasL in porcine peripheral blood lymphocytes (PBL) was induced by porcine interleukin-18 (IL-18). These results indicate that porcine FasL identified in this study is biologically functional and has the ability to induce apoptosis as reported in other species.

Amino Acid Sequence↗
PubMed2001

Efficient production of biologically active porcine interleukin-18 by coexpression with porcine caspase-1 using a baculovirus expression system.

We previously reported that the precursor form of porcine interleukin-18 (IL-18) expressed by the baculovirus system was able to be secreted efficiently into the supernatant of insect cells, whereas only small amounts of mature IL-18 were secreted from insect cells. As insect cells do not normally have the IL-1beta converting enzyme (caspase-1), which is required for processing of the precursor IL-18 into the mature IL-18, we recently cloned porcine caspase-1 cDNA. In this study, we constructed a recombinant baculovirus containing the cDNA encoding porcine caspase-1 and showed that the coexpression of caspase-1 and the precursor IL-18 enabled insect cells to secrete mature IL-18 into the culture supernatant efficiently. Moreover, inhibition of caspase-1 activity by its specific inhibitor prevented the processing of precursor IL-18 into the mature form. These results indicated that the processing and secretion of precursor IL-18 into the mature form in insect cells were enhanced by the artificial introduction of caspase-1 activity for cleavage.

Animals↗
PubMed2001

Porcine caspase-3: its cloning and activity during apoptosis of PK15 cells induced by porcine Fas ligand.

We cloned and sequenced cDNA that contained the coding sequence of porcine caspase-3. The open reading frame (ORF) of porcine caspase-3 cDNA was 834 base pairs (bp) in length and encoded 277 amino acids. The predicted amino acid sequence was 88.4%, 86.6%, and 87.7% homologous to the predicted human, murine, and rat amino acid sequences, respectively. The activity of caspase-3 in porcine renal tubular cell line PK15 after recombinant porcine Fas ligand (FasL) stimulation was examined. The enzymatic activity of caspase-3, but not that of caspase-1, was significantly increased after FasL treatment. Western blot analysis also showed that the processing of caspase-3 from proenzyme to mature subunits occurred after FasL treatment. The inhibition of caspase-3 by its specific inhibitor partially prevented the apoptotic cell death of PK15 cells caused by FasL. The porcine caspase-3 cDNA isolated in this study will be useful for the study of apoptotic cell death in pigs and will lead to the discovery of therapeutic uses of caspases and their inhibitors in the prevention of viral and bacterial diseases and tissue injury associated with xenotransplantation and allotransplantation.

Amino Acid Sequence↗
PubMed2000

Production of monoclonal antibodies to porcine interleukin-18 and their use for immunoaffinity purification of recombinant porcine interleukin-18.

We have recently reported the cloning and expression of porcine interleukin-18 (IL-18). In this study, we describe the production of anti-porcine IL-18 monoclonal antibodies (mAb) and their use in the purification of a large amount of recombinant porcine IL-18 by immunoaffinity column chromatography. Five monoclonal antibodies (2-2-B, 2-5-B, 2-13-C, 3-1-C and 5-3-B) were established and characterized. Three (2-2-B, 3-1-C and 5-3-B) of them were of IgG1 subclass, and the other two were IgMs. Epitope analysis of the three IgG1 mAbs showed that they recognized the same epitope. All five mAbs demonstrated reactivity with baculovirus generated porcine IL-18 by immunoblot analysis. Biologically active porcine IL-18 was obtained by immunoaffinity chromatography using anti-porcine IL-18 mAb at more than 85% purity from culture supernatants of Trichoplusia ni (Tn5) derived cells infected with recombinant baculovirus containing the coding sequence of porcine mature IL-18. These results suggest that the anti-porcine IL-18 mAbs established in this study are useful for one-step purification of porcine mature IL-18 as well as the detection of porcine IL-18 by immunoblotting.

Animals↗
PubMed2000

Porcine interleukin 18: cloning, characterization of the cDNA and expression with the baculovirus system.

We have isolated and sequenced a cDNA that contains the coding sequence of porcine interleukin 18 (IL-18) and the recombinant protein of porcine IL-18 was expressed using the baculovirus system. The open reading frame (ORF) of the porcine IL-18 cDNA is 579 base pairs (bp) in length and encodes 192 amino acids. The predicted amino acid sequence is 76.7%, 64.7% and 61.6% homologous to the predicted human, murine and rat amino acid sequences, respectively. The porcine precursor and mature IL-18 protein were expressed respectively in Trichoplusia ni -derived (Tn5) cells using the baculovirus Autografha californica nuclear polyhedorosis virus (AcNPV) as a vector. Tn5 cells infected with recombinant virus containing a whole IL-18 protein coding region sequence secreted porcine precursor IL-18 into the culture medium. On the other hand, Tn5 cells infected with recombinant virus containing a mature IL-18 protein coding region sequence expressed several proteins in the cell lysates, but did not secrete mature protein into the culture medium efficiently. Immunoblotting analysis of recombinant protein showed cross-reactivity with anti-human IL-18 polyclonal antibody. The mature form of porcine IL-18 protein induced IFN-gamma production in suboptimal doses of anti-CD3 antibody and concanavalin A- (ConA) stimulated porcine peripheral blood mononuclear cells (PBMC), but the precursor form had little effect.

Amino Acid Sequence↗
PubMed2000

Clinical features of sudden hearing loss associated with a high signal in the labyrinth on unenhanced T1-weighted magnetic resonance imaging.

We report on five patients with high signals in the labyrinth on unenhanced magnetic resonance imaging who developed sudden hearing loss and vertigo. Weissman et al. (1992) suggested the possibility that such high signals were caused by hemorrhage. We assessed these patients using audiograms, caloric tests, and auditory brainstem responses to investigate the possibility of inner ear hemorrhage. Most of the patients were found to have severe and irreversible impairment of both cochlear and vestibular function. These findings were consistent with the hypothesis that their symptoms were caused by inner ear hemorrhage.

Adolescent↗
PubMed2000

Detection of porcine interleukin-18 by sandwich ELISA and immunohistochemical staining using its monoclonal antibodies.

We describe here the development of sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunohistochemical staining for porcine interleukin-18 (PoIL-18) and their application to detection of PoIL-18 in vivo. Ten anti-PoIL-18 monoclonal antibodies (mAb), all of which were reactive with recombinant PoIL-18 by Western blotting, were established. Four (2-C-4, 9-H-6, 11-H-5, and 12-C-12) of 10 neutralized the biologic activity of PoIL-18 to induce interferon-y (IFN-gamma) from porcine peripheral blood mononuclear cells (PBMC). Four (2-C-4, 5-F-6, 9-H-6, and 12-C-12) of 10 were shown to be useful in immunohistochemical staining and detected PoIL-18 in Kupffer cells and macrophages in hepatic focal necrosis and macrophages in interstitial pneumonia in piglets with experimental endotoxemia using formalin-fixed, paraffin-embedded sections. A sandwich ELISA was developed using mAb 7-G-8 as a capture antibody and biotinylated mAb 5-C-5 as a detection antibody. This ELISA detected PoIL-18 with a minimum detectable concentration of 20 pg/ml and did not show cross-reactivity against PoIL-1beta, IL-8, IL-12, and IFN-gamma or murine and human IL-18. Using this ELISA, PoIL-18 was detected in the plasma and the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Actinobacillus pleuropneumoniae. The availability of this ELISA and immunohistochemical staining for PoIL-18 may contribute to a further understanding of the role of this cytokine in various porcine immune responses and diseases.

Animals↗
PubMed1999

Molecular cloning of porcine interleukin-1beta converting enzyme and differential gene expression of IL-1beta converting enzyme, IL-1beta, and IL-18 in porcine alveolar macrophages.

We have cloned and sequenced a cDNA that contains the coding sequence of porcine interleukin-1beta (IL-1beta) converting enzyme (ICE). Using degenerate oligonucleotide primers based on the amino acid sequences of the human, murine, and rat ICE, we performed the reverse transcription polymerase chain reaction (RT-PCR) with total RNA prepared from porcine alveolar macrophages stimulated with lipopolysaccharide (LPS) to clone the cDNA of porcine ICE. The open reading frame (ORF) of the porcine ICE cDNA is 1215 base pairs (bp) in length and encodes 404 amino acids. The predicted amino acid sequence is 72.5%, 62.6%, and 64.1% homologous to the human, murine, and rat amino acid sequences, respectively. The kinetics of mRNA expression of ICE, IL-1beta, and IL-18 in porcine alveolar macrophages after LPS stimulation revealed that ICE transcripts were weakly expressed in nonstimulated condition and upregulated after LPS stimulation. Moreover, IL-1beta and IL-18 transcripts were differently expressed after LPS stimulation.

Amino Acid Sequence↗
PubMed1993

[Computerized database system for pure tone audiometry].

The management and storage of pure tone audiometry data creates a major problem for otolaryngological clinics. We have devised a new computerized database system to overcome this problem. The hardware involved includes multiple audiometers and computers interfaced with a glassfiber local area network (Pi-Net). The software used is database 3 plus, a popular database language and utility software package. Programs were written for data entry, data transfer, reference, entry of patient ID, and evaluation of tympanoplasty. The following benefits of the system are noted. 1) Data entry is possible at every terminal (audiometer and computer). 2) Storage capacity is sufficient for more than 10 years of operation. 3) All data can be retrieved rapidly at any terminal. Evaluations of pre- and post-tympanoplasty hearing loss are facilitated by this system. This system is very effective for follow up of hearing disorders.

Audiometry, Pure-Tone↗