Résumé
The sequences of the internal transcribed spacer (ITS) and 5.8s ribosomal RNA gene (rDNA) from Fusarium solani and its four formae speciales belonging to section Martiella was determined to investigate intraspecific divergence of the ITS regions. The length of the 5.8S, a coding region, was equally 158 bp at all isolates, whereas the variable range of ITS region was shown at 147~152 bp (ITS1) and 148~174 bp (ITS2). According to the maximum-matching method, the matching percentage was 94~100 at 5.8s rDNA, 77~97 at ITS1, and 67~97 at ITS2, respectively. In dendrogram based on the alignment of the ITS sequence data, F.solani f. sp. piperis was distinguished from other isolates belonging to the same species and nucleotide identity was considerably low (41.5%).
Sujets)
Codage clinique , ADN ribosomique , FusariumRésumé
Many fungi including Penicillium, Aspergillus, Gliocladium, and Thermoascus produce an epipolythiodioxopiperazine class of fungal metabolite, gliotoxin, which contirbutes the pathogenesis of fungal infection as an immunomodulator and cytotoxic agent. This study is designed to define the mechanism by which gliotoxin exerts the cytotoxic effect of gliotoxin on human promyelocytic leukemic cells, HL-60. Gliotoxin induces the apoptosis of HL-60 cells which is characterized by the ladder pattern fragmentation of DNA. Gliotoxin induces the activation of DEVD-specific cysteine protease in a time- and dose-dependent rnanner. It also increases the phosphotransferase activities of c-Jun N-terminal kinase1 (JNK1) and p38 in gliotoxin-treated HL-60 cells. Furthermore, gliotoxin decreases the activation of transcriptional activator, actiating protein (AP-1) and NF-kB. These results suggest that gliotoxin induces the apoptotic death of HL-60 cells via activation of DEVD- specific caspase as well as mitogen activated protein kinases (MAP kinases) including JNK1 and p38, and inhibition of transcriptional activators, AP-1 and NF-kB.
Sujets)
Humains , Apoptose , Aspergillus , Caspase-3 , Cysteine proteases , ADN , Champignons , Gliocladium , Gliotoxine , Cellules HL-60 , Mitogen-Activated Protein Kinases , Facteur de transcription NF-kappa B , Penicillium , Thermoascus , Facteur de transcription AP-1 , Facteurs de transcriptionRésumé
Programrned cell death (PCD), or apoptosis, is a process by which cells die in response to specific physiological and toxicological signals. This genetically programmed form of cellular suicide is intirnately involved in many biological processes including growth, metamorphosis, embryogenesis, and oncogenesis. Cells undergoing PCD in normal and neoplasmic tissues display the following biochemical and morphological features: internucleosomal DNA fragmentation, reduced cell volume, condensed chromatin in nucleus, zeiosis and generation of apoptotic bodies containing intact organelles and plasma rnembrane. Hybridoma cell production, resulting from the fusion of myeloma cells with antibody producing spleen cells, is widely used in various fields of life science. This technique requires hypoxanthine guanine phosphoribosyl transferase (HGPRT) deficient mutant myeloma cell line as a fusion partner. When these mutants cell is treated with aminopterin plus hypoxanthine-thymidine (HAT) after the cell fusion they are selectively and efficiently eliminated remaining fused hybridoma celis. But there hasn't been any report regarding the selective elimination mechanism of this HGPRT mutant myeloma cell. By using HGPRT myeloma P3-X 63-Ag8.653 (V653) as a model system, this study demonstrated that PCD was induced by aminopterin treatment of this V653 cell line. And the sequential ultrastructural changes during this death process were observed by using electron microscope. When V653 cells were incubated with 0.4 uM aminopterin, DNA fragmentation was detectable after 3 hours and peaked between 12 and 18 hours of aminopterin treatment and the cell viability decreased in a time dependant manner. V653 cells incubated with amiopterin showed following ultrastructural changes during the death process. Dilatation of rough endoplasmic reticulum (RER) and detachment of ribosomes were the earliest ultrastructural changes and first seen after 30 minute incubation. Dilatation of perinuclear cisternae began to appear after 1 hour and deformation of nucleoplasm such as decreased electron density of perinuclear heterochromatin and increased electron density of euchromatin were seen after 3 hours. Increased electron density of cytoplasm, decreased cell volume, condensation of chromatin and apoptotic bodies were observed in many cells after 9 hours but vacuolation by severe dilatation of RER was seen in some cells. 24 hours after incubation with aminopterin, many cells showed typical form of apoptosis characterized by cell shrinkage, increased electron density of cytoplasm and apoptotic bodies. On the contrary, some cells showed different type of cell death characterized by increased cell volume, decreased electron density of cytoplasm, severely dilated RER and apoptotic bodies. In both types of cells, mitochondrial cristae and limiting membrane of mitochondria are comparatively well preserved. In other cells, nuclei did not show significant changes but there were deformations of mitochondria such as markedly increased electron density and formation of lamella bodies. The death process of V653 cell was not synchronized among cells. The results of this study proved that aminopterin-induced selective elimination of fusion partner V653 myeloma cell is due to PCD. The earliest ultrastructural changes observed in this process were dilatation of RER and detachment of ribosomes. And there were two distinct morphological types in the PCD.
Sujets)
Animaux , Femelle , Souris , Grossesse , Aminoptérine , Apoptose , Phénomènes biologiques , Disciplines des sciences biologiques , Carcinogenèse , Mort cellulaire , Fusion cellulaire , Lignée cellulaire , Taille de la cellule , Survie cellulaire , Chromatine , Cytoplasme , Dilatation , Fragmentation de l'ADN , Développement embryonnaire , Réticulum endoplasmique rugueux , Euchromatine , Guanine , Hétérochromatine , Hybridomes , Hypoxanthine , Hypoxanthine phosphoribosyltransferase , Membranes , Mitochondries , Organites , Plasma sanguin , Ribosomes , Rate , Suicide , TransferasesRésumé
Anthrax toxin consists of three separate proteins, protective antigen (PA), edema factor (EF), and lethal factor (LF). PA binds to the receptor on mammalian cells and facilitates translocation of EF or LF into its cytosol. PA is the primary component of anthrax vaccines. In this study we purified PA from culture filtrates of Bacillus anthracis. The purification involved sequential chromatography through hydroxylapatite, DEAE-Sepharose CL-4B, followed by Mono-Q. The purified PA was judged to be homogeneous on SDS-PAGE, and consisted of a single polypeptide chain with a relative molecular weight of 85,000.
Sujets)
Maladie du charbon , Vaccins anticharbonneux , Bacillus anthracis , Bacillus , Chromatographie , Cytosol , Durapatite , Oedème , Électrophorèse sur gel de polyacrylamide , Masse moléculaireRésumé
Bacillus anthracis is a soil pathogen capable of causing anthrax in animals and humans. To establish a method for specifically detecting B. anthracis, we used nested polymerase chain reaction. Outer and inner sets of oligonucleotide primers were designed from the protective antigen (pag) gene and from the cya gene of the plasmid pXO1. Ainplification of 482 bp or 208 bp DNA fragment obtained from a nested PCR method provided the basis for rapid and reliable assay for the detection and identification of B. anthracis.
Sujets)
Animaux , Humains , Maladie du charbon , Bacillus anthracis , Bacillus , ADN , Amorces ADN , Plasmides , Réaction de polymérisation en chaîne , SolRésumé
Homeostasis of multicellular organism is controlled by proliferation and differentiation of cells as well as by cell death. The defects in programmed cell death contribute to the pathogenesis of rheumatoid arthritis (RA) and systemic lupus erythematous. RA is considered to be a proliferating disorder of synovial tissue which is accompanied by inflammatory cell infiltration and bone erosion. The aim of the study was to find whether potent inducers of apoptosis could be induced apoptosis in RA synovial cells. We examined the effects of drugs, such as dexamethasone, methotrexate, hydrogen peroxide, and ceramide on induction of apoptosis in cultured RA synovial cells. Used drugs did not induced apoptosis in RA synovial cells. Finally Fas antigen-mediated apoptosis of RA synovial cells was investigated by the addition of anti-Fas antibody. To examine the ICE (interleukin-1p-convertase; caspase-1) expression in synovial cells, RT-PCR of caspase-1 gene was performed. In synovial cells of RA, Fas induces that caspase-1 activation cause apoptosis.