Several toxic compounds are known to induce apoptosis in mammalian cell lines.The human neuroblastoma cells(SHSH-SY5Y)were exposed to the phosphatase inhibiting toxin okadaic acid(OA)or hydrogen peroxide(H2O2)to induc...Several toxic compounds are known to induce apoptosis in mammalian cell lines.The human neuroblastoma cells(SHSH-SY5Y)were exposed to the phosphatase inhibiting toxin okadaic acid(OA)or hydrogen peroxide(H2O2)to induce apoptosis as well as generate reactive oxygen species(ROS).Mitoxantrone(MXT)was used as a positive control for apoptosis.The SHSH-SY5Y cells were transfected with eukaryotic expression plasmid pHyPer-dMito encoding mitochondrial-targeted fluorescent or pHyPer-dCito encoding cytoplasmic-targeted fluorescent sensor for hydrogen peroxide(HyPer).The ERp57,also called GRP58(Glucose-regulated protein 58),is a stress protein induced in conditions like glucose starvation and viral infection.Recently ERp57 was shown to translocate from the endoplasmatic reticulum to the cell surface in anthracycline-induced apoptotic cells.ERp57 co-translocation together with calreticulin has been suggested to be crucial for recognizing tumor cells to induce immunogenic cell death.ERp57 translocation after exposure to okadaic acid was studied using immunofluorescence and confocal microscopy.These studies indicated that okadaic acid has induced the translocation of ERp57 to the cellular membrane.展开更多
Primary and secondary antibody responses in blood of teleost fish are mainly IgM mediated, as they lack an IgG homolog and a class switch mechanism. Thus, the major serum immunoglobulin is tetrameric IgM. A unique ant...Primary and secondary antibody responses in blood of teleost fish are mainly IgM mediated, as they lack an IgG homolog and a class switch mechanism. Thus, the major serum immunoglobulin is tetrameric IgM. A unique antibody class in teleosts;named IgT, appears to be specialized for mucosal immunity and is present in low concentrations in serum. A third class;IgD was recently detected in serum of rainbow trout, but is less abundant than IgT. In the present study, relative quantification of total IgM showed that Atlantic salmon (Salmosalar) which had received an oil-adjuvanted commercial vaccine maintained about 2-fold higher levels of IgM;14 and 17 months after intraperitoneal injection, in comparison to unvaccinated fish kept in the same cage. Similar results were obtained by ELISA (serum IgM) and reverse transcription real time PCR (spleen mRNA). Analyses conducted in our lab have shown that several salmonid species possess two slightly different IgM isotypes as a result of ancestral tetraploidy. In Atlantic salmon, two distinct IgM subpopulations (A and B) can be separated by anion exchange chromatography. The IgM-B tetramer exhibits a higher degree of disulfide cross-linking than IgM-A, presumably due to an extra cysteine near the C-terminus of the heavy chain. The typical A/B ratio is approximately 40/60. Anion exchange elution profiles of serum IgM from vaccinated fish indicate that prolonged triggering of the immune system can lead to a skewed ratio of IgM-A/IgM-B. In the context of recent results from rainbow trout (Onchorhynchusmykiss), showing that high affinity antibodies are more highly polymerized and have a longer half life time, Atlantic salmon is an interesting model to elucidate these aspects further since tools are available to distinguish IgM-A and IgM- B in this species.展开更多
文摘Several toxic compounds are known to induce apoptosis in mammalian cell lines.The human neuroblastoma cells(SHSH-SY5Y)were exposed to the phosphatase inhibiting toxin okadaic acid(OA)or hydrogen peroxide(H2O2)to induce apoptosis as well as generate reactive oxygen species(ROS).Mitoxantrone(MXT)was used as a positive control for apoptosis.The SHSH-SY5Y cells were transfected with eukaryotic expression plasmid pHyPer-dMito encoding mitochondrial-targeted fluorescent or pHyPer-dCito encoding cytoplasmic-targeted fluorescent sensor for hydrogen peroxide(HyPer).The ERp57,also called GRP58(Glucose-regulated protein 58),is a stress protein induced in conditions like glucose starvation and viral infection.Recently ERp57 was shown to translocate from the endoplasmatic reticulum to the cell surface in anthracycline-induced apoptotic cells.ERp57 co-translocation together with calreticulin has been suggested to be crucial for recognizing tumor cells to induce immunogenic cell death.ERp57 translocation after exposure to okadaic acid was studied using immunofluorescence and confocal microscopy.These studies indicated that okadaic acid has induced the translocation of ERp57 to the cellular membrane.
文摘Primary and secondary antibody responses in blood of teleost fish are mainly IgM mediated, as they lack an IgG homolog and a class switch mechanism. Thus, the major serum immunoglobulin is tetrameric IgM. A unique antibody class in teleosts;named IgT, appears to be specialized for mucosal immunity and is present in low concentrations in serum. A third class;IgD was recently detected in serum of rainbow trout, but is less abundant than IgT. In the present study, relative quantification of total IgM showed that Atlantic salmon (Salmosalar) which had received an oil-adjuvanted commercial vaccine maintained about 2-fold higher levels of IgM;14 and 17 months after intraperitoneal injection, in comparison to unvaccinated fish kept in the same cage. Similar results were obtained by ELISA (serum IgM) and reverse transcription real time PCR (spleen mRNA). Analyses conducted in our lab have shown that several salmonid species possess two slightly different IgM isotypes as a result of ancestral tetraploidy. In Atlantic salmon, two distinct IgM subpopulations (A and B) can be separated by anion exchange chromatography. The IgM-B tetramer exhibits a higher degree of disulfide cross-linking than IgM-A, presumably due to an extra cysteine near the C-terminus of the heavy chain. The typical A/B ratio is approximately 40/60. Anion exchange elution profiles of serum IgM from vaccinated fish indicate that prolonged triggering of the immune system can lead to a skewed ratio of IgM-A/IgM-B. In the context of recent results from rainbow trout (Onchorhynchusmykiss), showing that high affinity antibodies are more highly polymerized and have a longer half life time, Atlantic salmon is an interesting model to elucidate these aspects further since tools are available to distinguish IgM-A and IgM- B in this species.
