AIM： To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory （ES） product. METHODS： NIH-3T3, mouse fibroblast cells were treated with O. viverrini...AIM： To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory （ES） product. METHODS： NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA （cDNA） array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semiquantitative SYBR-based real-time RT-PCR. RESULTS： Among a total of 15 000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serinethreonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O.viverrini ES product, The expression level of signal transduction genes, pkC, pdgfra, jak 1, eps 8, tgfβ 1/4,strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC,eps 8 and tgfβ3 1/4 which are the downstream signaling molecules of either epidermal growth factor （EGF） or transforming growth factor-β （TGF-β） showed statistical significance （P 〈 0.05）.CONCLUSION： O. viverrini ES product stimulates the significant changes of gene expression in several functional categories and these mainly include transcripts related to cell proliferation. The TGF-β and EGF signal transduction pathways are indicated as the possible pathways of O. viverrini-driven cell proliferation.展开更多
The origin, development and ultrastructure of secretory cavities in leaf of Ginkgo biloba and its relation to the secretory products were studied by optical and electron microscope. The results indicated that the form...The origin, development and ultrastructure of secretory cavities in leaf of Ginkgo biloba and its relation to the secretory products were studied by optical and electron microscope. The results indicated that the formation pattern of the secretory cavities was schizo-lysigenous in nature. First, some original central secretory cells in the center of secretory cavities expanded, dissolved then moved aside schizogeneously forming an intercellular space. Later the central and peripheral secretory cells continued to dissolve making the intercellular space larger. This process continued until the central secretory cells degenerated, autolyzed and separated off into cavities. It has been assumed that the secretory products in the secretory cavities of Ginkgo biloba were synthesized mainly in the plastids and the endoplasmic reticulum (ER). However it was our observation that the Mitochondrion, Golgi Body and Cytoplasm were also involved in the synthesis and translocation of the secretions. After synthesized in ER and plastids, the secretory products approached the plasmalemma and fused their membranes with the latter in the form of samll vesicles, and then was eliminated to the spaces between the plasmalemma and the wall. At last the secretory products percolated through the wall that faded into an even looser mesh of fibrillar material toward the cavity.展开更多
The comparative studies of diagnostic efficiency of excretory-secretory antigens of Trichinella spiralis and Trichinella nativa were performed using blood sera of rats from Wistar line experimentally infected with Arc...The comparative studies of diagnostic efficiency of excretory-secretory antigens of Trichinella spiralis and Trichinella nativa were performed using blood sera of rats from Wistar line experimentally infected with Arctic trichinellae. For animal infection and antigen preparation Trichinella from muscles of wild carnivorous mammals from Arctic regions of Russia were used. When antigen from T. nativa larvae was used to analyze titers of sera of rats experimentally infected with Arctic Trichinella, a significant increase in efficacy ofELISAwas detected. E.g., sera of rats infected with trichinellae from ringed seals retained in ELISA with T. nativa antigen values higher than diagnostic level at titers of 1:6400 - 1:12800, while titer of those same sera when using T. spiralis antigen was no higher than 1:200 - 1:400.展开更多
DNA fragments encoding the light chain and heavy chain genes of an anti-human HER II antibody, trastuzumab, fused with an egg-lysozyme signal peptide were synthesized based on the codon bias of the methylotrophic yeas...DNA fragments encoding the light chain and heavy chain genes of an anti-human HER II antibody, trastuzumab, fused with an egg-lysozyme signal peptide were synthesized based on the codon bias of the methylotrophic yeast Pichia pastoris. These fragments were inserted into a site between the AOX 1-promoter and -terminator in pPICZ A to be expressed by P. pastoris. The expression vector was linearized, and introduced into P. pastoris GS115 by electroporation. After the checking of several transformants with PCR to ensure a precise insertion, one was selected and cultured to examine antibody production. The level of production reached 10 mg/L in a flask with medium containing 1% methanol. The heavy chain and light chain of the product were assembled to form a hetero tetramer, as detected by dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal amino acid sequencing revealed that the signal peptides of both chains were well processed. The mobility of the product in SDS-PAGE after treatment with Peptide N-Glycosidase F indicated the heavy chain to be N-glycosylated. Further analysis of the N-glycans with a mass spectrometer revealed a mixture of Man9-GlcNAc2, Man10-GlcNAc2, Man11-GlcNAc2 and Man12-GlcNAc2, but no hyper-mannosylated glycans. ELISA, surface plasmon resonance, and flow cytometric studies showed the affinity curve and Kd value for the antigen, HER II, and reactivity to a HER2-overexpressing breast cancer cell-line, SK-BR-3, to be almost the same as for the clinically used trastuzumab produced by CHO.展开更多
基金the Thailand Research Fund, Grant No. TRG4880004 and the Grants of Khon Kaen University 2004 and 2006 Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan the Grant for Student of Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, 2003-2005
文摘AIM： To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory （ES） product. METHODS： NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA （cDNA） array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semiquantitative SYBR-based real-time RT-PCR. RESULTS： Among a total of 15 000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serinethreonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O.viverrini ES product, The expression level of signal transduction genes, pkC, pdgfra, jak 1, eps 8, tgfβ 1/4,strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC,eps 8 and tgfβ3 1/4 which are the downstream signaling molecules of either epidermal growth factor （EGF） or transforming growth factor-β （TGF-β） showed statistical significance （P 〈 0.05）.CONCLUSION： O. viverrini ES product stimulates the significant changes of gene expression in several functional categories and these mainly include transcripts related to cell proliferation. The TGF-β and EGF signal transduction pathways are indicated as the possible pathways of O. viverrini-driven cell proliferation.
文摘The origin, development and ultrastructure of secretory cavities in leaf of Ginkgo biloba and its relation to the secretory products were studied by optical and electron microscope. The results indicated that the formation pattern of the secretory cavities was schizo-lysigenous in nature. First, some original central secretory cells in the center of secretory cavities expanded, dissolved then moved aside schizogeneously forming an intercellular space. Later the central and peripheral secretory cells continued to dissolve making the intercellular space larger. This process continued until the central secretory cells degenerated, autolyzed and separated off into cavities. It has been assumed that the secretory products in the secretory cavities of Ginkgo biloba were synthesized mainly in the plastids and the endoplasmic reticulum (ER). However it was our observation that the Mitochondrion, Golgi Body and Cytoplasm were also involved in the synthesis and translocation of the secretions. After synthesized in ER and plastids, the secretory products approached the plasmalemma and fused their membranes with the latter in the form of samll vesicles, and then was eliminated to the spaces between the plasmalemma and the wall. At last the secretory products percolated through the wall that faded into an even looser mesh of fibrillar material toward the cavity.
文摘The comparative studies of diagnostic efficiency of excretory-secretory antigens of Trichinella spiralis and Trichinella nativa were performed using blood sera of rats from Wistar line experimentally infected with Arctic trichinellae. For animal infection and antigen preparation Trichinella from muscles of wild carnivorous mammals from Arctic regions of Russia were used. When antigen from T. nativa larvae was used to analyze titers of sera of rats experimentally infected with Arctic Trichinella, a significant increase in efficacy ofELISAwas detected. E.g., sera of rats infected with trichinellae from ringed seals retained in ELISA with T. nativa antigen values higher than diagnostic level at titers of 1:6400 - 1:12800, while titer of those same sera when using T. spiralis antigen was no higher than 1:200 - 1:400.
文摘DNA fragments encoding the light chain and heavy chain genes of an anti-human HER II antibody, trastuzumab, fused with an egg-lysozyme signal peptide were synthesized based on the codon bias of the methylotrophic yeast Pichia pastoris. These fragments were inserted into a site between the AOX 1-promoter and -terminator in pPICZ A to be expressed by P. pastoris. The expression vector was linearized, and introduced into P. pastoris GS115 by electroporation. After the checking of several transformants with PCR to ensure a precise insertion, one was selected and cultured to examine antibody production. The level of production reached 10 mg/L in a flask with medium containing 1% methanol. The heavy chain and light chain of the product were assembled to form a hetero tetramer, as detected by dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal amino acid sequencing revealed that the signal peptides of both chains were well processed. The mobility of the product in SDS-PAGE after treatment with Peptide N-Glycosidase F indicated the heavy chain to be N-glycosylated. Further analysis of the N-glycans with a mass spectrometer revealed a mixture of Man9-GlcNAc2, Man10-GlcNAc2, Man11-GlcNAc2 and Man12-GlcNAc2, but no hyper-mannosylated glycans. ELISA, surface plasmon resonance, and flow cytometric studies showed the affinity curve and Kd value for the antigen, HER II, and reactivity to a HER2-overexpressing breast cancer cell-line, SK-BR-3, to be almost the same as for the clinically used trastuzumab produced by CHO.
