We have designed a fusion gene encoding a chimeric mitochondrial precursor protein (avidin fusion protein) that consists of the mitochondrial presequence followed by mouse dihydrofolate reductase, a spacer segment, and streptavidin. The avidin fusion protein synthesized in vitro formed a tetramer at the avidin moiety on incubation with biotin during or after the translation reaction. The avidin fusion protein purified from the Escherichia coli overexpresser cells also formed the tetramer on dilution from 6M urea into buffer containing biotin. In in vitro import experiments with isolated yeast mitochondria, the tetramer of the avidin fusion protein became stuck across both mitochondrial membranes, with its N-terminal dihydrofolate reductase moiety in the matrix and its C-terminal avidin moiety exposed on the mitochondrial surface. Accumulation of the translocation intermediate of the fusion protein inhibited the import of a mitochondrial precursor protein, and allowed us to estimate the number of mitochondrial import sites.
lmidazoleglycerolphosphate dehydratase (ICPD; EC 4.2.1.1 9), which is involved in the histidine biosynthetic pathway of Arabidopsis fhaliana and wheat (Triticum aestivum), has been expressed in insect cells using the baculovirus expression vector system. Nterminal amino acid sequencing indicated that recombinant IGPDs (rlCPDs) were produced as mature forms via nonspecific proteolytic cleavages in the putative transit peptide region. The wheat rlCPD contained one M n atom per subunit, and the M n was involved in the assembly of the subunits to form active ICPDs. Protein-blotting analysis, using antibodies raised against the wheat rlCPD, indicated that ICPD was located in the chloroplasts of wheat. The rlCPDs of Arabidopsis and wheat, which were 86% identical in their primary structures deduced from the cDNAs, exhibited similar properties in terms of the molecular mass, pH optimum, and the K,,, for the substrate, imidazoleglycerolphosphate. However, the nonselective herbicides 3-amino-l,2,4-triazole and a newly synthesized triazole [(l R', 3Re)-[3-hydroxy-3-(2H-[1,2,4]triazole-3-yl)-cyclohexyl]-phosphonic acid], inhibited Arabidopsis and wheat ICPDs in a mixed-type and a competitive manner, respectively.
In mammals, M cells can take up antigens through mucosal surfaces of the gut and the respiratory tract. Since M cells are deficient of lysosomes and phagosomes, the antigens are directly delivered to the mucosa-associated lymphoid tissue (MALT) without degradation. In teleost fish, the entire body surface (gills, skin, and intestinal system) is covered by mucus; however, specific antigen-sampling cells have not yet been identified in their mucosal tissues. Here, we show that two phenotypes of antigen-sampling cells take up antigens through epithelial surfaces of the rainbow trout gill. One phenotype of antigen-sampling cells has features of monocyte/macrophage/dendritic cell-type cells; they have large vacuoles in the cytoplasm and express PTPRC (CD45), CD83, IL-1β, and IL-12p40b. The second phenotype exhibits similar characteristics to mammalian M cells; the corresponding cells bind the lectin UEA-1 but not WGA and show expression of M cell marker gene Anxa5. In contrast to mammalian M cells, teleost M-type cells were found to exhibit small vacuoles in their cytoplasm and to express almost all genes related to the “phagosome”, “lysosome,” and “antigen processing and presentation” pathways. Furthermore, MHC class II was constitutively expressed on a fraction of M-type cells, and this expression was significantly increased after antigen uptake, suggesting that the MHC class II is inducible by antigen stimulation. Here, we suggest that teleost M-type cells play a role in the phylogenetically primitive teleost immune system, similar to bona-fide M cells. In addition, the presence of MHC class II expression suggests an additional role in antigen presentation in the gills, which are an organ with high T cell abundance, especially in interbranchial lymphoid tissue. The present results suggest an unconventional antigen presentation mechanism in the primitive mucosal immune system of teleosts, which generally lack highly organized lymphoid tissues. Moreover, the results of this work may be valuable for the development of mucosal vaccines that specifically target M-type cells; mucosal vaccines significantly reduce working costs and the stress that is usually induced by vaccination via injection of individual fish.
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