Myelin-associated glycoprotein(MAG) inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially availabl...Myelin-associated glycoprotein(MAG) inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor(NgR) and paired immunoglobulin-like receptor B(PirB) was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase(ROCK) phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.展开更多
Amyotrophic lateral sclerosis(ALS)is a progressively fatal neuromuscular disorder classically characterized by loss of upper and lower motor neurons from the cortex to the spinal cord Diagnosed patients have a media...Amyotrophic lateral sclerosis(ALS)is a progressively fatal neuromuscular disorder classically characterized by loss of upper and lower motor neurons from the cortex to the spinal cord Diagnosed patients have a median survival of about 3 years and death usually results from eventual respiratory failure.展开更多
NIH/3T3 cells were infected with a cell-free extract from the mouse SRS ascltic tumor and propagated at vitro. Experimental results showed that type A and type C virus particles were observed in the cytoplasm of infec...NIH/3T3 cells were infected with a cell-free extract from the mouse SRS ascltic tumor and propagated at vitro. Experimental results showed that type A and type C virus particles were observed in the cytoplasm of infected NIH/ 3T3 cells by electron-microscopy, X-C assay and reverse transcriptase were both positive. Free proviral-DN A of the leukemia vims was found in the infected NIH/3T3 cell by Southern blot hybridization.Morphologically, the NIH/3T3 cells infected in vitro appeared spherical and formed monolayer cell colonies of various sizes after 24 - 48 hours. When the cultured cells were Inoculated into nude mice (BALB/ c, nu nu ) subcutaneously, flbrosarcoma was Induced, 100%. Inoculation of the cell-free extract of the same cultured cells into Inbred SW-1 newborn mice, resulted in the production of lymphocytic leukemia and lymphoma in 52. 3% within 191 days.展开更多
Rapid diagnostic methods for classifying avian leukosis subgroups in the field were needed for routine, large-scale screening. As a first step in method development, we inserted the avian leukosis virus subgroup A (A...Rapid diagnostic methods for classifying avian leukosis subgroups in the field were needed for routine, large-scale screening. As a first step in method development, we inserted the avian leukosis virus subgroup A (ALV-A) env gene into plasmid pcDNA3.1/Zeo (+) and used this construct to transfect DF-1 cells. Zeocin-resistant cells were obtained after 2 weeks of zeocin selection. Then, the cells were analyzed using PCR, immunofluorescence, and Western blot for expression of the envA-encoded envelope protein after 30 serial passages. The DF-1/A cell line was completely resistant to 104 TCIDso/0.1 mL (50% tissue culture infective dose)ALV-A and was partially resistant to 10~ TCIDs0/0.1 mL ALV-A viral particles. By comparing the DF-1/A and DF-1 cell lines, an ALV-A isolate was identified using a gag-specific ELISAfor capsid protein p27. Thus, we established a DF-1/A cell line that was resistant to ALV-A infection. This cell line will be useful as a diagnostic tool.展开更多
The hemolysin oligomer promotes the proliferation of B-1a cells and the expression of CD25, which is indicative of cell activation, on B-1a cells. The upregulation of CD86 induced by the oligomer showed its selective ...The hemolysin oligomer promotes the proliferation of B-1a cells and the expression of CD25, which is indicative of cell activation, on B-1a cells. The upregulation of CD86 induced by the oligomer showed its selective bias for the B7-2 member of B7 family while the monomer failed to induce these effects. The oligomer induced the expression of CXCR3, associated with B cell activation, while the monomer induced the expression of CXCL4, a powerful angiostatic chemokine. In conclusion, we found that B-1a cells responded to the apoptogenic monomer by expressing CXCL4, whereas oligomerization of the immunogen induced CXCR3 to shift the response towards activation.展开更多
Objective To study the action of brain angiotensin Ⅱ(Ang Ⅱ) receptors and underlying intracellular mechanism in the catecholaminergic system Methods Action potentials (APs) of the primary co cultured catecholamin...Objective To study the action of brain angiotensin Ⅱ(Ang Ⅱ) receptors and underlying intracellular mechanism in the catecholaminergic system Methods Action potentials (APs) of the primary co cultured catecholaminergic tumor (CATH.a) cells were recorded with the whole cell patch clamp configuration in current clamp mode. Expression of Ang Ⅱ receptors subtypes (AT 1 and AT 2 ) was detected by RT PCR technique. Results The differentiated CATH.a cells represented a neuron like characterization. All CATH.a cells expressed mRNA encoding both Ang Ⅱ AT 1 and AT 2 receptor subtypes. Ang Ⅱ increased the firing rate in the CATH.a cells, which was inhibited completely by addition administration of the AT 1 but not AT 2 receptor antagonist, and partially by using the inhibitors of signal molecules, U73122 (10 μmol·L -1 ), or KN 93 (10 μmol·L -1 ), or calphostin C (10 μmol·L -1 ). Conclusion Ang Ⅱ increases firing rate in CATH.a cells via AT 1 receptor. The CATH.a cells expressing functional AT 1 and AT 2 receptor subtypes may be of general utility for the study of the Ang Ⅱ receptor induced modulation of brain catecholaminergic system.展开更多
Activin A, which was first described in 1986, has been shown to maintain hippocampal neuronal survival. Activin A increases intracellular free Ca2+ via L-type Ca2+ channels. Our previous study showed that activin A ...Activin A, which was first described in 1986, has been shown to maintain hippocampal neuronal survival. Activin A increases intracellular free Ca2+ via L-type Ca2+ channels. Our previous study showed that activin A promotes neurite growth of dorsal root ganglia in embryonic chickens and inhibits nitric oxide secretion. The present study demonstrated for the first time that activin A could maintain cerebral cortex neuronal survival in vitro for a long period, and that activin A was shown to increase voltage-gated Na+ current (/Na) in Neuro-2a cells, which was recorded by patch clamp technique. The present study revealed a novel mechanism for activin A, as well as the influence of activin A on neurons by regulating expressions of vasoactive intestine peptide and inducible nitric oxide synthase.展开更多
Long-term quiescence or dormancy is a fundamental feature of cancer stem cells(CSCs)that are genetically identical to the malignant clone but constitute the only cells with tumor propagation potential within the overa...Long-term quiescence or dormancy is a fundamental feature of cancer stem cells(CSCs)that are genetically identical to the malignant clone but constitute the only cells with tumor propagation potential within the overall tumor population.These quiescent cells show significant resistance to radiation and antiproliferative chemotherapy due to distinctive properties that seem to be related to their stem cell-like character.Hence,successful anticancer therapy must consist of approaches that can target not only the differentiated cancer cells,but also the CSCs.Using serum-starved KG1a cell line as an experimental model system of quiescent leukemic cells(QLCs),the present study demonstrates that QLCs exposed to low concentrations of curcumin show high proliferative potential.Furthermore,when subjected to a combination therapy consisting of low concentrations of curcumin and 5-fluorouracil(5-FU),the QLCs displayed a high kill with an increase in the levels of nitric oxide(NO)and reactive oxygen species.These results were further consolidated with the observation of high caspase-3 activity in cells subjected to the combination therapy.This may suggest that low concentrations of curcumin stimulate the QLCs to become mitotically active,thereby sensitizing them to killing by the antimitotic drug,5-FU.展开更多
To study the expression activity of various vectors containing anti-caspase-3 ribozyme cassettes in vivo, and to further study the role of caspas-3 in the apoptotic pathway, we constructed anti-caspase-3 hammerhead ri...To study the expression activity of various vectors containing anti-caspase-3 ribozyme cassettes in vivo, and to further study the role of caspas-3 in the apoptotic pathway, we constructed anti-caspase-3 hammerhead ribozyme embedded into the human snRNA U6, and detected the activity of the ribozyme in vitro and in vivo. Meanwhile we compared it with the self-cleaving hammerhead ribozymes that we previously studied, and with the general ribozyme, cloned into RNA polymerase II expression systems. The results showed that the three ribozymes, p1.5RZ107, pRZ107 and pU6RZ107 had the correct structure, and that they could cleave caspase-3 mRNA exactly to produce two fragments: 143nt/553nt. p1.5RZ107 has the highest cleavage efficiency in vitro, almost 80%. However, the U6 chimeric ribozyme, pU6RZ107, has the highest cleavage activity in vivo, almost to 65%, though it has lower cleavage activity in vitro. The cleavage results demonstrated that the pU6RZ107, the U6 chimeric ribozyme, could more efficiently express and downregulate the level of caspase-3 in vivo, and the ribozyme could provide an alternative approach to the research into the mechanism of apoptosis and human gene therapy also.展开更多
基金supported by the National Natural Science Foundation of China,No.81171178
文摘Myelin-associated glycoprotein(MAG) inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor(NgR) and paired immunoglobulin-like receptor B(PirB) was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase(ROCK) phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.
基金supported by the NUS Graduate School for Integrative Sciences and Engineering
文摘Amyotrophic lateral sclerosis(ALS)is a progressively fatal neuromuscular disorder classically characterized by loss of upper and lower motor neurons from the cortex to the spinal cord Diagnosed patients have a median survival of about 3 years and death usually results from eventual respiratory failure.
文摘NIH/3T3 cells were infected with a cell-free extract from the mouse SRS ascltic tumor and propagated at vitro. Experimental results showed that type A and type C virus particles were observed in the cytoplasm of infected NIH/ 3T3 cells by electron-microscopy, X-C assay and reverse transcriptase were both positive. Free proviral-DN A of the leukemia vims was found in the infected NIH/3T3 cell by Southern blot hybridization.Morphologically, the NIH/3T3 cells infected in vitro appeared spherical and formed monolayer cell colonies of various sizes after 24 - 48 hours. When the cultured cells were Inoculated into nude mice (BALB/ c, nu nu ) subcutaneously, flbrosarcoma was Induced, 100%. Inoculation of the cell-free extract of the same cultured cells into Inbred SW-1 newborn mice, resulted in the production of lymphocytic leukemia and lymphoma in 52. 3% within 191 days.
基金The work was founded by the National Key R&D Program of China(2016YFD0501606)the Public Industry Research Program,the Ministry of Agriculture of China(201203055)+2 种基金the Program of Science and Technology Development of Guangdong Province,China(2015A020209145)the China Meat-Type Chicken Research System(CARS-42-G09)the Modern Agriculture Talents Support Program,Ministry of Agriculture of China([2012] no.160)
文摘Rapid diagnostic methods for classifying avian leukosis subgroups in the field were needed for routine, large-scale screening. As a first step in method development, we inserted the avian leukosis virus subgroup A (ALV-A) env gene into plasmid pcDNA3.1/Zeo (+) and used this construct to transfect DF-1 cells. Zeocin-resistant cells were obtained after 2 weeks of zeocin selection. Then, the cells were analyzed using PCR, immunofluorescence, and Western blot for expression of the envA-encoded envelope protein after 30 serial passages. The DF-1/A cell line was completely resistant to 104 TCIDso/0.1 mL (50% tissue culture infective dose)ALV-A and was partially resistant to 10~ TCIDs0/0.1 mL ALV-A viral particles. By comparing the DF-1/A and DF-1 cell lines, an ALV-A isolate was identified using a gag-specific ELISAfor capsid protein p27. Thus, we established a DF-1/A cell line that was resistant to ALV-A infection. This cell line will be useful as a diagnostic tool.
文摘The hemolysin oligomer promotes the proliferation of B-1a cells and the expression of CD25, which is indicative of cell activation, on B-1a cells. The upregulation of CD86 induced by the oligomer showed its selective bias for the B7-2 member of B7 family while the monomer failed to induce these effects. The oligomer induced the expression of CXCR3, associated with B cell activation, while the monomer induced the expression of CXCL4, a powerful angiostatic chemokine. In conclusion, we found that B-1a cells responded to the apoptogenic monomer by expressing CXCL4, whereas oligomerization of the immunogen induced CXCR3 to shift the response towards activation.
文摘Objective To study the action of brain angiotensin Ⅱ(Ang Ⅱ) receptors and underlying intracellular mechanism in the catecholaminergic system Methods Action potentials (APs) of the primary co cultured catecholaminergic tumor (CATH.a) cells were recorded with the whole cell patch clamp configuration in current clamp mode. Expression of Ang Ⅱ receptors subtypes (AT 1 and AT 2 ) was detected by RT PCR technique. Results The differentiated CATH.a cells represented a neuron like characterization. All CATH.a cells expressed mRNA encoding both Ang Ⅱ AT 1 and AT 2 receptor subtypes. Ang Ⅱ increased the firing rate in the CATH.a cells, which was inhibited completely by addition administration of the AT 1 but not AT 2 receptor antagonist, and partially by using the inhibitors of signal molecules, U73122 (10 μmol·L -1 ), or KN 93 (10 μmol·L -1 ), or calphostin C (10 μmol·L -1 ). Conclusion Ang Ⅱ increases firing rate in CATH.a cells via AT 1 receptor. The CATH.a cells expressing functional AT 1 and AT 2 receptor subtypes may be of general utility for the study of the Ang Ⅱ receptor induced modulation of brain catecholaminergic system.
基金the National Natural Science Foundation of China, No.30903123, 30901329the Project of Science and Technology of Jilin Province, No.20090741, 20090185
文摘Activin A, which was first described in 1986, has been shown to maintain hippocampal neuronal survival. Activin A increases intracellular free Ca2+ via L-type Ca2+ channels. Our previous study showed that activin A promotes neurite growth of dorsal root ganglia in embryonic chickens and inhibits nitric oxide secretion. The present study demonstrated for the first time that activin A could maintain cerebral cortex neuronal survival in vitro for a long period, and that activin A was shown to increase voltage-gated Na+ current (/Na) in Neuro-2a cells, which was recorded by patch clamp technique. The present study revealed a novel mechanism for activin A, as well as the influence of activin A on neurons by regulating expressions of vasoactive intestine peptide and inducible nitric oxide synthase.
文摘Long-term quiescence or dormancy is a fundamental feature of cancer stem cells(CSCs)that are genetically identical to the malignant clone but constitute the only cells with tumor propagation potential within the overall tumor population.These quiescent cells show significant resistance to radiation and antiproliferative chemotherapy due to distinctive properties that seem to be related to their stem cell-like character.Hence,successful anticancer therapy must consist of approaches that can target not only the differentiated cancer cells,but also the CSCs.Using serum-starved KG1a cell line as an experimental model system of quiescent leukemic cells(QLCs),the present study demonstrates that QLCs exposed to low concentrations of curcumin show high proliferative potential.Furthermore,when subjected to a combination therapy consisting of low concentrations of curcumin and 5-fluorouracil(5-FU),the QLCs displayed a high kill with an increase in the levels of nitric oxide(NO)and reactive oxygen species.These results were further consolidated with the observation of high caspase-3 activity in cells subjected to the combination therapy.This may suggest that low concentrations of curcumin stimulate the QLCs to become mitotically active,thereby sensitizing them to killing by the antimitotic drug,5-FU.
基金the High School Science Foundation of Shanghai (Grant No. 98QN60) and the Chinese Academy of Sciences (Grant No. KSCX2-2-04).
文摘To study the expression activity of various vectors containing anti-caspase-3 ribozyme cassettes in vivo, and to further study the role of caspas-3 in the apoptotic pathway, we constructed anti-caspase-3 hammerhead ribozyme embedded into the human snRNA U6, and detected the activity of the ribozyme in vitro and in vivo. Meanwhile we compared it with the self-cleaving hammerhead ribozymes that we previously studied, and with the general ribozyme, cloned into RNA polymerase II expression systems. The results showed that the three ribozymes, p1.5RZ107, pRZ107 and pU6RZ107 had the correct structure, and that they could cleave caspase-3 mRNA exactly to produce two fragments: 143nt/553nt. p1.5RZ107 has the highest cleavage efficiency in vitro, almost 80%. However, the U6 chimeric ribozyme, pU6RZ107, has the highest cleavage activity in vivo, almost to 65%, though it has lower cleavage activity in vitro. The cleavage results demonstrated that the pU6RZ107, the U6 chimeric ribozyme, could more efficiently express and downregulate the level of caspase-3 in vivo, and the ribozyme could provide an alternative approach to the research into the mechanism of apoptosis and human gene therapy also.