Kinesins are microtubule-based motors involved in various intracellular transports. Neurons, flagellated cells, and pigment cells have been traditionally used as model systems to study the cellular functions of kinesi...Kinesins are microtubule-based motors involved in various intracellular transports. Neurons, flagellated cells, and pigment cells have been traditionally used as model systems to study the cellular functions of kinesins. Here, we report silkworm posterior silkgland （PSG）, specialized cells with an extensive endomembrane system for intracellular transport and efficient secretion of fibroin, as a novel model for kinesin study. To investigate kinesindriven intracellular transport in PSG cells, we cloned five silkworm kinesin-like proteins （KLPs）, BmKinesin-1, BmKinesin-6, BmKinesin-7, BmKinesin-13, and BmKinesin-14A. We determined their expression patterns by relative real-time PCR and western blotting. Immunofluorescence microscopy verified their colocalization with microtubules. By combining pull-down assays, LC-MS/MS, and western blotting analysis, we identified many potential cargoes of BmKinesin-1 in PSG, including fibroin-containing granules and exuperantia-associated ribonucleoprotein （RNP） complexes. Moreover, BmKinesin-13 overexpression disrupted the microtubule network in BmN cells, which is consistent with a role of Kinesin-13 in regulating microtubule dynamics in other organisms. On the basis of these results, we concluded that PSG might have advantages in elucidating mechanisms of intracellular transport in secretory tissues and could serve as a potential model for kinesin studies.展开更多
OBJECTIVE: To determine whether affected reticulocytes could be a reliable marker for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH), we analyzed CD59-antigen expression on the membranes of reticulocytes a...OBJECTIVE: To determine whether affected reticulocytes could be a reliable marker for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH), we analyzed CD59-antigen expression on the membranes of reticulocytes and erythrocytes. METHODS: We studied 10 PNH patients and 5 healthy volunteers by two-color flow cytometry with a membrane permeable fluorescent dye, thiazole orange (TO), and anti-CD59 monoclonal antibodies (MoAb). TO was introduced to gate reticulocytes and anti-CD59 MoAb were used to identify glycosylphosphatidylinositol (GPI)-deficient cells. RESULTS: Cells from healthy individuals were only CD59 positive. However, in all PNH patients, CD59-antigen expression could be divided into 3 types: type I cells (CD59 normally positive), type II cells (CD59 partly positive) and type III cells (CD59 negative). The majority of reticulocytes belonged to type III cells, GPI-deficient cells (61.0%). In addition, the percentage of affected reticulocytes was higher than that of erythrocytes. CONCLUSIONS: Analyzing PNH reticulocytes was important, because most patients had elevated numbers of reticulocytes, which represent more closely the recent erythroid output of BM. However, circulating mature erythrocytes were subject to complement-mediated intravascular lysis. Therefore, the percentage of abnormal erythrocytes may not accurately reflect the proliferation rate of normal and abnormal erythroid progenitor cells. Thus, affected reticulocytes could be a more reliable indicator for the diagnosis of PNH than mature erythrocytes.展开更多
基金Acknowledgments We wish to thank Prof GZ Zhang and Prof ZF Zhang at the Sericultural Research Institute of the Chinese Academy of Agricultural Sciences for B. mori strain and silkworm artificial diet, respectively. This work was supported by the National Natural Science Foundation of China （30670659, 30771086, 30721064）, the Major State Basic Research Development Program of China （973 Program） （2006CB500700, 2006CB910700, 2010CB833705）, and the National High Technology Research and Development Program of China （863 Program） （2006AA10A119）.
文摘Kinesins are microtubule-based motors involved in various intracellular transports. Neurons, flagellated cells, and pigment cells have been traditionally used as model systems to study the cellular functions of kinesins. Here, we report silkworm posterior silkgland （PSG）, specialized cells with an extensive endomembrane system for intracellular transport and efficient secretion of fibroin, as a novel model for kinesin study. To investigate kinesindriven intracellular transport in PSG cells, we cloned five silkworm kinesin-like proteins （KLPs）, BmKinesin-1, BmKinesin-6, BmKinesin-7, BmKinesin-13, and BmKinesin-14A. We determined their expression patterns by relative real-time PCR and western blotting. Immunofluorescence microscopy verified their colocalization with microtubules. By combining pull-down assays, LC-MS/MS, and western blotting analysis, we identified many potential cargoes of BmKinesin-1 in PSG, including fibroin-containing granules and exuperantia-associated ribonucleoprotein （RNP） complexes. Moreover, BmKinesin-13 overexpression disrupted the microtubule network in BmN cells, which is consistent with a role of Kinesin-13 in regulating microtubule dynamics in other organisms. On the basis of these results, we concluded that PSG might have advantages in elucidating mechanisms of intracellular transport in secretory tissues and could serve as a potential model for kinesin studies.
文摘OBJECTIVE: To determine whether affected reticulocytes could be a reliable marker for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH), we analyzed CD59-antigen expression on the membranes of reticulocytes and erythrocytes. METHODS: We studied 10 PNH patients and 5 healthy volunteers by two-color flow cytometry with a membrane permeable fluorescent dye, thiazole orange (TO), and anti-CD59 monoclonal antibodies (MoAb). TO was introduced to gate reticulocytes and anti-CD59 MoAb were used to identify glycosylphosphatidylinositol (GPI)-deficient cells. RESULTS: Cells from healthy individuals were only CD59 positive. However, in all PNH patients, CD59-antigen expression could be divided into 3 types: type I cells (CD59 normally positive), type II cells (CD59 partly positive) and type III cells (CD59 negative). The majority of reticulocytes belonged to type III cells, GPI-deficient cells (61.0%). In addition, the percentage of affected reticulocytes was higher than that of erythrocytes. CONCLUSIONS: Analyzing PNH reticulocytes was important, because most patients had elevated numbers of reticulocytes, which represent more closely the recent erythroid output of BM. However, circulating mature erythrocytes were subject to complement-mediated intravascular lysis. Therefore, the percentage of abnormal erythrocytes may not accurately reflect the proliferation rate of normal and abnormal erythroid progenitor cells. Thus, affected reticulocytes could be a more reliable indicator for the diagnosis of PNH than mature erythrocytes.
